Gene Editing

Collateral effect / trans-cleavage

Newly discovered characteristics like “collateral effect” or trans-cleavage in CRISPR-Cas13 and CRISPR-Cas12 systems have enabled their usage in nucleic acid detection. The collateral RNA cleavage of Cas13a has been reported to be harmful for cell development. As a representative gene editor of CRISPR-Cas12 system, CRISPR-Cas12a (Cpf1) holds great potential for therapeutic applications in the future. However, when used for genome editing in mammalian cells, target-activated Cas12a has the risk to cleave transiently exposed ssDNA during replication, transcription and homology-directed repair processes (Fig. 1), raising the concern of its therapeutic applications.

Fig. 1 Collateral effect or trans-cleavage in CRISPR-Cas13 and CRISPR-Cas12 systems.

DNA replication, transcription, homology-directed repair and R loop structure would lead to the unwinding of double-stranded DNA (dsDNA) to ssDNA. Whether the indiscriminate ssDNA cleavage activity of Cas12a would induce genome-wide off-target effects in mammalian cells needs to be explored.

Recent studies reported that Cas13a-mediated targeting on massive copies of RNA generated substantial “collateral effect” in cultured cells and individual organisms. By contrast, Cas12a only targeted a limited number of gene copies in mammalian cells, and thus would not cause broad ssDNA cleavage. Besides, protective DNA repairing mechanism can repair the limited number of ssDNA cleavage . In addition, low-frequency trans cleavage off-target events and large scale deletions or insertions could be missed by our detection approach, resulting in the undetectable off-target effects in our study. Considering that LbCas12a and AsCas12a have comparable editing efficiencies, smaller size and lower mismatch tolerance comparing with spCas9, they hold great promise and competition for therapeutic application in the future.

Therefore, the potential off-target effects caused by the indiscriminate ssDNA cleavage activity of Cas12a need to be carefully investigated.

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