科研之友
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摘要
The development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology for mainstream biotechnological use based on its discovery as an adaptive immune mechanism in bacteria has dramatically improved the ability of molecular biologists to modify genomes of model organisms. The zebrafish is highly amenable to applications of CRISPR/Cas9 for mutation generation and a variety of DNA insertions. Cas9 protein in complex with a guide RNA molecule recognizes where to cut the homologous DNA based on a short stretch of DNA termed the protospacer-adjacent motif (PAM). Rapid and efficient identification of target sites immediately preceding PAM sites, quantification of genomic occurrences of similar (off target) sites and predictions of cutting efficiency are some of the features where computational tools play critical roles in CRISPR/Cas9 applications. Given the rapid advent and development of this technology, it can be a challenge for researchers to remain up to date with all of the important technological developments in this field. We have contributed to the armamentarium of CRISPR/Cas9 bioinformatics tools and trained other researchers in the use of appropriate computational programs to develop suitable experimental strategies. Here we provide an in-depth guide on how to use CRISPR/Cas9 and other relevant computational tools at each step of a host of genome editing experimental strategies. We also provide detailed conceptual outlines of the steps involved in the design and execution of CRISPR/Cas9-based experimental strategies, such as generation of frameshift mutations, larger chromosomal deletions and inversions, homology-independent insertion of gene cassettes and homology-based knock-in of defined point mutations and larger gene constructs.
- 出版日期2016-2-1
