AsCas12a tolerates insertions in target DNA.

ABSTRACT: CRISPR-Cas12a enzymes are RNA-guided nucleases widely used for programmable genome editing and diagnostics. Perfect complementarity between guide RNA and target DNA is essential for efficient binding and cleavage by Cas12a. However, we report that a particular ortholog of Cas12a, Acidaminococcus sp. Cas12a (AsCas12a), shows an unexpected tolerance to noncomplementary insertions at various positions in its DNA target. AsCas12a remains functional despite DNA bubbles or loops in the CRISPR-RNA (crRNA)-target DNA duplex, displaying both cis- and trans-cleavage activities even when the target harbors insertions of lengths 1-20 nucleotides in the crRNA-binding region. This activity is sequence-independent and works for ssDNA and is observed on dsDNA in vitro for specific insertion lengths/positi

SUBMITTER: Rananaware SR 

PROVIDER: S-EPMC12448901 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature