citations

                    Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.
                

Authors:

Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE.

In:

Source: Nat Biotechnol
Publication Date: (2016)
Issue: 1(1): 1-7

Research Area:

Gene Expression

Cells used in publication:

293: Species: human; Tissue Origin: kidney

Platform:

4D-Nucleofector® X-Unit

Experiment

2 × 10e5 HEK293 cells were harvested, washed once in PBS, and resuspended in 20 µL of SF nucleofection buffer (Lonza, Basel, Switzerland). 10 µL of RNP mixture (nuclease and sgRNA complex), 100 pmol of donor DNA, and cell suspension were combined in a Lonza 4D 16well strip nucleocuvette. Reaction mixtures were electroporated using setting DS150, incubated in the nucleocuvette at RT for 10 min, and transferred to culture dishes containing pre-warmed media.

Abstract

Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ~6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3' end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.

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