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

项目摘要 正向遗传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， 通过CRISPR/Cas9等技术进步更容易获得。因为绝大多数的突变 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进行研究。相比之下， 用于正向遗传筛选的诱变范例在过去保持相对不变 40年来，仍然依赖于几代近亲繁殖来使动物模型中的突变等位基因纯合。没有 已经开发了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， 双等位基因，通过支持新一代无偏见的询问， 在整个基因组中的基因功能。 我们提出的范例使用两步过程实现随机双等位基因诱变。第一，Prime Editor 酶（PE）stochemical重编程单链TALEN（scTALEN）的DNA结合结构域。Prime 编辑是独立于细胞发生的;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， 结合结构域，每个结合结构域将scTALEN募集到每个个体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。最终的结果是一个巨大的 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，这个过程可以部署在几乎任何模式生物， 细菌对哺乳动物我们将使用幼斑马鱼系统来说明这种方法如何缩短 用于正向遗传筛选的诱变时间轴从大约一年缩短到仅仅几周。我们 我将开发我们提出的范例，称为TERM（随机突变的TALEN编辑）：1）评估 不同scTALEN变体的双等位基因诱变效率和毒性; 2）优化引物编辑 反应，以实现scTALEN靶的有效随机化;和3）使用TERM进行正向遗传 在体外和体内筛选。 通过打破长期存在的饱和诱变筛选技术障碍，$TERM将打开 基因研究中最强大的技术之一的大门。具体而言，我们的方法将有助于 描述了约80%的脊椎动物基因仍然研究不足，抵消了长期存在的偏见， 研究编码区，促进药物靶点识别，并为疾病提供新的治疗靶点 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。

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.