Chemical approaches for precision genome editing

项目摘要 CRISPR-Cas9是一种RNA引导的核酸内切酶，被积极用于序列特异性DNA测序。 识别、基因组工程、靶向转录激活/抑制和基因组成像。Cas9是 作为一种基因治疗剂正在开发的多种病理，包括艾滋病毒，视力障碍，肌肉 营养不良和遗传性疾病。CRISPR-Cas9的特异性的精确控制是必需的， 在升高的活性下观察到靶向效应和染色体易位。此外，瞄准目标的容易性 催化受损的Cas9与任何基因组基因座的结合导致了变革性技术。比如说 无催化活性的Cas9（dCas 9）与转录激活因子或阻遏物的融合使基因 转录和阻遏;催化受损的Cas9与碱基修饰酶的融合允许碱基修饰酶的表达。 转换（例如，C→T）; dCas 9与GFP融合使基因组位点成像成为可能; 和dCas 9的乙酰转移酶或脱乙酰酶融合使得表观基因组编辑成为可能。我们建议申请 化学和遗传学方法开发了允许精确控制特异性的试剂和方法 CRISPR-Cas9 1

PROJECT SUMMARY CRISPR-Cas9 is an RNA-guided endonucleases that is being actively used for sequence-specific DNA recognition, genome engineering, targeted transcriptional activation/repression and genome imaging. Cas9 is being developed as a gene therapy agent for multiple pathologies, including HIV, vision disorders, muscular dystrophy, and hereditary disorders. The precision control of specificity of CRISPR-Cas9 is required as off- target effects and chromosomal translocations are observed at elevated activity. Further, the ease of targeting catalytically impaired Cas9 to any genomic locus has resulted in transformative technologies. For example, the fusion of catalytically inactive Cas9 (dCas9) to transcriptional activators or repressors has enabled gene transcription and repression; fusion of catalytically impaired Cas9 to base-modifying enzymes has allowed base conversion (e.g., C→T) at specific genomic sites; dCas9‒GFP fusion has made imaging genomic loci possible; and dCas9‒acetyltransferases or deacetylases fusion has enabled epigenome editing. We propose to apply chemical and genetic approaches develop reagents and methods that will allow precision control of specificity of CRISPR-Cas9. 1