Cas12a off-target genomic DNA sites (Cas12a off-targets)

Cas12a off-target genomic DNA sites are unintended loci within the genome where the Cas12a (formerly Cpf1) endonuclease binds and cleaves due to sequence similarity with the intended target. These sites typically possess a compatible Protospacer Adjacent Motif (PAM), such as TTTN, and exhibit partial complementarity to the CRISPR RNA (crRNA) guide sequence (Zetsche et al., 2015, Cell). While Cas12a is generally noted for higher specificity compared to Cas9, off-target activity remains a significant concern in gene editing applications as it can lead to double-strand breaks at non-target locations (Kim et al., 2016, Nature Biotechnology). Such unintended modifications can result in genomic instability, gene disruptions, or chromosomal translocations, potentially leading to cellular dysfunction or oncogenic transformation (Kleinstiver et al., 2016, Nature Biotechnology). The kinetics of Cas12a binding suggest that mismatches in the seed region of the crRNA are less tolerated than those in the distal region, though off-target cleavage can still occur at sites with multiple mismatches (Strohkendl et al., 2018, Molecular Cell). Monitoring and minimizing these off-target effects is a critical component of safety assessments for CRISPR-Cas12a-based therapeutics. Identifying these sites often requires specialized empirical methods like GUIDE-seq or CIRCLE-seq to ensure the safety and precision of the genetic intervention.

Other names

Cpf1 off-target sitesCas12a off-targetsUnintended genomic cleavage sitesNon-specific Cas12a binding sites

Unintended endonucleolytic cleavage of double-stranded DNA at genomic loci with partial complementarity to the guide RNA.