Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing

Abstract

To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a "core" sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of 'super' off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.

The development of genome editing tools as powerful techniques with versatile applications in biotechnology and medicine: CRISPR/Cas9, ZnF and TALE nucleases, RNA interference, and Cre/loxP

“…However, it might take more basic research to be able to apply CRISPR/Cas for therapeutic applications in humans and for biotechnology. As stated above, CRISPR/ Cas can result in severe off-target effects by targeting similar or identical sequences within the genome and it can result in activation of the error-prone non-homologous end joining DNA repair pathway also resulting in severe mutations [96,97]. Efforts were made to develop more specific Cas9•sgRNA systems and systems that activate homology-directed repair instead of non-homologous end joining [93,[98][99][100].…”

The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies

“…CRISPR-Cas systems have proven unique because of their repurposing into powerful diagnostic, therapeutical and experimental tools. While there are several aspects such as off-target effects and promiscuous DNA damage that need to be fixed by future research ( Haapaniemi et al., 2018 ; Wang et al., 2020 ), the days of personal precision genome medicine are a step closer with this powerful technology.…”

Development of CRISPR/Cas9 system for targeted DNA modifications and recent improvements in modification efficiency and specificity

“…Some of the Cas9 variants even shows to have increased specificity of targeting site, among other improvements such as PAM diversification and increase of efficiency ( Table 1 ). However, it should be noted that off-target should still be carefully considered, as a recent study using reporter activation assay to investigate editing efficiency shows that both CRISPR/Cas9 and CRISPR/Cpf1 tolerate off-target mismatch mutation, especially in PAM-distal region of investigated target ( 63 ).…”