Evidence map›Paper›PMID 42143446›Full record

ArticleBiochemical and biophysical research communications2026

Helicity of the bridge helix of Cas12a regulates on-target DNA cleavage efficiency and off-target cleavage propensity.

Lindsie Martin, Saadi Rostami, Isabelle Schuster, Peter Z Qin, Rakhi Rajan

Abstract read
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In one paragraph

Article in Biochemical and biophysical research communications, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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0citing papers in PubMed
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1 · What the graph read from it

What it found

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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

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3 · Its place in the literature

Who cites it

0 citing papers in PubMed.

No citing paper in PubMed yet.

4 · The record

Corrections and comments

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5 · Who and what money

Authors and funding

5 authors.

Lindsie MartinDepartment of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, OK, 73019, USA.
Saadi RostamiDepartment of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, OK, 73019, USA.
Isabelle SchusterDepartment of Chemistry, University of Southern California, 3430 S. Vermont Ave., Los Angeles, CA, 90089, USA.
Peter Z QinDepartment of Chemistry, University of Southern California, 3430 S. Vermont Ave., Los Angeles, CA, 90089, USA.
Rakhi RajanDepartment of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, OK, 73019, USA. Electronic address: r-rajan@ou.edu.

Funding

Structure-Function Studies of MsvR, a Methanogen-Specific Transcriptional RegulaP20GM103640 · NIGMS · UNIVERSITY OF OKLAHOMA · PI THOMAS, LEONARD M · 2012 to 2021
$20.4M
Protein Production and Characterization CoreP30GM145423 · NIGMS · UNIVERSITY OF OKLAHOMA · PI ANN H WEST · 2022 to 2026
$6.9M
Supplement: Acquisition of an NGC Discover 10 Pro Chromatography SystemR35GM145341 · NIGMS · UNIVERSITY OF SOUTHERN CALIFORNIA · PI Peter Z Qin · 2022 to 2026
$2.1M
NIGMS NIH HHS P20 GM103640NIGMS NIH HHS P30 GM145423NIGMS NIH HHS R35 GM145341
6 · The paper itself

Abstract

CRISPR-Cas systems comprise a CRISPR RNA (crRNA)-guided CRISPR-associated (Cas) nuclease for providing immune protection. The complementary base pairing between crRNA and the invader genome leads to the formation of an "R-loop", which triggers the nuclease activity of the Cas protein, effectively neutralizing the invasion. This molecular mechanism has been repurposed for genome applications using Cas9 and Cas12a. Cas12a has several favorable features for applications including its smaller size, crRNA processing ability, and creation of staggered double-stranded DNA (dsDNA) cleavage. Gene editing with these Cas proteins, however, has some setbacks due to off-target and non-specific DNA cleavages. To increase the specificity in DNA cleavage, we introduced proline/alanine substitutions at different positions along a conserved arginine/lysine-rich "bridge helix" (BH) of Cas12a that plays an integral role in mediating conformational changes needed for DNA cleavage. Cleavage kinetics analyses reveal that enhanced helical integrity of the BH of Francisella novicida Cas12a provided by alanine substitutions increases DNA cleavage efficiency, while reducing the ability of the variants to discriminate DNA mismatches. Proline substitutions demonstrate an opposite effect by reducing the efficiency of cleaving on-target DNA, but almost completely abolishing linearization of a target with a mismatch in the middle of the R-loop. These results parallel those reported for Cas9 and show that balancing the helicity of BH through rational amino acid substitutions can finetune Cas12a's off-target profiles. This may provide a strategy for enhancing specificity of Cas12a in genome manipulation.

Indexed as

Bacterial ProteinsCRISPR-Associated ProteinsDNADNA CleavageEndodeoxyribonucleasesAmino Acid SequenceAmino Acid SubstitutionCRISPR-Cas SystemsFrancisellaProtein Structure, SecondaryBacterial ProteinsCas12a proteinCRISPR-Associated ProteinsDNAEndodeoxyribonucleasesBridge helixCas12aLoop-to-helical transitionMismatch toleranceOff-target DNA cleavage

Identifiers

PMID42143446
PMCPMC13220989

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LicenceCC BY-NC-ND
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Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.