Genome engineering using the CRISPR-Cas9 system

Authors:
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
In:
Source: Nat Protocols
Publication Date: (2013)
Issue: 8(11): 2281–2308
Research Area:
Stem Cells
Basic Research
Cells used in publication:
293
Species: human
Tissue Origin: kidney
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Platform:
4D-Nucleofector™ X-Unit
Experiment
Summary (Lonza): This publication from the group of Feng Zhang gives comprehensive background information about CRISPR technology and provides a detailed protocol how to use Lonza’s 4D-Nucleofector™ X Unit for CRISPR-based genome editing in HUES9 (a human stem cell line) and HEK293 cells. It also includes protocols for functional analyses, tips for minimizing off-target effects and FAQs.
Abstract
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.