TERM: a novel mutagenesis paradigm enabling streamlined saturation forward genetics in vertebrate models

术语：一种新的诱变范例，可在脊椎动物模型中简化饱和正向遗传学

基本信息

批准号：
10477464
负责人：
JEFFREY MUMM
金额：
$ 20.47万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10477464
关键词：
Affect Alleles Animal Model Back Bacteria Biological Assay Biology CRISPR/Cas technology Cell Culture System Cells Code Collection Complex Cultured Cells DNA DNA Binding Domain Diploidy Disease Drug Targeting Eligibility Determination Embryo Enzymes Exhibits Generations Genes Genetic Diseases Genetic Research Genetic Screening Genetic study Genome Genomic DNA Genomic Segment Genomics Genotype Germ Cells In Vitro Inbreeding Individual Mammalian Cell Mammals Maps Measures Medicine Methodology Methods Modeling Modern Medicine Modernization Modification Mutagenesis Mutation Nature Outcome Performance Phenotype Pigmentation physiologic function Plague Positioning Attribute Process Randomized Rapid screening Reaction Refractory Reporter Research Personnel Research Technics Site System Techniques Technology Tertiary Protein Structure Testing TimeLine Toxic effect Transgenes Variant Writing Zebrafish base design embryo cell flexibility forward genetics gene function genetic analysis genetic approach genome editing genome-wide genomic locus in vivo interest mutant new therapeutic target next generation novel novel therapeutics nuclease off-target site prime editing prime editor process optimization recruit reverse genetics screening transcription activator-like effector nucleases virtual whole genome

项目摘要

PROJECT SUMMARY Forward genetic screening is a remarkably powerful research technique, but has been widely abandoned. Instead, modern genetics research relies heavily on reverse genetics approaches, revolutionized and made more accessible via technological advances such as CRISPR/Cas9. As the vast majority of mutations are recessive, CRISPR/Cas9 has enabled researchers to rapidly produce mutant phenotypes in reverse genetic studies by simultaneously disrupting both alleles of a known gene of interest. By contrast, the random mutagenesis paradigms used for forward genetic screening have remained relatively unchanged for the past 40 years, and still rely on generations of inbreeding to homozygose mutant alleles in animal models. No mutagenesis technique has ever been developed that is both random and biallelic. Here we propose to leverage modern genome-editing approaches to efficiently introduce mutations that are both random and biallelic, revolutionizing the field of forward genetics by supporting a new generation of unbiased interrogations of gene function across entire genomes. Our proposed paradigm achieves random biallelic mutagenesis using a two-step process. First, a Prime Editor enzyme (PE) stochastically reprograms the DNA-binding domain of a single-chain TALEN (scTALEN). Prime editing occurs cell-independently; thus a culture with millions of cells would generate millions of unique DNA- binding domains, each recruiting the scTALEN to a distinct genomic target site in each individual cell. Second, the scTALEN induces biallelic mutations at its newly programmed target site. The end result is a large collection of cells or embryos that each harbor unique biallelic mutations which can then be screened for phenotypes of interest. Most importantly, this process can be deployed in virtually any model organism from bacteria to mammals. Using the larval zebrafish system, we will illustrate how this approach shortens the mutagenesis timeline for forward genetic screening from approximately one year down to just a few weeks. We will develop our proposed paradigm, called TERM (TALEN Editing for Random Mutagenesis) by: 1) evaluating biallelic mutagenesis efficiency and toxicity of different scTALEN variants; 2) optimizing the prime-editing reaction to achieve efficient randomization of scTALEN targets; and 3) using TERM to perform forward genetic screens in vitro and in vivo. By breaking down the longstanding technical barriers to saturation mutagenesis screening, TERM will open doors to one of the most powerful techniques in genetic research. Specifically, our approach will help to characterize the ~80% of vertebrate genes that remain understudied, counteract longstanding bias towards studying coding regions, facilitate drug target identification and highlight new therapeutic targets for disease treatments, and expand genome-wide genetic analysis to models beyond cell-culture systems.

项目概要正向基因筛选是一种非常强大的研究技术，但已被广泛放弃。相反，现代遗传学研究在很大程度上依赖于反向遗传学方法，彻底改变并制造了通过 CRISPR/Cas9 等技术进步，更容易获得。由于绝大多数突变是 CRISPR/Cas9 使研究人员能够在反向遗传中快速产生突变表型通过同时破坏已知感兴趣基因的两个等位基因来进行研究。相比之下，随机用于正向遗传筛选的诱变范式在过去相对保持不变 40 年来，仍然依赖动物模型中纯合突变等位基因的几代近交。不已经开发出随机和双等位基因的诱变技术。在此我们建议利用现代基因组编辑方法有效地引入随机突变和双等位基因，通过支持新一代的公正审讯彻底改变正向遗传学领域整个基因组的基因功能。我们提出的范例使用两步过程实现随机双等位基因诱变。首先，Prime Editor 酶 (PE) 随机重新编程单链 TALEN (scTALEN) 的 DNA 结合域。主要的编辑独立于细胞进行；因此，具有数百万个细胞的培养物将产生数百万个独特的DNA- 结合域，每个结合域将 scTALEN 招募到每个单独细胞中的不同基因组靶位点。第二， scTALEN 在其新编程的靶位点诱导双等位基因突变。最终结果是一个大细胞或胚胎的集合，每个细胞或胚胎都具有独特的双等位基因突变，然后可以对其进行筛选感兴趣的表型。最重要的是，这个过程几乎可以部署在任何模型生物中细菌到哺乳动物。使用幼虫斑马鱼系统，我们将说明这种方法如何缩短正向基因筛查的诱变时间表从大约一年缩短到几周。我们将通过以下方式开发我们提出的范例，称为 TERM（TALEN 随机突变编辑）：1）评估不同 scTALEN 变体的双等位基因诱变效率和毒性； 2）优化prime编辑实现 scTALEN 靶点有效随机化的反应； 3）使用TERM进行正向遗传体外和体内筛选。通过打破饱和诱变筛选长期存在的技术障碍，TERM将开放通向基因研究中最强大技术之一的大门。具体来说，我们的方法将有助于描述了约 80% 的脊椎动物基因尚未得到充分研究，抵消了长期以来对基因的偏见研究编码区，促进药物靶点识别并突出疾病的新治疗靶点治疗，并将全基因组遗传分析扩展到细胞培养系统之外的模型。