Genome engineering using CRISPR-Cas9 system.

Methods Mol Biol
Authors
Keywords
Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

Year of Publication
2015
Journal
Methods Mol Biol
Volume
1239
Pages
197-217
Date Published
2015
ISSN
1940-6029
URL
DOI
10.1007/978-1-4939-1862-1_10
PubMed ID
25408407
Links