The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms.
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Here, the scientists describe a Cas9D10A-based screening approach that combines an All-in-One Cas9D10A nickase vector with minimal off-target effects. They 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. 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.