Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity

Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F
Source: Cell
Publication Date: (2013)
Issue: 15(6): 1380-1389
Research Area:
Stem Cells
Gene Expression
Basic Research
Molecular Biology
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
4D-Nucleofector® X-Unit
hES cells HUES62 were transfected with Amaxa P3 Primary Cell 4D Nucleofector Kit (Lonza). Summary (Lonza): The authors tested modifications of the CRISPR/cas9 genome editing system with the aim to reduce off-target effects. They evaluated the modified components in HEK293FT cells and the human embryonic stem cell line HUES62. While using a lipid reagent for transfection of HEK293FT, the 4D-Nucleofectorâ„¢ System was used for HUES62.
Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.