Or other statistics course with approval of the Graduate Group.
The degree and major requirements displayed are intended as a guide for students entering in the Fall of 2024 and later. Students should consult with their academic program regarding final certifications and requirements for graduation.
Code | Title | Course Units |
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Year 1 | ||
Fall | ||
Cell Biology | ||
CAMB First Year Seminar | ||
Molecular Basis of Genetic Therapies | ||
Lab Rotation | ||
Spring | ||
Regulation of the Genome | ||
Immunology for CAMB | ||
Immune Mechanisms | ||
Lab Rotation | ||
Lab Rotation | ||
Summer | ||
Pre-Dissertation Lab Rot | ||
Year 2 | ||
Fall | ||
Foundations in Statistics | ||
Pre-Dissertation Lab Rot | ||
Spring | ||
Scientific Writing | ||
Pre-Dissertation Lab Rot | ||
Year 3 and Beyond | ||
Dissertation |
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Excel in a number of different scientific careers
The Graduate Program in Cell & Developmental Biology (CDB) provides outstanding doctoral training in fundamental aspects of cell and developmental biology, ranging from stem cells to regenerative medicine and organogenesis to cancer biology. Research teams in CDB employ high-resolution microscopy, live single cell imaging, biochemistry-based methods, sophisticated genetics including gene editing, and systems-based strategies to understand the basic building block of life, the cell.
Research activities in CDB place a strong emphasis on quantitative and computational skills, cover gametes as well as highly differentiated and specialized cell types, and involve a broad array of model systems including budding yeast, flies, mice and human samples. CDB graduate students thus receive rigorous and comprehensive training and are integral members of a highly collaborative and collegial environment. Together these elements provide exceptional preparation for graduate students to excel in a number of different scientific careers.
Apply through our PIBS application
CDB faculty work in a very broad range of research areas:
Our faculty and students are extremely interactive and collaborative. Many CDB students have interdisciplinary projects involving multiple laboratories.
CDB is home to several special programs and research facilities:
The Microscopy and Image Analysis Laboratory (MIL) , a facility of more than 3,000 square feet housing state-of-the-art microscopy and imaging equipment, is housed within the Department of Cell and Developmental Biology.
The Center for Organogenesis is an interdisciplinary group of scientists working on basic mechanisms by which organs and tissues are formed and maintained, using this knowledge to create long lasting artificial organs, stem cell therapies or organ transplantation systems that will correct genetic and acquired diseases. The Center has run an NIH-supported training grant for over 15 years.
The Michigan Center for hES Cell Research was established in 2002, with funding from the Medical School and the NIH, for the study of human embryonic stem cells. The Consortium for Stem Cell Therapies is now part of the Taubman Institute and provides training and resources for students and faculty interested in ESC and iPSC stem cell research.
Course requirements are flexible; the goal is to establish a basic working knowledge of current concepts in molecular biology, cell biology, biochemistry, genetics, and neurobiology while allowing students to pursue more advanced coursework related to their research interests, such as developmental biology, bioinformatics, gene expression, signal transduction, etc. In your first year, you will take the PIBS curriculum, which also fulfils CDB requirements. When you choose a research laboratory in CDB, you will enroll in our Seminars in Cell and Developmental Biology course, CDB 801. You will also select additional program courses and electives from the rich offerings at the University of Michigan.
The preliminary exam consists of two checkpoints. The first is the preliminary exam in the form on a paper discussion. For students in good academic standing, this will take place in August at the end of their first year in order to advance to PhD candidacy. The second checkpoint takes place in the second year and is a vital component of the first thesis committee meeting. This checkpoint is comprised of two steps, the writing of a 6-page fellowship (NIH format) and the oral presentation of the proposed research to the thesis committee.
As part of their professional training, CDB students serve as graduate student instructors (GSIs) in a graduate-level course for one semester.
The usual time to degree is approximately 5 to 5 1/2 years.
CDB students run the course CDB 801 where they take turns presenting their research, give and get feedback on their research and presentations, explore career development opportunities, and interact with their fellow students in a supportive and cohesive environment. CDB students also participate in department committees, invite and host seminar speakers, help organize the annual departmental retreat, and plan/attend monthly happy hours.
CDB students have also expanded their skills and knowledge through university certificate programs such as those in teaching and translational science.
In recent years, a number of CDB students have been heavily involved in an outreach program called Developing Future Biologists (DFB). This is a graduate student-led educational organization that organizes and facilitates a week-long summer course aimed at teaching the next generation of biologists the fundamental concepts of developmental biology regardless of race, gender, or socioeconomic status.
In addition to scientific discovery, our mission is to train future leaders in cell and developmental biology, by encouraging high-impact research and providing teaching and mentorship of the highest quality.
Our students have received University and national recognition for their scholarship, research, and teaching, and their work is frequently published in high-impact journals (including recent papers in Nature Cell Biology, Science Signaling, Neuron, PLoS Biology, Current Biology, Developmental Cell, Molecular Cell, PLoS Pathogens, J Neurosci, J Virology, PNAS, J Cell Sci, EMBO J, Cell Metabolism, MCB, JBC, Blood, J Clin Invest, Development, etc.).
CDB graduates have gone on to successful careers in academic science, medical research, scientific consulting, and biotechnology.
Learn more about the Department of Cell & Developmental Biology.
We transform lives through bold discovery, compassionate care and innovative education.
Founding Director: Krzysztof Palczewski, PhD
Introduced just a decade ago, gene editing has swiftly emerged as one of the most promising frontiers in the quest for innovative approaches to restore DNA sequences.
At UC Irvine, our investigators have unlocked the ability to reverse and forestall cell degeneration. Currently, our researchers are tirelessly working to refine a method for directing this therapy to specific cells in need of correction. The pioneering approaches spearheaded by the Genome Editing Research Program, once realized, hold the potential to revolutionize not only the treatment of genetic disorders affecting vision but also a broader spectrum of inherited conditions.
The program has been designated as one of the 12 high-impact research programs to be housed in the Falling Leaves Foundation Medical Innovation Building.
Current world-renowned scientists and clinicians , alongside newly recruited experts, will collaborate in custom-designed labs to translate discoveries into cures.
Interdisciplinary teams will collaborate closely to model and comprehend inherited disorders, then develop and test therapies for the benefit of patients.
Future leaders will receive training from the forefront of science and medicine.
Indoor and outdoor gathering spaces will facilitate events, speakers and presentations, fostering engagement and dialogue to expedite and share discoveries.
Empowered by the distinctive One Health approach of UCI Health Affairs that transcends disciplinary boundaries, the Genome Editing Research Program comprises faculty and staff from the Susan & Henry Samueli College of Health Sciences (encompassing schools of medicine, nursing, pharmacy and pharmaceutical sciences, population and public health) and UCI Health, our regional healthcare delivery system which includes the trailblazing Susan Samueli Integrative Health Institute. By nurturing collaborative efforts among brilliant minds, our teams can surpass the limitations of working in isolation. This collaborative approach enables us to push the boundaries of innovation and make fundamental discoveries in the field of gene therapy.
The strength of the Genome Editing Research Program lies in a critical mass of multifaceted research laboratories, each focusing on complementary aspects of function and disease, ranging from structural biology and genetics to physiology and pharmacology. Dedicated investigators collaborate to model and understand disorders, subsequently developing and testing therapies to benefit patients.
In the realm of retinal research, our program is addressing the urgent need for effective treatments for inherited retinal diseases, which currently have limited therapeutic options. Our innovative use of base and prime editing has led to the correction of mutations responsible for conditions such as Leber Congenital Amaurosis and Retinitis Pigmentosa, not only restoring vision from the retina to the visual cortex but also offering a protective shield for photoreceptors against degeneration. These groundbreaking advancements have been published in prestigious journals such as Cell, Nature Biotechnology, Nature Biomedical Engineering, Nature Communications and PNAS . We are dedicated to refining our genome engineering techniques and developing new delivery methods to ensure our genome editing is safer, more effective and more precise. This effort demonstrates our steadfast commitment to advancing the field of genetic therapy.
Glaucoma is the second leading cause of irreversible blindness worldwide. We are paving the way to innovative treatments through studies that successfully target the mutant MYOC gene using the CRISPR-Cas9 system, significantly reducing mutant myocilin levels in the trabecular meshwork and preventing the onset of glaucoma in models. This research is instrumental in developing targeted delivery systems for various mutations associated with glaucoma. Our holistic approach, combining cutting-edge research with compassionate patient care, ensures our work goes beyond laboratory experiments to make a significant difference in patients’ lives. Through collaborative efforts, we are creating a unique research environment conducive to discovering breakthroughs in the treatment of ocular diseases.
We have assembled, and will seek to expand, an exceptional group of scientists with a keen understanding of genome editing to optimize research collaborations. Faculty leadership includes:
Founding Director of the Genome Editing Research Program; Director of the Center for Translational Vision Research
Professor, Ophthalmology, Biomedical Engineering
Associate Professor, Ophthalmology, Biomedical Engineering
Professor, Biomedical Engineering, Molecular Biology and Biochemistry
Professor, Chemistry, Molecular Biology and Biochemistry, Pharmaceutical Sciences
Professor, Ophthalmology, Physiology & Biophysics
We invite philanthropic partners to collaborate with us in establishing a new home for the UCI Genome Editing Research Program within the Falling Leaves Foundation Medical Innovation Building, and in providing essential support for our programmatic funding to drive transformative discoveries.
The architectural design of the Falling Leaves Foundation Medical Innovation Building is tailored to facilitate vibrant collaborations that translate inspiration into tangible results. You have the exclusive opportunity to name the new headquarters for the Genome Editing Research Program and custom-designed laboratories and gathering spaces. As a philanthropic partner, your contribution will ensure that our scientists have the necessary space, resources and facilities to advance their life-changing work.
Research endowments play a pivotal role in sustaining our investigators, who are at the forefront of shaping the future of science and healthcare. External grants cover only a fraction of associated expenses, underscoring the importance of endowed funding in supporting innovative discoveries and pioneering approaches. Endowed grants bear special significance, offering the flexibility to pursue scientific avenues that lead to breakthroughs. Your generous support will enable this vital work to be carried out under the auspices of our philanthropic partners. Additionally, named endowed scholarships will further enrich the training of future generations of leading investigators and healthcare providers in perpetuity.
By partnering with us as a philanthropic sponsor to name an endowed chair, you will establish a lasting connection between your legacy and UCI’s esteemed experts. Ultimately, advancements in biomedical research will drive the development of innovative therapies. Philanthropic contributions towards endowed chairs, research and scholarships ensure sustained support, facilitating the recruitment and retention of top-tier faculty who are instrumental in maintaining UCI’s status as a world-class center of excellence in emerging therapies. Endowed chairs are particularly impactful as they establish a perpetual annual funding stream, empowering the chair-holder to focus their efforts on groundbreaking research initiatives.
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genes • gene editing • heredity• evolution • genetic variability • phenotypic variability • horizontal gene transfer • meiosis • recombination • epigenetics • DNA repair and replication • chromosome segregation • cell division • gene regulation • development • aging • pathogenesis • cancer • disease
Iain m. cheeseman, olivia corradin, gerald r. fink, mary gehring, alan d. grossman, leonard p. guarente, michael t. hemann, h. robert horvitz, david housman, siniša hrvatin, tyler jacks, chris a. kaiser, kristin knouse, eric s. lander, michael t. laub, ruth lehmann, troy littleton, david c. page, peter reddien, francisco j. sánchez-rivera, anthony j. sinskey, graham c. walker, yukiko yamashita.
David Bartel studies molecular pathways that regulate eukaryotic gene expression by affecting the stability or translation of mRNAs.
School of medicine, ph.d. program.
The Johns Hopkins Human Genetics and Genomics Training Program provides training in all aspects of human genetics and genomics relevant to human biology, health and disease.
Advances in human genetics and genomics continue at an astounding rate and increasingly they are being integrated into medical practice. The Human Genetics and Genomics Program aims to educate highly motivated and capable students with the knowledge and experimental tools that will enable them to answer important questions at the interface between genetics and medicine. Ultimately, our trainees will be the leaders in delivering the promise of genetics to human health.
The overall objective of the Human Genetics program is to provide our students with a strong foundation in basic science by exposure to a rigorous graduate education in genetics, genomics, molecular biology, cell biology, biochemistry and biostatistics as well as a core of medically-related courses selected to provide knowledge of human biology in health and disease.
This program is also offered as training for medical students in the combined M.D./Ph.D. program. Students apply to the combined program at the time of application to the M.D. program. (See section entitled Medical Scientist Training Program).
Research laboratories are well equipped to carry out sophisticated research in all areas of genetics. The proximity to renown clinical facilities of the Johns Hopkins Hospital, including the Department of Genetic Medicine, and Oncology Center provides faculty and students with access to a wealth of material for study. Computer and library facilities are excellent. Laboratories involved in the Human Genetics Program span Johns Hopkins University; consequently supporting facilities are extensive.
The program is supported by a training grant from the National Institute of General Medical Sciences. These fellowships, which are restricted to United States citizens and permanent United States residents, cover tuition, health care insurance and a stipend during year one. Once a student has joined a thesis lab, all financial responsibilities belong to the mentor. Students are encouraged, however, to apply for fellowships from outside sources (e.g., the National Science Foundation, Fulbright Scholars Program, Howard Hughes Medical Institute) before entering the program.
Applicants for admission should show a strong academic foundation with coursework in biology, chemistry and quantitative analysis. Applicants are encouraged to have exposure to lab research or to data science. A bachelor's degree from a qualified college or university will be required for matriculation. GREs are no longer required.
The Human Genetics and Genomics site has up-to-date information on “ How to Apply .” For questions not addressed on these pages, please access the contact information listed on the program page: Human Genetics and Genomics Training Program | Johns Hopkins Department of Genetic Medicine .
The program includes the following required core courses: Advanced Topics in Human Genetics, Evolving Concept of the Gene, Molecular Biology and Genomics, Cell Structure and Dynamics, Computational Bootcamp, Pathways and Regulation, Genomic Technologies, Rigor and Reproducibility in Research, and Systems, Genes and Mechanisms of Disease. Numerous elective courses are available and are listed under sponsoring departments.
Our trainees must take a minimum of four electives, one of which must provide computational/statistical training.
The HG program requires the “OPTIONS” Career Curriculum offered by the Professional Development and Career Office. OPTIONS is designed to provide trainees with the skills for career building and the opportunity for career exploration as well as professional development training
Human Genetics trainees also take a two-week course in July at the Jackson Labs in Bar Harbor, Maine entitled "Human and Mammalian Genetics and Genomics: The McKusick Short Course" which covers the waterfront from basic principles to the latest developments in mammalian genetics. The faculty numbers about 50 and consists roughly in thirds of JAX faculty, Hopkins faculty and “guest” faculty comprising outstanding mammalian geneticists from other US universities and around the world.
The courses offered by the faculty of the program are listed below. All courses are open to graduate students from any university program as well as selected undergraduates with permission of the course director.
Trainees must complete three research rotations before deciding on their thesis lab. They must also participate in the Responsible Conduct of Research sessions offered by the Biomedical Program; starting at year 3, students must attend at least two Research Integrity Colloquium lectures per year.
Our trainees participate in weekly journal clubs, department seminars, monthly Science & Pizza presentations as well as workshops given twice a year on diversity, identity and culture.
At the end of the second year, trainees take their Doctoral Board Oral Examination. Annual thesis committee meetings must be held following successful completion of this exam.
Average time for completion is 5.3 years.
Code | Title | Credits |
---|---|---|
Advanced Topics in Human Genetics | 1.5 | |
Introduction to Rigor and Reproducibility in Reseach | ||
Evolving Concepts of the Gene | 5 | |
Introduction to Responsible Conduct of Research | 1 | |
Human Genetics Boot Camp | 2 | |
Cell Structure and Dynamics | 1.5 | |
Molecular Biology and Genomics | 1.5 | |
Independent Research | 1 - 18 | |
Systems, genes and mechanisms in disease | 3 | |
Genomic Technologies: Tools for Illuminating Biology and Dissecting Disease | 1.5 | |
Understanding Genetic Disease | 0.5 | |
Pathways and Regulation | 2 |
Graduates from the Human Genetics program pursue careers in academia, medicine, industry, teaching, government, law, as well the private sector. Our trainees are encouraged to explore the full spectrum of professional venues in which their training my provide a strong foundation. Driven by curiosity and a desire for excellence, our trainees stand out as leaders in the chosen arenas of professional life. They are supported in the development of their career plans by a program faculty and administration who are dedicated to their success, and by a myriad of support networks across the Johns Hopkins University, many of which are provided by the Professional Development Career Office of the School of Medicine.
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at the same time they enhance soil quality. With the support of Azolla Biotech Ltd. and Cranfield University the student will use CRISPR-Cas9 gene editing and Agrobacterium-mediated transformation methods
models and gene editing . Our goal is to design novel protein-based gene writers with improved efficacy and safety. The candidate will train large language and diffusion models to provide new protein
Parkinson’s Disease. The researcher will endeavor to solve some of the biggest problems in the field, using cutting-edge technologies and concepts, e.g. CRISPR gene editing , stem cell technology, human brain
-leading research in several fields. Notably, the groundbreaking discovery of the CRISPR-Cas9 gene - editing tool, which was awarded the Nobel Prize in Chemistry, was made here. At Umeå University, everything
cell technology. Experience with RNA sequencing, gene editing (CRISPR-Cas9), single particle imaging, and large data set analysis. Proficiency in programming languages like R, python, Matlab Online
University of Iowa Stead Family Department of Pediatrics seeks a faculty member at the rank of Research Assistant Professor (non-tenure track) level in the research area of gene therapy
to as STING (stimulator of interferon genes ) that is activated by DNA based microbes such as viruses, bacteria, and parasites. He is interested in developing viral oncolytic agents and immunotherapeutic
Phd student in computing science with a focus on efficiently monitoring and managing of large-scale cloud edge systems, phd position in chemistry, searches related to gene editing.
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Hematopoietic specification using single-cell in vivo crispr screen (mok_u25dtp1), phd research project.
PhD Research Projects are advertised opportunities to examine a pre-defined topic or answer a stated research question. Some projects may also provide scope for you to propose your own ideas and approaches.
This project is in competition for funding with other projects. Usually the project which receives the best applicant will be successful. Unsuccessful projects may still go ahead as self-funded opportunities. Applications for the project are welcome from all suitably qualified candidates, but potential funding may be restricted to a limited set of nationalities. You should check the project and department details for more information.
Funded phd project (students worldwide).
This project has funding attached, subject to eligibility criteria. Applications for the project are welcome from all suitably qualified candidates, but its funding may be restricted to a limited set of nationalities. You should check the project and department details for more information.
Self-funded phd- understanding the molecular mechanism of a bacterial genome defence system and its synergy with crispr-cas, self-funded phd students only.
This project does not have funding attached. You will need to have your own means of paying fees and living costs and / or seek separate funding from student finance, charities or trusts.
Investigate the molecular mechanism controlling feme (fast endophilin mediated endocytosis) in directed cancer cell migration using crispr, biochemistry, and advanced microscopy methods., using crispr in ips cells to modify platelet function, decoding the epigenetic mechanisms of drug resistance in aggressive breast cancers, in vivo reprogramming of extracellular vesicles for targeted drug delivery of genome editors to the central nervous system, using single-cell nanopore long-read sequencing to identify drivers of neurodegenerative disorders, understanding the physiological roles and pathological impacts of er-autophagy, mechanism evaluation of synthetic lethality in cancers of mesenchyme, decoding prokaryotic adaptive immunity and pathways to therapeutic innovation, transcriptional kinases in cancer and developmental disorders, dfg-funded phd position (f/m/x) in protein biochemistry.
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Engineered lipid nanoparticles drive durable gene editing in lungs of mice.
Gene-editing therapies —techniques that modify DNA to treat or prevent disease—have the potential to transform the field of drug development. By making precise edits to the genome, problematic genes could be modified or eliminated, representing long-lasting therapies for genetic disorders that currently have no treatment.
Few gene-editing therapies currently exist. Last year, the FDA approved a gene-editing therapy for sickle cell disease, a landmark treatment that uses CRISPR to modify blood stem cells. But like most experimental gene-editing treatments, this therapy requires a stem cell transplant, a costly and time-consuming procedure that involves removing stem cells from a patient, modifying them to correct defects causing the disease, and reintroducing them back to the patient. Finding a way to modify a patient’s stem cells in vivo —without having to remove them from the body—could revolutionize the field of genome editing.
Researchers at UT Southwestern are working to bring in vivo gene editing to the fore. Through rational engineering of lipid nanoparticles, this collaborative team developed a way to effectively target specific organs in the body to precisely deliver therapeutic cargo, including gene-editing molecules. Their research demonstrated that a one-time treatment with their nanoparticles resulted in durable gene editing in mouse lungs for nearly two years. Further, their technique showed promise in correcting a mutation present in a currently untreatable form of cystic fibrosis in several models of the disease. The research was recently published in Science .
“There is a real desire to imagine a one-time injection of a medicine that could correct mutations that are causing and driving diseases,” said senior study author Daniel Siegwart, Ph.D., a professor at UT Southwestern Medical Center. “Our preclinical platform illustrates a potential method to achieve long-term gene editing in the lungs, representing a new treatment approach for a variety of genetic respiratory conditions.”
Lipid nanoparticles, a popular drug delivery method thanks to the success of mRNA COVID-19 vaccines, are typically comprised of four different lipids (fats) that envelop their therapeutic cargo. While these traditional lipid nanoparticles are effective at transporting and protecting their payload, when infused into a patient, they accumulate in one specific organ: the liver.
“Traditional lipid nanoparticles are remarkably similar to low-density lipoprotein (LDL) in terms of their size and chemical composition,” explained Siegwart. “LDL particles naturally travel to the liver to be broken down, so it makes sense that lipid nanoparticles accumulate there as well.”
To enable delivery to tissues other than the liver, Siegwart and colleagues previously developed a new class of particle, termed selective organ targeting (SORT) nanoparticles. In addition to the four conventional lipids found in traditional lipid nanoparticles, SORT nanoparticles contain a fifth lipid that directs the particles to a specific organ. This fifth lipid affects the physiochemical properties of the nanoparticle and attracts distinct plasma proteins to its surface, two factors that influence uptake by different types of tissues in the body.
“We knew that we needed to break the rules of traditional lipid nanoparticle formulations to target tissues other than the liver,” Siegwart said. “Using SORT, we’ve shown that we can direct nanoparticles specifically to the liver, spleen, and lungs of mice. Whether this technique could result in durable, tissue-specific gene editing remained an open question.”
Getting the lipid nanoparticles to the right organ is an important first step. But for effective gene editing, it’s also important to target the right type of cell—specifically, stem cells and progenitor cells, or cells that can become different types of cells.
“It’s estimated that the cells in the lungs of rodents turn over and regenerate every few months or so,” said Siegwart. “If you achieve genome correction in mature cells, the effects will be temporary: as soon as the new cells are born, they will no longer have those corrected events, and malfunctioning genes will be produced again.”
To understand if their SORT nanoparticles could achieve durable genome editing in the lungs, Siegwart and colleagues used a genetically engineered mouse that has the capacity to make a red fluorescent protein—but only if its genome is edited in a specific way. “It’s a beautiful model because it allows us to quantify which specific cell types have been edited by the nanoparticles, and we can easily visualize where these nanoparticles are effective,” he said.
The researchers administered SORT nanoparticles filled with gene-editing molecules to mice and then evaluated lung tissues at ten different time points, ranging from two days to 22 months after the injections. They found that the red fluorescence was uniformly spread throughout the lungs at every time point. Further, cell-specific analyses revealed that genome editing was achieved in multiple different types of cells, and that the editing was sustained over the course of the study, nearly two years after treatment with the nanoparticles.
“When we tracked the animals over time, we found that these genome editing events were completely persistent, and almost two years later, the animals were equally edited as they were on the second day,” Siegwart said. “This indicates that the nanoparticles can successfully edit stem and progenitor cell populations that then differentiate over time into healthy, edited cells.”
With a method to achieve long-term gene editing in hand, Siegwart and colleagues turned their attention to a genetic lung disease: cystic fibrosis.
Cystic fibrosis is caused by mutations in a chloride pump, a protein that can regulate the concentration of salt inside and outside of the cell, Siegwart explained. “When this protein malfunctions, there’s an improper salt balance, leading to thick and sticky mucus building up in the lungs. This can lead to a whole host of different respiratory issues, infections, and eventually persistent lung damage that may lead to the necessity of having a lung transplant.”
Roughly 90 percent of patients with cystic fibrosis can be treated with a breakthrough medicine that helps this chloride pump function more efficiently. However, some patients have mutations in their genome that lead to a truncated, non-functional form of this protein—or the protein is not made at all. These patients currently have no approved treatment options.
In this backdrop, Siegwart and colleagues focused on one of these “undruggable” cystic fibrosis mutations. They filled their lung SORT nanoparticles with a gene editor that corrects the mutation, turning the truncated chloride pump into the normal version of the protein. Then they evaluated how well their system performed in cystic fibrosis models.
In experiments using patient-derived cystic fibrosis lung cells, the researchers found that lung SORT could correct the faulty gene, effectively restoring the function of the chloride pump by more than 50%. What’s more, in a mouse model carrying this undruggable human cystic fibrosis mutation, the researchers found that lung SORT could achieve gene correction in nearly 50% of lung stem cells.
“Our early results suggest that this technique could someday correct dysfunctional proteins in the lungs, which would absolutely be transformative in the daily lives of cystic fibrosis patients,” Siegwart said.
Jermont Chen, Ph.D., a program director in the Division of Discovery Science and Technology at NIBIB, agreed: “Through clever modifications of standard lipid nanoparticles, this team has laid the groundwork for an in vivo gene editing platform in the lungs, which could potentially be translated to other tissues down the line. While future work in relevant animal models will be required before this technique can be evaluated in humans, the method described here has the potential to lead to long-lasting treatments for patients with genetic conditions.”
Note: Siegwart is a co-founder of the company ReCode Therapeutics, a partner in this study.
This study was supported in part by a grant from NIBIB (R01EB025192).
This science highlight describes a basic research finding. Basic research increases our understanding of human behavior and biology, which is foundational to advancing new and better ways to prevent, diagnose, and treat disease. Science is an unpredictable and incremental process—each research advance builds on past discoveries, often in unexpected ways. Most clinical advances would not be possible without the knowledge of fundamental basic research.
Study reference: Yehui Sun et al., In vivo editing of lung stem cells for durable gene correction in mice. Science 384 ,1196-1202(2024). DOI: 10.1126/science.adk9428
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The National Institutes of Health has awarded a $14 million grant to the Perelman School of Medicine and the Children’s Hospital of Philadelphia for gene-editing research.
This funding, provided through the NIH’s Somatic Cell Genome Editing Program , will support gene-editing research aimed at developing treatments for rare metabolic diseases. Researchers will aim to target developing therapies for urea cycle disorders, which affect approximately one in every 35,000 children, according to the Penn Medicine announcement.
Central to the research is a new, more advanced form of CRISPR technology known as prime editing. This technology allows for more precise changes in the genome, allowing for the correction of any genetic mutation rather than simply replacing DNA bases.
With prime editing, there is potential for the personalized treatment of many rare metabolic diseases, including life-threatening disorders like type I citrullinemia, ASA lyase deficiency, and CPS1 deficiency. Prime editing would allow permanent genetic corrections, mitigating previous challenges of the body’s immune response to treatments.
Derek Griffith named Penn Integrates Knowledge with appointments in Penn Nursing, Penn Med
Penn Health System revenue passes $10 billion for first time, marking nearly a 10% increase
Kiran Musunuru , a professor of cardiovascular medicine and director of Penn Cardiovascular Institute's Genetic and Epigenetic Origins of Disease Program, spoke with Penn Med about prime editing.
“With this technology, we hope to not just manage symptoms, but offer a durable, potentially lifelong cure for these children,” he said.
The SCGE program is specifically designed to tackle diseases resulting from genetic mutations, focusing on developing innovative genome-editing tools and therapies. The four-year grant aims to develop a method that can support the creation of personalized gene-editing therapies for a broad spectrum of rare genetic disorders.
Now entering its second phase, the program is looking to translate gene-editing advancements from the laboratory into clinical practice. During its initial phase, spanning 2018 to 2023, SCGE focused on developing the fundamental tools required for genome editing in non-reproductive cells in the body.
Rebecca Ahrens-Nicklas , an attending physician with the Metabolic Disease Program and the Division of Human Genetics at CHOP, told Penn Med that the program's scope is broader than focusing on one more specific disease.
"We’re focusing on the patient in front of us, whatever variant they have,” Ahrens-Nicklas said. “This approach enables us to treat a wider array of patients who’ve previously had no options.”
Having previously received a grant from the SCGE program, the team is now focused on advancing research toward clinical trials, which it hopes to begin within the next four years.
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SHANGHAI and MIDDLETOWN, Del. , Oct. 21, 2024 /PRNewswire/ -- HuidaGene Therapeutics (“HuidaGene”), a global clinical-stage biotechnology company pioneering CRISPR-based programmable genome medicines, today announced the appointment of Dr. TJ Cradick as Chief Technology Officer. In this role, Dr. Cradick will further drive innovation and development of delivery vectors and gene editing tools through computational biology, artificial intelligence (AI), machine learning (ML), and other tools and methodologies.
A recognized leader in genome editing technologies, Dr. Cradick has made significant contributions to the development of nucleases and gene therapy methods, particularly for CRISPR/Cas nucleases, and before that, TAL Effector Nucleases (TALENs) and Zinc Finger Nucleases (ZFNs). His gene editing industry experiences began at Sangamo Therapeutics in 2000, with recent roles at CRISPR Therapeutics and Excision BioTherapeutics. When he was Chief Scientific Officer (CSO) at Excision, Dr. Cradick led the development of the first in-vivo CRISPR-based systemic treatment targeting latent HIV DNA reservoirs, currently under evaluation in clinical trials in the United States . Additionally, as Head of Genome Editing at CRISPR Therapeutics, he contributed to the approval of the groundbreaking therapy Casgevy™, the first-ever approved CRISPR-based treatment.
“We are excited to welcome TJ to the leadership team at this pivotal moment in our growth,” stated Alvin Luk , PhD., MBA, CCRA, co-founder and CEO of HuidaGene. “I have known TJ since before at UCSF, when we were colleagues. I have had the pleasure of working alongside Prof. Yang, who has been instrumental in developing our gene editing tools. TJ will now take the baton to optimize and enhance these tools to further advance our clinical programs. His vast expertise in gene editing will be key in advancing our mission to bring life-changing genomic medicines to patients worldwide.”
Prof. Hui Yang , co-founder and Chief Scientific Advisor at HuidaGene added, “I am delighted to welcome TJ to the team. His remarkable track record applying gene editing technologies to a range of diseases aligns perfectly with HuidaGene’s vision. I am confident that TJ’s leadership will further accelerate our efforts to develop cutting-edge tools and therapies that could transform the lives of patients around the world.”
Dr. Cradick’s achievements include developing gene editing assays, bioinformatics tools, and concepts that are widely used in genome editing research. He holds an undergraduate degree from MIT , an M.A. in Microbiology and Immunology from the University of California, San Francisco , and a PhD in Molecular and Cell Biology from the University of Iowa .
“I am thrilled to join HuidaGene at such an exciting time,” commented Dr. TJ Cradick. “The innovative work being done here has the potential to revolutionize how we approach gene editing. I look forward to collaborating with the talented team on new means to develop genomic medicines.”
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SOURCE HuidaGene Therapeutics
Selangor, Malaysia, & Biopolis, Singapore 18/09/2024 – 19/09/2024 9:00 am – 4:45 pm
Technological change is occurring at an unprecedented rate in the global agri-food value chain. New biotechnologies are a major part of this transformational change, including precision agriculture technology and new plant breeding techniques such as gene editing for targeted modification of plant genomes. In combination with innovative practices for monitoring and managing food and agricultural systems, they are opening up new pathways to delivering sustainable food production systems of the future.
It is, thus, important that key stakeholders along the value chain and regulatory authorities share the latest scientific knowledge on the
safety assessment, updates on status and advancement as well as regulatory approach towards potential adoption of these technologies. There is also increasing recognition of needs to engage and educate the broader community and consumers through innovative communication tools to impart knowledge and mitigate any misperception of these technologies.
This seminar to be held back-to-back in Singapore and Malaysia aims to bring together key stakeholders to share scientific updates and facilitate stakeholders’ engagement, addressing issues and perspectives on new biotechnologies, safety assessment, regulatory development and communication strategies.
Note: Topic and title of presentations are subject to change upon final confirmation with invited speakers.
Dato' Dr Mohamad Zabawi bin Abdul Ghani, Director General, Malaysian Agricultural Research and Development Institute (MARDI), Malaysia
Mr Timothy Harrison, Regional Agricultural Counselor, USDA Foreign Agricultural Service (FAS)
Mr Geoffry Smith, ILSI SEA Region
Prof Wayne Allen Parrott, Professor, University of Georgia, USA
Mr Amin Asyraf Tamizi, Research Officer, Agri-Omics and Bioinformatics Programme, Biotechnology and Nanotechnology Research Centre, MARDI, Malaysia
Dr Kumitaa Theva Das, Senior Lecturer, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia
Dr Tan Yong Quan, Covering Specialist Team Lead, Bioengineering, National Centre for Food Science, Singapore Food Agency, Singapore
Dr Gabriel Romero, Executive Director, Philippines Seed Industry Association (PSIA), Philippines
Dr Mahaletchumy Arujanan, Executive Director, Malaysian Biotechnology Information Center (MABIC), Malaysia
Prof Dr Rofina Yasmin Othman, Honorary Professor, Institute for Advanced Studies, Universiti Malaya, Malaysia
Ms Li Xin Kang, Genetic Modification Advisory Committee (GMAC) Secretariat, Singapore
Prof Paul Teng, Dean and Managing Director, National Institute of Education International (NIEI), Nanyang Technology University, Singapore
Prof. Teng has won numerous awards for his work such as the Eriksson Prize in Plant Pathology (Royal Swedish Academy of Science), an Honorary Doctor of Science (from Murdoch University, Australia) and is a Fellow of the American Phytopathological Society, the International Society of Plant Pathology, and The World Academy of Sciences (TWAS). He is a Past-Chair, Genetic Modification Advisory Committee, Singapore and is also Immediate Past Chairman, International Service for the Acquisition of Agri-biotech Applications (ISAAA) Inc. He has edited/co-edited fifteen books, authored/co-authored four books and published over 250 technical papers. His latest edited book (2024) is “Food Security Issues in Asia”.
Assoc Prof Tan Meng How, Associate Professor, Nanyang Technological University (NTU) & Genetic Modification Advisory Committee (GMAC), Singapore
Dr Andrew D. Powell,CEO, Asia BioBusiness Pte. Ltd., Singapore
Ms Mazlina Banu Jaikubali, Genetic Modification Advisory Committee (GMAC) Secretariat, Singapore
If you have any inquiries, please do not hesitate to contact the following:
For Malaysia: Dr. Rogayah Sekeli and/or Dr. Zulkifli Ahmad Seman Malaysian Agricultural Research and Development Institute (MARDI) Email: [email protected] ; [email protected]
For Singapore: ILSI Southeast Asia Region Singapore Tel: 65 6352 5220 Email: [email protected]
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Ruslan kalendar, alexandr v shustov, ilyas akhmetollayev, ulykbek kairov.
Edited by: Pier Paolo Piccaluga , University of Bologna, Italy
Reviewed by: Alsamman M. Alsamman , Mohammed VI Polytechnic University, Morocco
Nosheen Masood , Fatima Jinnah Women University, Pakistan
*Correspondence: Ruslan Kalendar, [email protected]
ORCID: Ruslan Kalendar, orcid.org/0000-0003-3986-2460 ; Alexandr V. Shustov, orcid.org/0000-0001-9880-9382 ; Ilyas Akhmetollayev, orcid.org/0000-0002-6219-4002 ; Ulykbek Kairov, orcid.org/0000-0001-8511-8064
This article was submitted to Molecular Diagnostics and Therapeutics, a section of the journal Frontiers in Molecular Biosciences
Received 2021 Sep 10; Accepted 2022 Feb 2; Collection date 2022.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Polymerase chain reaction (PCR) is a simple and rapid method that can detect nucleotide polymorphisms and sequence variation in basic research applications, agriculture, and medicine. Variants of PCR, collectively known as allele-specific PCR (AS-PCR), use a competitive reaction in the presence of allele-specific primers to preferentially amplify only certain alleles. This method, originally named by its developers as Kompetitive Allele Specific PCR (KASP), is an AS-PCR variant adapted for fluorescence-based detection of amplification results. We developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. In addition, the tool allows discrimination of up to four-allelic variants at a single SNP site. To increase both the reaction specificity and the discriminative power of SNP genotyping, each allele-specific primer is designed such that the penultimate base before the primer’s 3′ end base is positioned at the SNP site. The tool allows design of custom FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user-friendly and powerful Java-based software that is freely available ( http://primerdigital.com/tools/ ). Using the FastPCR environment and the tool for designing AS-PCR provides unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, which translates into a greater likelihood of research success.
Keywords: genotyping assay design software, polymerase chain reaction-based markers, diagnostic system, genotyping system, single nucleotide polymorphism, insertion-deletion polymorphism
High-throughput technologies for nucleotide sequence analysis and detection of sequence variation have been increasingly used for plant and animal genotyping, forensics, genetic medicine, and other fields of genetic testing. The most frequently encountered sequence variations in any genome are single-nucleotide polymorphisms (SNP) ( Clevenger et al., 2015 ). The primary consideration for selecting a marker type for genotyping is the information content of the polymorphisms and the informative capacity of the test. In this regard, SNPs are very effective markers. A variety of SNP genotyping methods are available, and new methods appear regularly with the aim of reducing cost and increasing throughput. PCR can be adapted for rapid detection of single-base changes in genomic DNA by using a family of closely related methods, such as allele-specific PCR (AS-PCR) ( Ugozzoli and Wallace, 1991 ; Bottema and Sommer, 1993 ; Spierings et al., 2006 ), PCR-amplification of specific alleles (PASA) ( Sommer et al., 1989 ; Sarkar et al., 1990 ), allele-specific amplification (ASA), and amplification refractory mutation system (ARMS) ( Newton et al., 1989 ; Nichols et al., 1989 ; Wu et al., 1989 ). All these methods use specifically designed PCR-primer sets containing allele-specific primers (ASP) in which the allele specificity is determined by the base at or near the 3′ end. In principle, AS-PCR assays can be developed to analyse almost any allelic variation ( Sommer et al., 1989 ; Ye et al., 1992 ).
There currently exists an enormous number and variety of established methods applicable for SNP analysis and genotyping based on distinctly different platforms and approaches ( Kim and Misra, 2007 ). FRET (Fluorescence Resonance Energy Transfer) is one such method and is based on dual fluorescence that is quantified during ligation. Another method is allele-specific PCR with SNP targeting ( Didenko, 2001 ; Kaur et al., 2020 ). Single-plex PCRs for SNP analysis, such as TaqMan (Life Technologies, USA) and SimpleProbe (Roche Applied Science, USA) have been described as valuable additions for marker-assisted selection in plant breeding ( Makhoul et al., 2020 ; Della Coletta et al., 2021 ). Further improvements for AS-PCR include using fluorescence to detect the amplification of a specific allele. Kompetitive Allele Specific PCR (KASP) (LGC Biosearch Technologies, Teddington, UK) and PCR Allele Competitive Extension (PACE) (Integrated DNA Technologies, Inc.) genotyping represent developments to the original AS-PCR approach. These techniques use fluorescence resonance energy transfer (FRET) for signal generation and both allow accurate bi-allelic discrimination of known SNPs and insertion-deletion polymorphisms (InDels). The KASP method has great potential for expanded utilization, as it requires only a slight modification to commonly used procedures for designing ASPs, offers convenient FRET detection, and master-mixes are commercially available ( Wang et al., 2015 ; Ryu et al., 2018 ; Brusa et al., 2021 ). In KASP, PCR amplification is performed using a pair of allele-specific (forward) primers and a single common (reverse) primer. In addition to the common-core primers, the reaction mixture is supplemented with a FRET cassette. This is a duplex of two synthetic complementary oligonucleotides; one is labelled with a fluorescent dye and the other carries a fluorescence quencher. Further, each ASP has a unique 5′ terminal extension (tail), which is complementary to the sequence in the FRET cassette. The oligonucleotides in the FRET cassette are modified such that they do not participate in polymerase-mediated extension steps. The dye-labelled oligonucleotide is capable of annealing to the reverse-complement of the tail sequence in PCR fragments containing one selected allele. During amplification of the allele with participation of the tailed ASP, an amount of DNA increases to which the dye-labelled component anneals. This disrupts the integrity of the FRET cassette and the fluorescent dye is spatially separated from the quencher and thus able to emit fluorescence. Unlike other PCR-based genotyping assays, KASP/PACE requires no labelling of the target-specific primers/probes, which provides additional flexibility in the assay design. Both methods use two reporting cassettes. If a genotype at a given SNP is homozygous, only one of the two possible fluorescent signals will be generated. If the genotype is heterozygous, both fluorescent signals will be generated. KASP/PACE technology is especially suitable for high-volume screening projects, such as in plant breeding. KASP/PACE technology has a key feature, which is utilizing a universal FRET cassette reporter system that eliminates the need for dual-labelled probes. Commercial companies produce PCR additives, called master mix, which contains one or more ‘universal’ FRET cassette(s). In theory, this additive can be used to upgrade existing AS-PCR assays to KASP/PACE, provided that new ASPs are used that have the described cassette-specific tails. For example, a protocol upon which this work is based, uses chemistry consisting of the following two parts. These are the assay mix (template-specific, contains target DNA, two ASPs [for binary SNP] and a single reverse primer) and master mix (not template-specific, combines all reagents required for PCR and in addition contains two different FRET cassettes, one cassette labelled with FAM dye and the other with HEX dye). Each ASP carries a unique tail sequence that corresponds to a FRET cassette. In other FRET AS-PCR methods, such as Amplifluor ( Rickert et al., 2004 ; Fuhrman et al., 2008 ) and STARP ( Rasheed et al., 2016 ; Long et al., 2017 ; Li et al., 2019 ; Wu et al., 2020 ), these two parts are completely separated into non-labelled ASPs such that the last base of the primer’s 3′ end base is positioned on the SNP site, and labelled universal probes (UPs) that carries a fluorophore at the 5′ terminus and a quencher attached in the middle of the universal probes ( Nazarenko et al., 1997 ). Simple and inexpensive ASPs can be designed and ordered for each SNP separately, while the relatively expensive UPs with fluorophores and quenchers are ordered just once for a stock that can be used over a very long time in many different SNP analyses. The principle of ASP-UP is similar to that of Molecular Beacons, with the addition of specialized identical “tags” at the 5′ end of the ASP and the 3′ end of the UP. In this particular case, ASPs are slightly longer ( Myakishev et al., 2001 ). In KASP-related methods, a set of non-labelled ASPs includes two forward primers and a single common reverse primer that act on a competitive basis in conjunction with one of two corresponding UPs with “hair-pin” FRET structures that end with either FAM or HEX/VIC fluorophores. This approach allows for great flexibility in assay design, which translates into a higher overall success rate for SNP genotyping and detection of InDels. This principle of separated ASPs and UPs is used in various methods, including commercially produced Amplifluor (Merck KGaA) and KASP markers (LGC Biosearch Technologies) for fluorescent signal generation that enable bi-allelic discrimination and genotyping of SNPs or InDels.
Developing high-throughput, multiplex genotyping assays require using computerized approaches to design primers and probes and select reaction conditions. In recent years, several software tools have been developed to aid AS-PCR assay development, including GSP ( Wang et al., 2016 ), PrimerSearch-EMBOSS ( http://emboss.open-bio.org/rel/rel6/apps/primersearch.html ), WASP ( Wangkumhang et al., 2007 ), PolyMarker ( Ramirez-Gonzalez et al., 2015 ), KASPspoon ( Alsamman et al., 2019 ), and PUNS ( Boutros and Okey, 2004 ). However, not all of these programs are readily available or broadly applicable; some are no longer actively updated whilst others are extremely narrow in their applicability.
Here we describe a software tool and a modified KASP method that further increases the convenience and power of the genotyping protocol. We have named this allele-specific quantitative PCR (ASQ). The software facilitates the development of assays using KASP and PACE technology and works with SNP and InDel polymorphisms. For all polymorphisms taken into the study, the program produces the thermodynamically optimal combination of ASPs for single-plex or multiplex assays. During ASP design, a user can add a tail sequence (which can be a custom sequence) or a sequence that matches a commercially available aster mix (e.g., manufactured by LGC Biosearch Technologies, or Merck KGaA). If a commercial FRET cassette is not optimal, the program allows use of custom FRET cassettes. The program also computes the optimal reaction conditions to perform KASP. This software will be most useful for multiplex genotyping in a high-performance environment. Furthermore, potential uses are not limited to genotyping. The program can produce tests for selecting mutants, e.g., after genome editing or gene knockout ( Lee et al., 2016 ). Other possible applications include analysis of genetic variations in microorganisms, strain identification, detecting genetic markers of resistance or virulence, and tracing pathogens in epidemiology and medical diagnostics. The ASQ method described here is a tool for assessing the relative amount of different allelic variants in a sample. This method can be useful for a variety of tasks. For example, ASQ can be used to measure a fraction of admixed GMO material in samples of agricultural products or to measure a portion of malignant cells in samples from cancer patients. Distinctive features of the presented program make it unique in its class. These features include computing of multiplex reactions for simultaneous identification of up to four alleles (e.g., 3-state or 4-state polymorphisms); use of input polymorphisms of mixed type (overlapping SNP and InDel sites); and absence of restrictions on the length difference between InDel alleles during genotyping of InDels. The program automatically calculates primers for possible amplification in both directions from the polymorphic site. The program allows the user to include in the protocol commercially available master mixes and FRET cassettes or to create unique FRET cassettes and use custom reagents in designing other assays. The software described herein is integrated into and works inside the FastPCR environment. FastPCR is an integrated software space for developing PCR-based processes. The FastPCR program, including the presented AS-PCR-designing tool, may be used online or downloaded and used in Microsoft Windows ( Kalendar et al., 2011 ; Kalendar et al., 2017 ) and is freely available at http://primerdigital.com/tools/ . This AS-PCR tool simplifies and automates design of genotyping assays, resulting in a greater likelihood of success.
Selection of highly informative snp candidates.
SNP data in the SNPforID browser ( http://spsmart.cesga.es/snpforid.php ) were employed in the present study for SNP genotyping of humans in forensic studies. The allelic frequencies were used for screening to select highly informative SNP candidates. As markers with even allelic distributions have high observed heterozygosity and are more informative, 7 of the SNPs were selected with a common 40:60-60:40 allelic distribution in European and Central Asian populations. All of the selected markers are located on autosomal chromosomes ( Supplementary Table S1 ). The marker also known as “C/T(-13910)” located in the MCM6 gene but with influence on the lactase LCT gene (rs4988235) is one of two SNPs that is associated with the primary haplotype associated with hypolactasia, or more commonly known as lactose intolerance in European populations.
This work was discussed by the institutional review board and was approved by the ethical committee of the Center for Life Sciences, National Laboratory Astana, Nazarbayev University (protocol #21, 10 October 2017). Institutional written informed consent about nationality declaring, DNA extraction and for further investigation was signed and obtained from the participated individuals.
Informed consent was obtained for each participant. DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). The extracted DNA pellet was diluted in TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA), and DNA concentration was measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). Quality was assessed using 1 mg of DNA visualised after running in a 1% agarose gel.
A QuantStudio-7 Real-Time PCR instrument (Thermo Fisher Scientific, USA) and CFX96 Real-Time PCR Detection System (Bio-Rad, USA) were used. These instruments have detection systems with filters for FAM, VIC/HEX, Cy3, Cy5, and ROX fluorophores. While SNP identity calls were made automatically using software accompanying the instruments, amplification curves were checked for each genotype manually for final allele discrimination. SNP genotyping experiments used at least three to eight biological replicates and were repeated three times.
The PCR conditions employed an altered PCR cocktail composition ( Table 1 ). PCR plates with 96-wells were used with a 15 μl total reaction volume in each well. The PCR mix consisted of the following reagents: 1x OneTaq buffer (total 3 mM MgCl 2 ), 0.2 mM of each dNTP, 0.2 μM of each UP, 0.5 μM quencher oligo, 0.1 μM of each AS primer, 0.3 μM of reverse primer, and 0.5 units of Taq DNA polymerase (NEB). Half of the PCR volume was genomic DNA, adjusted to 5 ng/μl.
PCR cocktail mix composition for the proposed ASQ method of SNP genotyping.
Component | Concentration | Volume (µl) | Final concentration |
---|---|---|---|
5x OneTaq Buffer (with 9 mM MgCl ) | 5× | 20 | 1× |
MgCl | 25 mM | 4.8 | 1.2 mM |
DNTP | 10 mM | 2 | 0.2 mM |
ASP-F1 | 5 µM | 2 | 0.1 µM |
ASP-F2 | 5 µM | 2 | 0.1 µM |
ASP-R | 5 µM | 6 | 0.3 µM |
UP-FAM | 10 µM | 2 | 0.2 µM |
UP-HEX | 10 µM | 2 | 0.2 µM |
Uni-Q | 50 µM | 1 | 0.5 µM |
Taq DNA Polymerase | 5 units/µl | 0.8 | 0.04 units/µl |
Milli-Q water | 37.4 | ||
DNA template | 10 ng/μl | 20 | 2 ng/μl |
Total | 100 |
The PCR program was optimised and consisted of 95°C, 120 s; 10 cycles of 10 s at 95°C; 20 s at 55°C; 20 s at 68°C; 30 cycles of 10 s at 95°C; 30 s at 68°C; 30 s at 55°C ( Table 2 ). Fluorescence was monitored during the last step at a second annealing. ASQ was performed to simultaneously detect two alleles in a single tube. Each well was examined for the characteristic fluorescent emissions of both fluorescein (FAM channel) and HEX (HEX channel).
PCR protocol for genotyping using advanced ASQ method for SNP genotyping.
Step | Temperature | Duration (sec) | Notes |
---|---|---|---|
1 | 95°C | 120 | Initial denaturation |
2 | 95°C | 10 | First-round denaturation |
3 | 55°C | 20 | First-round annealing |
4 | 68°C | 20 | First-round extension |
5 | 10 cycles repeat for steps 2–4 | ||
6 | 95°C | 10 | Second-round denaturation |
7 | 68C | 30 | Second-round annealing and extension |
8 | 55°C | 30 | FRET cassette annealing and signal read |
9 | 30 cycles repeat for steps 6–8 |
Genotyping with SNP calling was determined automatically. Each experiment was repeated twice and technical replicates confirmed a confidence of SNP calls.
Standard KASP allows genotyping of only two alternative alleles at any specific site. This is because there are two only FRET cassettes, and thus two fluorochromes, present in commercially available kits. However, advances in real-time PCR equipment allow detection of up to 6 spectrally separated dyes.
In addition, there are approaches to overcome physical spectral dye channels and expand the potential of spectral channels to practically detect an unlimited number of independent targets ( Marras et al., 2019 ). Theoretically, the number of different multiplex targets or alternative alleles that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colours for the identification of each target allele ( Marras et al., 2019 ). The novel tool described here allow detection of up to 4 spectrally separated dyes ( Figure 1 ). Moreover, the program calculates even more assay possibilities because the ASPs are searched for on both strands and at both sides from a polymorphism site ( Figure 2 ). The user has the possibility to use the single most suitable set of primers from the output or, if desired, alternative sets of primers may be used to target the same site. The tool also allows design combinations of tail sequences to create a multiplex PCR targeting several different polymorphic sites simultaneously. There is a need to incorporate flexibility in a primer-designing algorithm to select AS primers, as placing the 3′-terminal nucleotide on the polymorphic site may not be an optimal solution for some sites. Briefly, to achieve optimal amplification, the tool automatically selects the primer length and the position of its 3′ terminus for each ASP. This kind of optimization is also necessary when working with InDels. For example, for a null-site polymorphism (i.e., one or more nucleotide[s] absent in one allele or there are insertions in other alleles), the ASP sequence to target this allele will be positioned such that the primer’s 3′-terminal sequences are sufficient for annealing. Thus, ASP sequences are properly adjusted for each allelic variant. In case of short InDels (<4 nucleotides), the better solution is to individually design the sequence of the ASP 3′ termini for each variant, which can be achieved with this tool. Thus, the algorithm allows the generation of ASPs with 3′-terminal sequences that are unique for each allelic variant. This tool works within the FastPCR environment, which contains numerous functions that are necessary for the development of PCR primers for most applications. These include complex PCR designs, such as multiplex PCR or Loop-mediated Isothermal Amplification (LAMP). Regardless of the intended application, PCR primer design using FastPCR’s algorithms attempts to generate sets of primers with a high likelihood of success. For this purpose, there is an automatic check for unwanted binding sites (non-specific priming control) in input sequences to prevent the generation of side PCR products. Inputs may be linear or circular sequences. Moreover, the FastPCR environment is expandable, allowing addition of specialized functions for specific tasks.
Four-plex fluorescent ASQ assay genotyping system compared to the standard two-plex KASP technology (LGC Biosearch Technologies). The main differences in these approaches are associated with the potential number of simultaneously detectable polymorphic sites (4 in ASQ, 2 in KASP) and the structure of the primers that compose the FRET cassette. For ASQ, the FRET cassette consists of 2 or more of allele-specific primers (ASP) and a fluorescently labelled universal probe (UP) with a single universal quencher probe (Uni-Q). Differences in the tail sequence ASPs and UPs are determined by a unique 6-nt barcode sequence that is not part of the universal tail of the Uni-Q sequence. KASP technology includes 2 variants of ASPs and fluorescently labelled UPs (UP-1/2) with each UP requiring a specific quencher (SP1/2-Q). In addition, the ability to design an allele-specific primer with the SNP site at the penultimate or antepenultimate 3′ base of each allele-specific primer is characteristic of the ASQ method. (A) Both allele-specific primers query the SNP locus. Denaturation of DNA template and annealing ASP to the target, PCR round 1. (B) Formation of a PCR product containing a specific tail sequence that is complementary to allele-specific primers. This PCR product will be used in subsequent PCR cycles as a template for amplification using a specific fluorescently labelled UP (C) . During the first two amplification cycles, a tail sequence is incorporated into the amplicon that is subsequently recognized by a universal, probe-based reporter system.
Flowchart of the main steps in the AS-PCR process. Data can be uploaded into FastPCR, which accepts a single sequence or multiple separate DNA sequences in FASTA, tabulated format, multiple alignment, or from clipboard. SNP sequence data are pre-processed by FastPCR prior to execution to collect the SNP position and also to remove non-DNA symbols from the entire sequence. The user sets the various parameters for primer design for the DNA or SNP sequences, such as primer maximum and minimum lengths and T m . Repetitive or low-complexity sequences are excluded from primer design by default (this can be changed by the user). The initial primer design attempts both directions and a reverse primer is designed at the upstream accordingly. An allele-specific primer with the SNP site is designed at the second (penultimate) position of the primer’s 3′ terminus or to any other position in manual or automatic mode. Once the allele-specific primers are designed, an individual 5′-tail sequence is added to the 5′ end of each allele-specific sequence. This combined whole primer sequence is reanalysed for self- and cross-dimerization. Finally, results are returned to the text-editor window.
Components of FastPCR are programmed in Java and thus the program is intrinsically multiplatform. Entering inputs and execution and post-processing of the output can be performed with ease. The selection of the optimal target for KASP primer sets is performed in the same way as for PCR primers. The thermodynamic stability of hairpins is evaluated primarily for the 3′-terminal sequence and, if desired, for the primer’s 5′ terminus as well. The user may select thermodynamic options that will be used to design primer sets (within different templates) or individually set the options for each template in a set of several ones. The program calculates several primer pairs or sets from which the user may choose the best. The user can request a desired product size or instruct the software to search for primer pairs for diagnostic PCR, in which case a whole input sequence will be used to search for the best primer pair. By default, the program selects primers sets with compatible melting temperatures. Alternatively, the user may choose primer sets with similar Gibbs free energy (dG). The tool checks for self-dimer and cross-dimer interactions for all primers. Additionally, users may design compatible primer sets for a predetermined primer (probe) or a list of predetermined primers (probes). This feature can be useful when designing a multiplex assay for different targets. FastPCR allows avoiding non-specific amplification by choosing the best paired primer for a given oligonucleotide to achieve the highest likelihood of success.
To this end, the program chooses primers that avoid repeats or other stable motifs, like potential G-quadruplex sequences. Results with program-suggested primers and primer sets may be exported in common exchange formats (e.g., MS Excel). The following parameters may be placed into spreadsheets: primer name, sequence, location on target, sequence direction, length, melting temperature, GC content, molecular weight, molar extinction coefficient, linguistic complexity (LC), and primer quality (PQ).
For primer pairs, the recommended temperature of the annealing step, product size (in bp), and the T m of a PCR product will be provided. For pairs, the results include primers for individual sequences, primers compatible with each other, product sizes, and annealing temperatures. For all selected primer pairs, the program provides (in tabular form) the compatibility of the two primers in one reaction, including primer-dimers, cross-hybridization, and product size overlaps ( Figure 2 ).
Users will find examples for entering sequences encoding polymorphic sites in the File menu of the FastPCR software. Input sequences can be in FASTA, in tabular formats, or as multiple sequence alignment as given in the examples. Square brackets are used to specify borders of a polymorphic site of interest. The alternative alleles of a SNP are indicated by brackets, e.g., [C/G] (alternative way to code—[S]). Individual allelic variants in a polymorphic site are thus listed as [variant1/variant2] for binary polymorphism or [variant1/variant2/variant3/variant4] for quaternary polymorphism. Alternatively, IUPAC notation can be used for a degenerate base (e.g., FASTA format) ( Supplementary material ). When a polymorphic site contains InDels, the same format with square brackets ([variant1/variant2/variant3/variant4]; two or more different variants per site) is used to describe individual variants. Only differences between the variants must be shown within the brackets ( Supplementary material ). The program allows primers to be designed with the SNP placed at any position with respect to the 3’ end. To do this, the user must specify the desired SNP position using the command with the position number. By default, the program automatically specifies the SNP position at a penultimate base (-aspcr). In addition, for InDel sites, the number of discreditable nucleotides at the 3′ end of each ASP can be automatically determined depending on the polymorphism variant. For InDels, the potential of 3′-end generation for each ASP design option is wider, thus offering the possibility of a more effective discrimination of the various alleles. The program will also determine the minimum length of the 3′ end of each ASP for effective detection of all polymorphisms. The user can define the position of the polymorphic site in any of the positions, such as at a first or penultimate or another base in 3′ end of each ASP to increase reaction specificity and allele discrimination. The 3′-end sequence of each ASP should be designed such that each ASP is unique and specific only to its target. The program uses the sequence from the insertion to generate the 3′-end of ASP sequence for a null-site polymorphism. In the case of a single- or two-base InDel, the following sequence after target site will be used to generate the 3′ end of each ASP. The maximum length that the program can use to develop unique 3′ ends of ASP sequences in the program is 12 nucleotides. This should be sufficient for any InDel polymorphism to generate unique sequences for all 3′ ends of ASPs. The primer sequences will be optimized for similar thermodynamic parameters ( T m , dG) ( Peyret et al., 1999 ; Bommarito et al., 2000 ). To align the thermodynamic parameters of each ASP, the program extends the length for the low- T m primer sequence to approximate the T m for a primer with more G/C groups at the 3′ end.
In our experiments, we used four UPs with types of specific tails. Sequences of the four UPs are presented in Table 3 ; an example of one such ASP for the human SNPs is shown. All SNP-specific primers and UPs labelled with FAM, HEX, Cy3, and Cy5 were synthesized locally (National Center for Biotechnology, Nur-Sultan, Kazakhstan). A stock of each probe and a single quencher (Uni-Q) was prepared by dissolving each in TE buffer. A working stock of all four probes and a quencher was used for assays. A Uni-Q was conjugated with BHQ1 at the 3′ end. The sequence of Uni-Q was complementary to all corresponding probes. The quenchers were shorter in nucleotide length (13 nt) than the UPs (19 nt). The T m of this quencher oligonucleotide and the complementary probe was 55°C (for oligonucleotide concentration of 500 nM in 55 mM KCl with 2.2 mM Mg 2+ (3 mM Mg 2+ minus 0.8 mM dNTP) ( Owczarzy et al., 2008 ) as calculated by FastPCR ( Kalendar et al., 2017 ). The UPs were designed to have a higher T m (66–68°C). At an annealing-extension temperature of 60–68°C, the AS primers and UPs can bind the target and induce polymerization without much interference from the lower T m of the quencher oligo. When the temperature is subsequently decreased to 55°C, the Uni-Q binds the tail of the free, single-stranded UPs, but not the double-stranded PCR product. Because the Uni-Q concentration is two- to four-fold higher than that of each UP, most of the free UPs are expected to bind the quencher oligo at 55°C, thus strongly quenching the UP fluorescence. Since the 5′ end of the UP tail is opposite to the 3′ end of the quencher oligo, the interaction is mediated via direct contact-quenching between the 5′ fluorophore and the 3′ quencher present on the tail and quencher oligo, respectively, which for most fluorophores provides stronger quenching than fluorescence resonance energy transfer (FRET) ( Fiandaca et al., 2001 ; Li et al., 2006 ). The 3′ BHQ1 was used as it has a wide range of absorbance wavelengths and is appropriate for quenching multiple fluorophores simultaneously, including the FAM and Cy5 used for multiplex PCR.
Fluorescent probes (UP) and a quencher oligonucleotide (Uni-Q) for Allele-Specific Quantitative PCR (ASQ).
Primer ID | Sequence (5′-3′) | nt | (°C) | dG (kcal/mole) | GC (%) | LC (%) | Type and fluorescent label |
---|---|---|---|---|---|---|---|
Uni-Q | Accgttcagctgg | 13 | 53.3 | −16.7 | 61.5 | 100 | Universal 3′ Quencher (Eclipse Quencher or 3′ Black Hole Quencher 1) |
UP1 | ccagctgaacggtACGGCA | 19 | 67.4 | −26.4 | 63.2 | 86 | UP1: 5′-FAM |
UP2 | ccagctgaacggtCGTTGC | 19 | 66.5 | −26.4 | 63.2 | 95 | UP2: 5′-HEX/JOE/VIC |
UP3 | ccagctgaacggtAGCCGA | 19 | 66.9 | −26.1 | 63.2 | 89 | UP3: 5′-Cy3/TAMRA |
UP4 | ccagctgaacggtGCGTCA | 19 | 67.9 | −26.8 | 63.2 | 92 | UP4: 5′-Cy5/Liz |
Capital letters used for a unique barcode sequence; underlined letters used for the universal tail in UPs, and Uni-Q.
Melting temperature ( T m ) calculated for oligonucleotide concentration of 200 nM in 55 mM KCl with 2.2 mM Mg 2+ .
Linguistic Complexity (%).
The SNP site in the AS primers of each locus were designed to directly flank the site in both the sense and antisense orientation. The position of the polymorphic site was located in the penultimate base in the 3′ end of each ASP. Amplification primers were designed such that the amplicons ranged in size from 100 to 200 bases and encompassed the SNP site.
The remaining allele-specific region of ASPs was selected to obtain a T m close to 63°C (range 62–64°C, calculated for oligonucleotide concentration of 200 nM, 55 mM K + , in the present of 2.2 mM Mg 2+ using FastPCR software) ( Kalendar et al., 2014 ). The initial primer design was first attempted for the current strain and then a complement strain direction was attempted and a reverse primer was designed at the upstream accordingly. Once the allele-specific primers were designed, an individual 5′-tail sequence was added to the 5′ end of each allele-specific sequence. This combined whole primer sequence was reanalysed for self- and cross-dimerization.
The validation of the proposed ASQ method was performed using SNP for genotyping humans. We designed eight ASQ sets for informative SNP candidates that could be used for genotyping humans and tested their genome specificity using PCR assays. Validation tests were performed for ASQ with quantitative PCR-based experimentation. The corresponding primer sequences are listed in Supplementary Table S1 . PCR reactions using primers designed by our tool were performed and the resulting products are presented in Figure 3 . In each case, bands of the correct size were obtained from each PCR. These ASQ sets showed genome specificity and identified SNP alleles in the human genome ( Supplementary Table S1 ). SNP allele discrimination for 16 human genotypes is presented in Figure 3 . These results for SNP for genotyping of humans validated our conclusion that the proposed ASQ method is very accurate and effective for SNP genotyping; we expect similar results in other animal species. In addition, we developed ASQ sets and applied SNP genotyping in the barley genes HvSAP16 and HvSAP8 (controlling stress-associated proteins); these are validated examples and have been accepted for publication ( Baidyussen et al., 2021 ; Kalendar et al., 2022 ). Our tool and the ASQ method are suitable for two-, three-, or four-allelic uniplex reactions but can potentially be used for different SNPs in a multiplex format in a range of applications, including medical or forensic studies or other studies involving SNP genotyping.
Validation and testing of ASQ method. Probes for ASQ assays were designed using FastPCR to human SNPs (rs7520386 and rs1454361) that were employed for SNP genotyping of humans in forensic studies and PCR products from a series of PCR reactions using these primers were examined by the agarose gel electrophoresis was used without staining (A) . M—Thermo Scientific GeneRuler DNA Ladder Mix (100–10,000 bp) stained with SYBR Green I. Primer sequences are displayed in Supplementary Table S1 . PCR bands of the correct size (123 bp for rs7520386 and 168 bp for rs1454361, respectively) were obtained from each qPCR. (B) qPCR amplification plot for SNP (rs1454361). FAM plot (green) shows amplifications of a A-allele, whereas HEX plot (blue) shows amplifications of a T-allele.
We compared the functionality of the tool described here with other web-based software available online. Only software programs capable of AS-PCR designs were compared ( Table 4 ). One example of an AS-PCR-directed program is WASP ( https://bioinfo.biotec.or.th/WASP ) ( Wangkumhang et al., 2007 ). We used example allele sequences published on the WASP website to produce ASPs (primers were computed with WASP and with the KASP/PACE tool) ( Supplementary Data S2 ). It appeared that the primer design in WASP has some limitations, e.g., WASP does not allow addition of user-defined 5′ tail sequences to ASPs. Additionally, WASP searches for ASP binding sites from only one side (upstream) of a polymorphism site. Furthermore, it appeared that when attempting to target an ASP on an SNP site, WASP places an additional deliberate mismatch at the penultimate base of AS primers. This peculiarity in the primer-design algorithm was proposed by WASP developers as they sought to increase specificity of allele discrimination. However, using ASPs with deliberate mismatches in the 3′ terminal region is controversial, at least because such mismatches are known to decrease overall PCR efficiency and may lead to complete PCR failure.
Comparison between the KASP tool (in the FastPCR suite) and other AS-PCR programs (web-based).
Feature | WASP | PolyMarker | FastPCR |
---|---|---|---|
Web site | |||
Platform | Web server | Web server | Java Web Start (Oracle) |
Primer-designing algorithm | Primer3 | Primer3 | FastPCR |
Detection limit of SNP/InDel alleles | 2 | 2 | 2-(4)-any |
SNP genotyping | yes | yes | yes |
InDel genotyping | no | no | yes |
Primer-binding site selection to one side or both sides from the polymorphic site | One side | One side | Both sides |
Variable base positioning in ASPs (for SNP targeting) related to the primer’s 3′ terminus | At the first base, an additional deliberate mismatch is introduced at the penultimate base | At the first base | At the first, second, or third base, and automatic selection based on thermodynamic calculations |
Uses multiple sequence alignment of alleles as input | no | yes | yes |
Allows user-defined 5′ tails in ASPs | no | no | yes |
Multiplex reaction design | no | no | yes |
Inclusion of commercial (e.g. LCG or Merck KGaA) or custom FRET cassette | no | no | yes |
Analysis for primer self-dimers and cross-dimers in all multiplexed primer sets | no | no | yes |
/dG selection for thermodynamic comparisons | no | no | yes |
Probe design (TaqMan, MGB) | no | no | yes |
Identification of low- and high-complexity sequences with automatic adjustment of primer positions | no | no | yes |
BLAST test | no | yes | no |
Ability to automatically adjust algorithm to the sequence complexity of an input template | no | no | yes |
Potential for wide range applications beyond genotyping (such as diagnostics, quantitation of alleles) | no | no | yes |
In contrast, when targeting SNPs our AS-PCR tool automatically places a variable base at the second (penultimate) position of the primer’s 3′ terminus. The selection of an exact position (of the variable base) is driven by thermodynamic considerations. In our opinion, the latter solution provides greater flexibility in equilibrating thermodynamic parameters for all ASPs targeting existing alleles. In this regard, it was published that 3′-terminal and internal oligonucleotide mismatches differ on the effect on duplex stability. Mismatches at the oligonucleotide’s very 3′ terminus actually stabilize its duplex with a template, whereas internal mismatches can be either stabilizing or destabilizing. A destabilizing effect for internal mismatches occurs when there are unfavourable constraints on the geometry of hydrogen bonding in a DNA duplex ( Santalucia and Hicks, 2004 ). In general, the destabilizing effect is more pronounced for mismatches in the penultimate or third position (at the 3′ terminus) and when A or T is a terminal base. These considerations work in our AS-PCR algorithm in a way that the tool generates various candidate ASPs that carry one intended mismatch at different positions and compares their thermodynamic stability. As a result, the program automatically shifts the 3′ terminus of the ASP to produce the best performing ASP. The best performing ASP does not decrease the efficiency of amplification. In our experience, the described approach increases the specificity of allele detection. Importantly, ASPs designed with our AS-PCR tool can be used in PCR driven by either proofreading or non-proofreading DNA polymerases. In our experience, proofreading DNA polymerases (e.g., brands Phire, Phusion from Thermo Scientific) also produce better results when DNA sources for genotyping are not of the highest quality (direct PCR protocol).
A program capable of designing primers for KASP is PolyMarker ( http://www.polymarker.info ) ( Ramirez-Gonzalez et al., 2015 ). We used example template sequences published on the PolyMarker website to produce primers using PolyMarker and our AS-PCR tool. However, for two example sequences (Cadenza1697_chr1A_12142209 1A; BA00343846 5A), PolyMarker did not perform the expected KASP analysis, as it identified two copies of the target region in the hexaploid wheat genome. In contrast, our AS-PCR tool does not impose similar restrictions because our tool is not only able to check for repeats but can also position ASPs in non-repeated regions. Thus, our AS-PCR tool is more suitable for work with polyploid genomes. When our AS-PCR tool analysed a template in the example above, the tool found a non-repeated region at one side of a polymorphic site of interest and generated ASPs. With two different example templates (BA00591935 3B; BA00122841 7D), PolyMarker and our AS-PCR design tool generated ASPs of the same sequences. However, the computed common reverse primers were different. Our AS-PCR tool’s output with the primers’ T m illustrates that this program can design reverse primers with thermodynamic properties very similar to ASPs.
The other differences between these programs (WASP, PolyMarker) favour our AS-PCR design tool, as both programs do not allow for addition of predefined 5′ tails to ASPs and do not check primer thermodynamic compatibility if the ASPs have 5′ tails. PolyMarker searches for primer-binding sites at only one side of a polymorphic site, whereas our tool searches in both directions.
It should be mentioned that the primer-design algorithm in software from other developers (WASP and PolyMarker) is significantly different from ours. This difference becomes evident when a sequence surrounding a polymorphic site has low (linguistic) complexity, as in cases of perfect or imperfect microsatellites (simple sequence repeats, SSRs) or G-rich sequences capable of G/C-quadruplex formation. The algorithm in software from other groups ignores the linguistic complexity and sometimes target primers on sequences with low linguistic complexity. During PCR, such primers will generate unexpected amplifications and may hamper identification of specific alleles ( Kalendar, 2022 ).
An important condition for successful PCR is that all primers in one multiplex reaction must have similar thermodynamic properties ( T m , dG) and be compatible in terms of possible primer-dimers. Our AS-PCR tool is arguably the most sophisticated instrument in these regards, as the tool automatically checks the compatibility of all primers in a multiplex design.
Thus, the main differences between these programs (WASP, PolyMarker) and our AS-PCR tool are that probe sequences are designed in both directions and both SNPs and InDels may be targeted. Our tool automatically places a variable base at the second (penultimate) position of the primer’s 3′ terminus or to any other position in manual or automatic mode. In addition, our tool allows discrimination of up to four-allelic variants at a single SNP site. Finally, our tool allows design of custom FRET cassette reporter systems for fluorescence-based assays.
We propose a modification to the KASP method to an improved and universal ‘Allele-specific qPCR’ (ASQ) for designing target-specific primers for KASP genotyping assays. When compared with KASP, this proposed ASQ method also contains two separate components: an allele-specific part (two or more AS primers targeting the SNP/InDel with identity in the penultimate 3′ nucleotide and specific 5′ tags) and a universal part. There are two or more universal probes (UP-1-4) with corresponding tags and different fluorescent dyes at the 5′ end and a single common UP with a quencher at the 3′ end (Uni-Q) ( Table 3 ). A single common UP Uni-Q at the 3′ terminus carries a universal 3′ quencher (Eclipse with quenching range 390–625 nm; Black Hole Quencher [BHQ1] with quenching range 450–580 nm, Black Hole Quencher [BHQ2] with quenching range 520–650 nm, or Tide Quencher 3 [TQ3] with quenching range 510–620 nm) that effectively quenches a broad range of fluorescent dyes for most fluorescently labelled UPs. The identical sequence of the 13-bp tag-fragment in each UP fully complements those in the Uni-Q. Differences in the tail sequences are determined by short barcode 6-nt sequences that are not part of the universal tag sequence and are located between it and ASP sequence ( Table 3 ). The proposed modified KASP method based on FRET is suitable for single- or multiplex applications and can be used in various approaches for SNP/InDel genotyping ( Figure 1 ). During thermal cycling, the relevant ASP binds to a template and elongates, thus attaching its tail sequence to a newly synthesized strand. The complement of the allele-specific tail sequence is generated during subsequent rounds of PCR, enabling the dye-containing component in a FRET cassette to bind to DNA. Through binding, the dye is no longer quenched and emits fluorescence. Separating of the reactions into template-specific and not template-specific parts allows screening of multiple templates in one run. ASQ protocols use FRET master mixes for generating fluorescent signals with different templates in development of various genotyping assays.
The wide use of sequencing has shown that sequence variations (SNPs and InDels) between individuals of a given species is very common; for example, millions of SNP differences are found between two wheat varieties. It is not always necessary to have access to all this information for comparative studies. For many tasks, a limited number of polymorphic DNA sites is sufficient, and the use of high-density microarrays and NGS is unjustified and excessively costly. Improved AS-PCR techniques (e.g., KASP-related technology) are entirely appropriate in such situations. A limitation of the originally described KASP method is that only two alternative alleles at any specific site can be detected because of the use of individual quencher oligos for each ASP at a FRET cassette. However, planning KASP for the detection of three or four alleles at the same site and for allele identification at different polymorphic sites is possible in one multiplexed reaction. The latter possibility is valuable for those who require multiplexing to decrease sample number and increase throughput. Using a four-plexed fluorescent KASP assay, output during SNP genotyping will be doubled without additional labour or a significant increase in costs.
Here we describe a convenient alternative to originally described fluorescent reporters (FRET cassettes) that employs one universal quencher oligonucleotide. The quencher oligonucleotide is complementary to the UP 5′ tails. The quencher oligonucleotide is also complementary to the 5′ proximal sequence in the fluorescent probes. The quencher oligonucleotide at the 3′ terminus carries a universal quencher that effectively quenches a broad group of fluorescent dyes (such as Eclipse, BHQ1 or BHQ2 or Tide Quencher 2/3). Any of the known non-fluorescent dark quenchers can potentially be used ( Fiandaca et al., 2001 ; Li et al., 2006 ), as this quenching occurs in non-dual-labelled probes through non-FRET (static) quenching mechanisms ( Johansson et al., 2002 ; Marras et al., 2002 ) in situations where the dyes are held close together through hybridization. Static quenching occurs through the formation of a ground-state complex and can be important in dual-labelled “linear” probes ( Parkhurst and Parkhurst, 1995 ; Bernacchi and Mely, 2001 ). Efficient quenching can be obtained via static quenching without the use of molecular beacon stem-loop structures. The oligonucleotide presumably acts as a tether, effectively increasing the relative fluorophore quencher concentration, promoting heterodimer formation.
Differences in the tail UP sequences are determined by a short barcode 6-nt sequence that is not part of the universal tail sequence. The improved KASP makes AS-PCR-based genotyping into a flexible, easy-to-plan, high-throughput, and cost-effective technology. Modern qPCR machines are emerging that allow simultaneous use of four or more optical channels to detect fluorescent signals. Such equipment is poised to be utilized with the improved KASP as it is possible to genotype more SNPs or include more-than-two-variant SNPs.
In this work, we describe a tool working in the FastPCR environment for developing PCR-based genotyping assays. The tool helps design assays for detection of SNPs and InDels. The FastPCR software was originally created with the intention to allow great flexibility when designing PCR assays. Now with KASP capabilities, FastPCR allows for even more sophisticated genotyping assays, which translates into a higher overall success rate and provides the unique ability to genotype mix-type SNP/InDel polymorphisms. Increasing the reaction specificity and discriminative power of an assay, the ASPs are positioned automatically such that the penultimate base at the primer’s 3′ end is placed over the variable base. This feature of the FastPCR algorithm permits the use of proofreading enzymes, which is necessary when working with low-quality DNA templates and with a direct PCR assay. FastPCR expands detection options as it makes possible detection of all four variants at a single SNP site and any form of InDel. Theoretically, it also allows genotyping of several different SNP targets in a multiplexed reaction and genotyping mix-type polymorphisms. This contrasts with the limited ability of standard KASP assays to detect only two variants at a SNP site. FastPCR generates primers to target a polymorphic site from either strand. Custom FRET cassette reporter systems may be devised, and a universal FRET cassette system from LGC Biosearch Technologies may be used in planning the assays.
The AS-PCR design tool may be used to plan high-throughput screening assays to rapidly identify mutant strains of microorganisms, particular genetic lines of higher organisms, or cell lines, such as those produced with genome editing using Transcription Activator-Like Effector Nucleases (TALEN) or Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) ( Lee et al., 2016 ; Koonin et al., 2017 ; Shmakov et al., 2017 ). An important prospective use of our AS-PCR design tool is rapid testing for specific variations in pathogen genomes. For example, the four-way genotyping assay may provide information on genetically encoded drug resistance or the presence of pathogenicity determinants in a clinical isolate. It may also be possible to rapidly design and deploy genotyping assays for epidemiological studies of circulating pathogens. Another entity presented in this manuscript is a KASP modification for quantitative measurement of the presence of allelic variants. The ASQ method is thus a highly multiplexed alternative to traditional quantitative PCR.
The authors wish to thank Derek Ho (The University of Helsinki Language Centre, Finland) for outstanding editing and proofreading of the manuscript.
The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding author.
The studies involving human participants were reviewed and approved by the Center for Life Sciences, National Laboratory Astana, Nazarbayev University (protocol #21, 10 October 2017). The patients/participants provided their written informed consent to participate in this study.
RK conceived the idea and provided bioinformatics analyses to design primers, tags, and barcode sequences; RK developed the methods and software; RK and AS performed the experiments and analysed the data. IA performed oligonucleotide synthesis and performed PCR analysis. RK prepared the initial draft of the manuscript. RK, UK, and AS wrote the main manuscript text and RK prepared all figures. UK provide the technical support, IA conceived the experiment. All authors reviewed and approved the final manuscript.
This research was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP08855353 and AP09058660). This study was also partially funded by Primer Digital Ltd. ( https://primerdigital.com ). Open access funding provided by University of Helsinki (Finland), including Helsinki University Library, via RK.
Author RK is the owner and director of PrimerDigital. Author RK is an employee of PrimerDigital. This affiliation does not alter our adherence to publication policies on sharing data and materials.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmolb.2022.773956/full#supplementary-material
ASP, allele-specific primer; AS-PCR, allele-specific PCR; ASQ, allele-specific qPCR; dG, Gibbs free energy; FRET, fluorescence resonant energy transfer; InDel, insertion-deletion polymorphism; KASP, kompetitive allele-specific PCR; SNP, single-nucleotide polymorphism; Uni-Q, universal quencher probe; UP, fluorescently-labeled Universal probe.
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SHANGHAI and MIDDLETOWN (DE), October 21, 2024 –HuidaGene Therapeutics (“HuidaGene”), a global clinical-stage biotechnology company pioneering CRISPR-based programmable genome medicines, today announced the appointment of Dr. TJ Cradick as Chief Technology Officer. In this role, Dr. Cradick will further drive innovation and development of delivery vectors and gene editing tools through computational biology, artificial intelligence (AI), machine learning (ML), and other tools and methodologies.
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SHANGHAI and MIDDLETOWN, Del., Oct. 21, 2024 /PRNewswire/ -- HuidaGene Therapeutics ("HuidaGene"), a global clinical-stage biotechnology company pioneering CRISPR-based programmable genome medicines, today announced the appointment of Dr. TJ Cradick as Chief Technology Officer. In this role, Dr. Cradick will further drive innovation and development of delivery vectors and gene editing ...
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