Screening for dual sgRNAs with comparable indel efficiencies enhances CRISPR-mediated large-fragment deletion

CRISPR-mediated large-fragment deletion provides a powerful approach for gene clusters, noncoding regions and structural variants, but its broader application is limited by low and variable deletion efficiency. Here, we systematically designed and evaluated 78 sgRNAs targeting nine representative gene clusters (ttn.1-ttn.2 cluster, 7 hox clusters and nppb-nppa cluster), containing 31 large fragments (5 kb-340 kb) to investigate the determinants of deletion efficiency. We found two key rules for achieving high deletion efficiency: (i) using dual sgRNAs with similar indel efficiencies, and (ii) applying a single sgRNA pair rather than multiple sgRNAs. Based on those rules, a 340 kb deletion is detected in the progenies of 95% of founders. Whereas the deletion size showed no significant linear correlation with deletion efficiency within the tested range. Implementing these rules resulted in an average of 70% of founders transmitting deletions across all tested sgRNA pairs. Therefore, screening sgRNAs can effectively enhance CRISPR utility in deletions, thereby facilitating the application of genomic manipulation in vertebrates and other species.