The Challenge Facing CRISPR/Cas9 System: Off-Target Effects and Their Optimization

Abstract

The field of genome editing has undergone a profound revolution with the emergence of CRISPR-Cas9 technology, which enables precise modifications to the genetic code. However, a critical concern is the possibility for the appearance of off-target, where the modifications induced by Cas9 nuclease are at non-intended targets. The mismatches of the seed sequence with the single guide RNA (sgRNA) and the inappropriate length of it could induce off-target effects. Moreover, the inflammatory response triggered by virus-mediated delivery methods may also be responsible for off-target. For the expected events, a large number of deletions of the sequences of the targeted sequence induced by CRISPR-Cas9 may bring the uncertainty of safety. The random changes of genes induced by CRISPR-Cas9 may affect future generations through gene drives, which still lacks long-term studies on long-lived organisms. The off-target effects could be detected by some sequencing methods such as GUIDE-seq, and ITR-seq; however, the suitable ways are situation-dependent. Eventually, the specificity of the technique may be improved through the predictions implemented by Deep Learning, enhancing fidelity and lowering the binding affinity of the Cas9, binding of dual sgRNAs, and delivery method of RNP complexes. This review introduced the causes, effects, and detection methods of the off-target and further discussed of several ways that can mitigate the off-target effects.