Use of two gRNAs for CRISPR/Cas9 improves bi-allelic homologous recombination efficiency in mouse embryonic stem cells

Targeted genome editing in mouse embryonic stem cells (ESCs) is a powerful resource to functionally characterize genes and regulatory elements. The use of the CRISPR/Cas9 genome editing approach has remarkably improved the time and efficiency of targeted recombination. However, the efficiency of this protocol is still far from ideal when aiming for bi-allelic homologous recombination, requiring at least two independent targeting recombination events. Here we describe an improved protocol that uses two gRNAs flanking the selected targeted region, leading to highly efficient homologous recombination in mouse ESCs. The bi-allelic recombination targeting efficiency is over 90% when using two gRNAs together with the inhibition of non-homologous end-joint repair. Moreover, this technique is compatible with the generation of knocked-in mice and the use of ESC-derived differentiation protocols, therefore facilitating and accelerating the gene targeting in mice and ESCs.