Chemical approaches for precision genome editing

项目总结 CRISPR-Cas9是一种RNA引导的内切酶，目前正被活跃地用于序列特异性DNA 识别、基因组工程、靶向转录激活/抑制和基因组成像。CAS9 IS 被开发为多种病理疾病的基因治疗剂，包括艾滋病毒、视力障碍、肌肉 营养不良和遗传性疾病。CRISPR-CAS9特异性的精密度控制要求为Off-Off。 在活性升高时观察到靶向效应和染色体易位。此外，瞄准目标的简单性 对任何基因组位置的催化损伤的Cas9导致了变革性的技术。例如， 催化失活的Cas9(DCas9)与转录激活物或抑制物的融合使能基因 转录和阻遏；催化受损的Cas9与碱基修饰酶的融合使碱基 在特定基因组位置的转换(例如，C-→T)；dCas9-绿色荧光蛋白融合使基因组基因座成像成为可能； 而dCas9-乙酰转移酶或脱乙酰酶的融合使表观基因组编辑成为可能。我们建议申请 化学和遗传方法开发的试剂和方法将允许精确控制特异性 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