清脆的
Cas9
基因组编辑
斑马鱼
计算生物学
生物
基因组
遗传学
引导RNA
突变体
基因
作者
Charles E. Vejnar,Miguel A. Moreno-Mateos,Daniel Cifuentes,Ariel Bazzini
出处
期刊:CSH Protocols
[Cold Spring Harbor Laboratory]
日期:2016-10-01
卷期号:2016 (10): pdb.prot086850-pdb.prot086850
被引量:63
标识
DOI:10.1101/pdb.prot086850
摘要
This protocol describes how to generate and genotype mutants using an optimized CRISPR–Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan algorithm is based on the results of an integrated analysis of more than 1000 sgRNAs in zebrafish, which uncovered highly predictive factors that influence Cas9 activity. Next, we describe how to easily generate sgRNAs in vitro, which can then be injected in vivo to target specific loci. The use of highly efficient sgRNAs can lead to biallelic mutations in the injected embryos, causing lethality. We explain how targeting Cas9 to the germline increases viability by reducing somatic mutations. Finally, we combine two methods to identify F 1 heterozygous fish carrying the desired mutations: (i) Mut-Seq, a method based on high-throughput sequencing to detect F 0 founder fish; and (ii) a polymerase chain reaction–based fragment analysis method that identifies F 1 heterozygous fish characterized by Mut-Seq. In summary, this protocol includes the steps to generate and characterize mutant zebrafish lines using the CRISPR–Cas9 genome engineering system.
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