ADVANCING GENE-EDITING NUCLEASES FOR DIVERSE ZEBRAFISH APPLICATIONS

总结 CRISPR-Cas9和相关技术极大地改变了我们操纵基因组的能力。 无数生物的基因组，产生疾病模型，迅速发现基因的功能， 为遗传疾病创造新的治疗方法。然而，该领域的全部潜力尚未实现。 在过去的15年里，彼得森和叶实验室合作开发了基因组， CRISPR-Cas9是一种新的编辑工具，并为几项关键进展做出了贡献，包括首次使用CRISPR-Cas9来修饰 任何动物的基因组，第一次在斑马鱼中使用prime editing，以及第一项高- 在脊椎动物中进行CRISPR高通量筛选。在目前的融资期内，我们成功地 完成了我们的所有三个目标，并产生了新的技术，TICIT和MIC-Drop，提出了令人兴奋的新 进一步发展和应用的机会。在此竞争性续期申请中，我们建议 扩展TICIT和MIC-Drop，开发几种有用的新基因编辑方法。 目的1建立在CRISPR-Cas9和整合酶技术（TICIT）的靶向整合基础上， 利用位点特异性DNA重组酶-phiC 31整合酶-将DNA插入基因组靶位点， 已经通过CRISPR-Cas9修饰预先指定。与传统的转基因方法不同， 随机基因组插入，TICIT能够将质粒精确整合到基因组中预先指定的基因座， 避免无意的基因破坏和位置效应。在TICIT的基础上，我们现在建议扩大 整合酶用于位点特异性基因组编辑的效用。在更新申请中，我们提出新的 技术，使TICIT更有效，并允许多种整合酶在各种安全， 携带基因组位点。此外，我们将使用优化的TICIT平台开发高通量 用于分离具有增强的细胞状态或细胞类型特异性的合成启动子的筛选系统。 目标2建立在多重混合CRISPR液滴（MIC-Drop）的基础上， CRISPR sgRNA，Cas9蛋白和DNA条形码进入纳升体积的微流体液滴，然后可以被 注射到成千上万的斑马鱼体内。MIC-Drop能够快速有效地破坏 数百或数千个基因，并且在鉴定感兴趣的表型之后，可以将致病基因 通过条形码恢复快速识别。在MIC-Drop的基础上，我们现在建议扩大 MIC-Drop用于分子表型分析。此前，我们已经通过目视检查来识别斑马鱼 具有感兴趣的表型的突变体，然后基于PCR回收DNA条形码。我们现在计划 联合收割机MIC-Drop技术与强大的分子表型分析工具（如单细胞RNA测序）相结合 （scRNAseq）和代谢组学。在这个目标中，我们将开发新的方法，条形码和工作流程， 能够以前所未有的规模进行有效的基因破坏和分子表型分析。

Summary CRISPR-Cas9 and related technologies have dramatically transformed our ability to manipulate the genomes of countless organisms, generate disease models, rapidly discover the functions of genes, and create new treatments for genetic disorders. Nevertheless, the full potential of the field has yet to be realized. Over the past fifteen years, the Peterson and Yeh labs have collaborated in the development of genome editing tools and have contributed to several key advances, including the first use of CRISPR-Cas9 to modify the genome of any animal, the first use of prime editing in zebrafish, and the first technology for high- throughput CRISPR screening in a vertebrate. During the current funding period, we have successfully completed all three of our aims and produced new technologies, TICIT and MIC-Drop, that raise exciting new opportunities for further development and application. In this competitive renewal application, we propose to expand on TICIT and MIC-Drop, developing several useful new gene-editing approaches. Aim 1 builds upon Targeted Integration by CRISPR-Cas9 and Integrase Technologies (TICIT), which utilizes the site-specific DNA recombinase – phiC31 integrase – to insert DNA into genomic target sites that have been pre-specified by CRISPR-Cas9 modification. Unlike traditional transgenic methods which lead to random genome insertion, TICIT enables precise integration of plasmids into the genome at prespecified loci, avoiding inadvertent gene disruption and positional effects. Building upon TICIT, we propose now to expand the utility of integrases for site-specific genome editing. In the renewal application, we propose new technologies to make TICIT more efficient and permit combinatorial uses of multiple integrases in various safe- harbor genomic loci. Additionally, we will use the optimized TICIT platform to develop high-throughput screening systems for isolating synthetic promoters with enhanced cell-state or cell-type specificity. Aim 2 builds upon Multiplexed, Intermixed CRISPR Droplets (MIC-Drop), which combines multiplexed CRISPR sgRNAs, Cas9 protein, and DNA barcodes into nanoliter-volume microfluidic droplets that can then be injected from a single needle into thousands of zebrafish. MIC-Drop enables rapid and efficient disruption of hundreds or thousands of genes, and after identification of phenotypes of interest, the causative gene can be quickly identified by barcode recovery. Building upon MIC-Drop, we propose now to expand the utility of MIC-Drop for molecular phenotyping. Previously, we have used visual inspection to identify zebrafish mutants with interesting phenotypes, followed by PCR-based recovery of the DNA barcodes. We now plan to combine MIC-Drop technology with powerful molecular phenotyping tools, such as single-cell RNA sequencing (scRNAseq) and metabolomics. In this aim, we will develop new methods, barcodes, and workflows that will enable efficient gene disruption and molecular phenotyping at unprecedented scales.