Improving one-step scarless genome editing in by combining co-CRISPR selection with sgRNA target site masking.

The precise and rapid construction of alleles through CRISPR/Cas9-mediated genome engineering renders a powerful animal system for molecular structure-function analyses and human disease models. Application of the co-selection method offers expedited generation and enrichment of scarlessly edited alleles without the need for linked transformation markers, which specifically in the case of exon editing can impact allele usability. However, we found that knockin procedures by homology-directed repair (HDR) under co-selection resulted in low transformation efficiency. This is likely due to repeated rounds of Cas9 cleavage of HDR donor and/or engineered genomic locus DNA, as noted for other CRISPR/Cas9 editing strategies before, impeding the recovery of correctly edited alleles. Here we provide a one-step protocol to improve the generation of scarless alleles by -co-selection with single-guide RNA (sgRNA) binding site masking. Using this workflow, we constructed human disease alleles for two genes, and . We show and quantify how a known countermeasure, the insertion of silent point mutations into protospacer adjacent motif (PAM) or sgRNA homology regions, can potently suppress unintended sequence modifications during CRISPR/Cas9 genome editing of under co-selection. This strongly increased the recovery frequency of disease alleles.