Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency

CRISPR-Cas9 is a powerful genome editing technology, yet with off-Target effects. Truncated sgRNAs (17nt) have been found to decrease off-Target cleavage without affecting on-Target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ∼95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60-70%) and MSCs (65-75%). Furthermore, we observed a decrease of 10-20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-Target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-Target mutations. Of interest, we occasionally observed off-Target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-Target gene cleavage potency with off-Target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable.