Kinetic basis for Cas12a off-target discrimination.

Yuyoung Kim, You Hee Choi, Minji Kim, Yunsu Jang, Sanghwa Lee

Abstract read

In one paragraph

Article in BMB reports, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.

0numbers the graph read from it

0cells of the map it votes in

1citing papers in PubMed

–field-weighted citation impact

1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

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

The trial behind it

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Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

1 citing paper in PubMed.

AAV-based gene therapies for neovascular AMD.Gene therapy · 2026
Review

4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

5 authors.

Yuyoung KimDepartment of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591; Department of Medical Sciences, Graduate School of The Catholic University of Korea, Seoul 06591, Korea.

You Hee ChoiKorea Institute of Medical Microrobotics, Gwangju 61011, Korea.

Minji KimDepartment of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591; Department of Medical Sciences, Graduate School of The Catholic University of Korea, Seoul 06591, Korea.

Yunsu JangAdvanced Photonics Research Institute, Gwangju Institute of Science and Technology, Gwangju 61005, Korea.

Sanghwa LeeDepartment of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591; Department of Medical Sciences, Graduate School of The Catholic University of Korea, Seoul 06591, Korea.

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

CRISPR-Cas12a proteins are RNA-guided endonucleases classified as type V-A effectors that function similarly to Cas9, but possess distinct biochemical features. Previous studies have reported that compared to Cas9, Cas12a exhibits reduced off-target activity, yet the mechanistic origin of this high specificity remains unclear. In this study, we used singlemolecule fluorescence assays to investigate the kinetic basis for the reduced off-target effects of Cas12a. Introducing double mismatches at various positions within the target DNA enabled systematic analysis of the off-target effects on individual reaction steps in the Cas12a-mediated DNA cleavage reaction: seeding, stable R-loop formation, and DNA cleavage. Our results show that mismatches within a 17 bp PAM-proximal seed region significantly impair stable R-loop formation and subsequent cleavage, whereas mismatches in the PAM-distal region exert minimal or negligible effects. These results suggest that the low off-target tolerance of Cas12a and the resulting high on-target selectivity arise from the high sensitivity of the R-loop formation rate to DNA mismatches in the PAM-proximal region, which strongly correlates with cleavage efficiency. This work establishes R-loop formation as a conformational checkpoint for specific target cleavage, and provides a mechanistic framework to improve the fidelity of genome editing. [BMB Reports 2025; 58(8): 364-368].

Indexed as

Bacterial ProteinsCRISPR-Associated ProteinsEndodeoxyribonucleasesCRISPR-Cas SystemsDNADNA CleavageGene EditingKineticsRNA, Guide, CRISPR-Cas SystemsBacterial ProteinsCas12a proteinCRISPR-Associated ProteinsDNAEndodeoxyribonucleasesRNA, Guide, CRISPR-Cas Systems

Identifiers

PMID40754777

PMCPMC12402690

What OpenQuestion holds

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LicenceCC BY-NC

Read underepoch 390

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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.