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Analysis of homolog-based CRISPR editing in somatic cells

体细胞中基于同源物的 CRISPR 编辑分析

基本信息

批准号：
10343429
负责人：
ETHAN BIER
金额：
$ 31.6万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-15 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10343429
关键词：
Adult Affect Agriculture Alleles Bacteria Biology CRISPR gene drive Cell Cycle Cell Line Cells Chromosome Mapping Chromosome Pairing Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Culicidae Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator DNA DNA Repair DNA cassette Disease model Drosophila genus Ectopic Expression Elements Endonuclease I Event Frequencies G2 Phase Gene Conversion Genes Genetic Genome Genomics Grant Guide RNA Homologous Gene Human Human Cell Line Induced Mutation Insect Control Insecta Mammalian Cell Mammals Mediating Medicine Meiosis Mitotic Modeling Mutation Nonhomologous DNA End Joining Oncogenes Organism Outcome Pathway interactions Phenotype Pigmentation physiologic function Population Process Reporter Resistance S phase Sequence Homologs Site Somatic Cell System Technology Testing Variant Visualization Yeasts base base editing design developmental genetics disease-causing mutation expectation experimental study fly gastrointestinal epithelium gene correction gene drive allele gene drive system gene therapy genetic element genetic variant genome editing genome integrity genomic locus improved in vivo insect disease vector insight mutant next generation notch protein novel novel strategies nuclease portability precise genome editing precursor cell repair model repair strategy repaired stem cells tool transgene expression

项目摘要

Recently developed CRISPR-based systems permit precise genome editing by inducing targeted DNA breaks at specific sites in the genome. Cellular DNA repair machinery can restore genome integrity by copying information from the intact homologous chromosome at the cleavage site via homology directed repair (HDR). While precise HDR-mediated DNA repair is the predominant pathway active during meiosis, the competing and potentially mutagenic non-homologous end-joining pathway (NHEJ) is typically thought to prevail in somatic cells. One reason for this bias is that the NHEJ pathway is active throughout somatic cell cycles, while HDR is primarily restricted to post-replicative S and G2 phases. Thus, achieving efficient HDR-based gene editing in somatic cells has proven challenging, which limits the in vivo use of this technology for human gene therapy. My group has contributed to developing the first CRISPR-based gene-drive (or active genetic) systems in flies, mosquitoes, mammals, and bacteria that bias germline inheritance of genetic elements programmed to cut the genome at their site of insertion. We also pioneered allelic-drive systems designed to promote biased inheritance of a favored allelic variant at a separate genetic locus. These germline drive systems also produce somatic phenotypes, which have generally been attributed to mutations induced by the NHEJ pathway. Recently, we developed genetic elements we refer to as “CopyCatchers” that permit visualization of HDR- mediated copying of gene cassettes. These studies have revealed an unexpectedly high frequency of somatic gene conversion (SGC) events in Drosophila (30-50%) wherein the chromosome homolog serves as a DNA repair template. Rates of SGC can be improved further by optimizing delivery of CRISPR components, or by reducing the expression of various genes encoding factors involved in DNA repair or chromosome pairing. Preliminary experiments indicate that interhomolog SGC can also take place in human cells and point to untapped strategies for repairing disease-causing mutations using intact sequences from the homologous chromosome. In this grant we propose to explore SGC repair mechanisms mediated by Cas9 and Nickase in somatic cells of Drosophila and then extend analysis of this interhomolog repair process to human cells. First, we will analyze the mechanisms underlying CRISPR dependent copying of gene cassettes or allelic variants to optimize their activities. Next, we will develop and optimize Drosophila models for homolog-based repair of disease-causing mutations in the Notch locus affecting mitotically active stem cells or in post-mitotic cells in the adult gut epithelium using a humanized Drosophila CFTR–/– disease model. Finally, we will assess whether insights gained in Drosophila are portable to human somatic cell lines, and whether interhomolog SGC can restore native gene activity in human cell-based models for cystic fibrosis. Enhancing homolog-based repair in mammalian cells could offer transformative possibilities for next-generation gene therapy strategies.

最近开发的基于crispr的系统可以通过诱导目标DNA进行精确的基因组编辑