Efficient creation and characterization of multi-kilobase knockins by CRISPR/Cas9 and multiple rAAVs
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We report the successful creation of multi-kilobase knockin mice and cell lines using three rAAV donors and CRISPR/Cas9 via consecutive homology-directed repair and reliable generation of over 120 KI mice with rAAV donors. For comprehensive analysis of the post-edit genome, we combined target capture and long-read sequencing for a multiplexable and high-throughput, all-in-one method to detect on-target insertion and as importantly, various undesired editing outcomes in the genome. Together, the two novel methods greatly improve the efficiency and accuracy of challenging large KI mouse and cell models.
