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

项目总结 正向基因筛查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，这些方法是革命性的， 通过CRISPR/CAS9等技术进步更容易获得。因为绝大多数突变都是 隐性，CRISPR/Cas9使研究人员能够在反向遗传中快速产生突变表型 通过同时干扰已知的感兴趣基因的两个等位基因进行的研究。相比之下，随机的 用于正向遗传筛查的突变范例在过去相对保持不变。 40年来，仍然依靠世代近亲交配来纯合子突变等位基因的动物模型。不是 诱变技术曾经被开发出来，它既是随机的，也是双等位的。在此，我们建议 利用现代基因组编辑方法高效地引入随机和 双等位基因，通过支持新一代无偏见的询问，彻底改变了正向遗传学领域 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突变。首先，一位顶级编辑 酶(PE)对单链TALEN(ScTALEN)的DNA结合域进行随机重编程。素数 编辑独立于细胞发生；因此，拥有数百万个细胞的培养将产生数百万独特的DNA- 结合结构域，每个结构域将scTALEN招募到每个单个细胞中不同的基因组靶点。第二, ScTALEN在其新编程的靶点诱导双等位基因突变。最终的结果是一个大的 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，然后可以进行筛选 感兴趣的表型。最重要的是，这一过程几乎可以部署在任何模型生物体中 细菌对哺乳动物的影响。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建议的范例，称为TERM(TALEN Editing For Random Mutagenesis)：1)评估 不同SCTALEN变异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；以及3)使用TERM执行正向遗传 在体外和体内进行筛选。 通过打破长期存在的饱和突变筛选的技术壁垒，术语将打开 通往基因研究中最强大的技术之一的大门。具体来说，我们的方法将有助于 描述仍未得到充分研究的~80%的脊椎动物基因，抵消长期存在的偏见 研究编码区，促进药物靶点识别，突出疾病治疗新靶点 治疗，并将全基因组遗传分析扩展到细胞培养系统以外的模型。

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.