Abstract
The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system’s specificity. Recently a “double-nicking” strategy using catalytic mutant Cas9D10A nickase has been developed to minimise off-target effects. Here, we describe a Cas9D10A-based screening approach that combines an All-in-One Cas9D10A nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.
Highlights
Significant attention has focused on Cas[9] nickases (RuvCD10A or HNHH840A) which, unlike wild-type Cas[9] that generates blunt double-strand breaks (DSBs), cut only one strand of the DNA, generate single-strand breaks (SSB) that can be repaired faithfully, without inducing indels
We found significant off-target mutagenesis caused by wild-type Cas[9] at ten off-target sites (Fig. 1d, lanes 2), which in some cases were comparable to on-target mutagenic efficiency
In agreement with a previous study[31], we consistently found that flow-activated cell sorting (FACS) enrichment substantially increased knockout efficiency in all three cell lines tested in our work
Summary
Significant attention has focused on Cas[9] nickases (RuvCD10A or HNHH840A) which, unlike wild-type Cas[9] that generates blunt DSBs, cut only one strand of the DNA, generate single-strand breaks (SSB) that can be repaired faithfully, without inducing indels. In order to create DSBs, a double-nicking strategy that involves paired nickases targeting adjacent regions has recently been developed, meaning that the potential for off-target DSBs is very much minimised. Co-transfection of multiple plasmids, including a Cas[9] nickase, two sgRNAs and a fluorescent marker (or a drug selection homology vector) may compromise transfection efficiency and targeting mutagenesis, which has so far discouraged the widespread use of this approach. We establish an approach based on an All-in-One plasmid encoding dual sgRNAs and fluorescent protein-coupled Cas9D10A nickase that circumvents these issues and thereby allows efficient genome engineering with minimal off-target effects
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