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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

AsCas12a tolerates insertions in target DNA.

Santosh R Rananaware¹, Grace M Shoemaker^1,2, Brianna L M Pizzano¹

¹Department of Chemical Engineering, University of Florida, Gainesville, FL 32611, United States.

Nucleic Acids Research

|September 18, 2025

Summary

This summary is machine-generated.

Acidaminococcus sp. Cas12a (AsCas12a) uniquely tolerates insertions in its DNA targets, unlike other CRISPR-Cas12a enzymes. This flexibility, linked to a specific alpha-helix, enables novel applications in diagnostics and genome editing.

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

CRISPR-Cas12a enzymes are crucial for genome editing and diagnostics.
Efficient Cas12a function typically requires perfect guide RNA-DNA complementarity.
Variations in Cas12a orthologs may lead to unique functional properties.

Purpose of the Study:

To investigate the target DNA binding and cleavage activity of different CRISPR-Cas12a orthologs.
To characterize the insertion tolerance of Acidaminococcus sp. Cas12a (AsCas12a).
To elucidate the structural basis for AsCas12a's unique insertion tolerance.

Main Methods:

Comparative analysis of 12 Cas12a orthologs' activity with insertion-containing DNA targets.
In vitro cleavage assays using single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).
Structural analysis of AsCas12a, including domain-specific deletions (e.g., WED domain α-helix).

Main Results:

AsCas12a demonstrated significant tolerance to insertions (1-20 nucleotides) in its target DNA, unlike other tested orthologs.
This tolerance was observed for both cis- and trans-cleavage activities on ssDNA and specific dsDNA configurations.
A unique α-helix in AsCas12a's WED domain was identified as critical for insertion tolerance; its deletion abolished this property.

Conclusions:

AsCas12a possesses a unique, sequence-independent insertion tolerance mechanism.
This property is structurally dependent on a specific α-helix within the WED domain.
AsCas12a's insertion tolerance offers potential for advanced applications like SNP detection and PAM-flexible DNA cleavage.