Improving CRISPR-Cas nuclease specificity using truncated guide RNAs

Yanfang Fu, Jeffry D. Sander, Deepak Reyon, Vincent M. Cascio, J. Keith Joung
Source: Nat Biotechnol
Publication Date: (2014)
Issue: 32(3): 279-84
Research Area:
Cancer Research/Cell Biology
Basic Research
Molecular Biology
Cells used in publication:
U-2 OS
Species: human
Tissue Origin: bone
4D-Nucleofector® X-Unit
To assay for disruption of EGFP expression, 2E+5 U2OS.EGFP cells were transfected in duplicate with gRNA expression plasmid or an empty U6 promoter plasmid as a negative control, Cas9 expression plasmid (pJDS246)4, and 10 ng of td-Tomato expression plasmid (to control for transfection efficiency) using a LONZA 4D-Nucleofectorâ„¢, with SE solution and DN100 program according to the manufacturer\\\\\\\'s instructions. We used 25 ng/250 ng, 250 ng/750 ng, 200 ng/750 ng, and 250 ng/750 ng of gRNA expression plasmid/Cas9 expression plasmid for experiments with EGFP site #1, #2, #3, and #4, respectively. Two days following transfection, cells were trypsinized and resuspended in Dulbecco\\\\\\\'s modified Eagle medium supplemented with 10% (vol/vol) fetal bovine serum (FBS) and analyzed on a flow cytometer.
Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.