设计和评估工程化的 CRISPR-Cpf1 及其在基因组编辑中的应用。
Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing.
机构信息
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, USA.
出版信息
Nat Protoc. 2018 May;13(5):899-914. doi: 10.1038/nprot.2018.004. Epub 2018 Apr 5.
Cpf1, a CRISPR endonuclease discovered in Prevotella and Francisella 1 bacteria, offers an alternative platform for CRISPR-based genome editing beyond the commonly used CRISPR-Cas9 system originally discovered in Streptococcus pyogenes. This protocol enables the design of engineered CRISPR-Cpf1 components, both CRISPR RNAs (crRNAs) to guide the endonuclease and Cpf1 mRNAs to express the endonuclease protein, and provides experimental procedures for effective genome editing using this system. We also describe quantification of genome-editing activity and off-target effects of the engineered CRISPR-Cpf1 in human cell lines using both T7 endonuclease I (T7E1) assay and targeted deep sequencing. This protocol enables rapid construction and identification of engineered crRNAs and Cpf1 mRNAs to enhance genome-editing efficiency using the CRISPR-Cpf1 system, as well as assessment of target specificity within 2 months. This protocol may also be appropriate for fine-tuning other types of CRISPR systems.
Cpf1 是一种在 Prevotella 和 Francisella 1 细菌中发现的 CRISPR 内切酶，为基于 CRISPR 的基因组编辑提供了一种替代平台，超越了最初在链球菌中发现的常用的 CRISPR-Cas9 系统。本方案能够设计工程化的 CRISPR-Cpf1 组件，包括引导内切酶的 CRISPR RNA（crRNA）和表达内切酶蛋白的 Cpf1 mRNA，并提供使用该系统进行有效基因组编辑的实验程序。我们还描述了使用 T7 内切酶 I（T7E1）测定法和靶向深度测序来定量分析工程化的 CRISPR-Cpf1 在人细胞系中的基因组编辑活性和脱靶效应。本方案能够快速构建和鉴定工程化的 crRNA 和 Cpf1 mRNA，以提高使用 CRISPR-Cpf1 系统的基因组编辑效率，并在 2 个月内评估目标特异性。该方案也可能适用于微调其他类型的 CRISPR 系统。
Zotero 插件