AsCas12a tolerates insertions in target DNA

Sep 18, 2025Nucleic acids research

AsCas12a can work even when extra DNA is inserted into its target

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Abstract

Acidaminococcus sp. Cas12a (AsCas12a) can tolerate noncomplementary insertions of 1-20 nucleotides in its DNA target.

AsCas12a maintains functionality despite DNA bubbles or loops in the CRISPR-RNA-target DNA duplex.
The enzyme displays both cis- and trans-cleavage activities on single-stranded and double-stranded DNA, though activity is reduced in cellular environments.
Among 12 tested Cas12a orthologs, only AsCas12a demonstrates this insertion tolerance.
A unique α-helix in AsCas12a's WED domain is necessary for its ability to tolerate DNA insertions.
Deletion of this α-helix results in loss of insertion tolerance in AsCas12a.

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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/positions and DNA topologies but is strongly diminished in cells. Among 12 Cas12a orthologs tested, only AsCas12a exhibits this tolerance, making it a unique member of the Cas12a family. Structural analysis suggests a distinctive α-helix in AsCas12a's WED domain is required for this flexibility. Upon deleting this α-helix, AsCas12a loses its ability to tolerate insertions. This discovery can be utilized to detect single-nucleotide polymorphisms and enable protospacer adjacent motif (PAM)-flexible DNA cleavage with Cas12a. Our findings expand our fundamental understanding of CRISPR-Cas12a systems. In conclusion, we uncover and characterize a unique property of AsCas12a to tolerate insertions in its target.

Key numbers

20 nt

Tolerance

Maximum length of DNA tolerated by AsCas12a.

10.3%

Indel Formation Rate

Average indel formation rate with perfectly matched guide RNA in HEK293T cells.

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AsCas12a tolerates insertions in target DNA

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