How to reduce off-target effects and increase CRISPR editing efficiency?

CRISPR has proven to be a highly versatile tool for gene editing with tremendous potential in a wide range of problems such as gene therapy, drug discovery, and genetic modification in plant technology. However, the accuracy and reliability of the CRISPR technology are severely hampered by the off-target effects, namely, the unintended cleavage of DNA at sites whose sequences show mismatches with the guide RNA (gRNA or sgRNA). Therefore, reducing the off-target effect becomes a timely critical issue in CRISPR genome editing technology.

various strategies have been reported to reduce RGEN off-target effects.First, the sgRNA sequence can be altered. Truncation of the 3′ end of sgRNA (derived from tracrRNA domain that interacts with Cas9), shortening the region complementary to the target site at the 5′ end of the sgRNA by as many as 3 nt (tru-gRNA) or addition of two guanine nucleotides to the 5′ end of the sgRNA (just before the 20-nt complementary region) improves target specificity, decreasing undesired mutagenesis at some off-target sites by 5,000-fold.

Second, one potential strategy for minimizing off-target effects is to control the concentration of the Cas9-sgRNA complex by titrating the amount of Cas9 and sgRNA delivered. However, increasing specificity by reducing the amount of transfected DNA also leads to a reduction in on-target cleavage. Therefore, a balance between on-target cleavage efficiency and off-target effects has to be considered. Nonetheless, future optimization of both Cas9 and sgRNA design may improve Cas9 specificity without sacrificing cleavage efficiency.