Evidence map›Paper›PMID 40273916›Full record

ArticleMolecular cell2025

Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.

Honglue Shi, Noor Al-Sayyad, Kevin M Wasko, Marena I Trinidad, Erin E Doherty, Kamakshi Vohra, Ron S Boger, David Colognori, Joshua C Cofsky, Petr Skopintsev and 2 more

Abstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Molecular cell, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 20 papers.

0numbers the graph read from it
0cells of the map it votes in
20citing 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

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

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

20 citing papers in PubMed.

  1. Article
  2. Article
  3. Review
  4. Article
  5. Article
  6. Article
  7. Review
  8. Article
  9. Fully computational design of PAM-relaxedbioRxiv : the preprint server for biology · 2026
    Article
  10. Article
  11. Stepwise DNA unwinding gates TnpB genome-editing activity.bioRxiv : the preprint server for biology · 2026
    Article
  12. Structure and evolution-guided design of minimal RNA-guided nucleases.bioRxiv : the preprint server for biology · 2025
    Article
  13. Article
  14. Article
  15. Article
  16. Article
  17. Article
  18. Article
  19. Rational Design of Enhanced Nme2Cas9 and Nme2bioRxiv : the preprint server for biology · 2024
    Article
  20. Article
4 · The record

Corrections and comments

5 · Who and what money

Authors and funding

12 authors.

Honglue ShiInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Noor Al-SayyadDepartment of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Physics, Stanford University, Stanford, CA 94305, USA.
Kevin M WaskoInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Marena I TrinidadInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.
Erin E DohertyInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Kamakshi VohraInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Ron S BogerInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720, USA.
David ColognoriInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Joshua C CofskyInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Petr SkopintsevInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
Zev BryantDepartment of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Structural Biology, Stanford University Medical Center, Stanford, CA 94305, USA. Electronic address: zevry@stanford.edu.
Jennifer A DoudnaInnovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Li Ka Shing Center for Genomic Engineering, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA 94158, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA. Electronic address: doudna@berkeley.edu.

Funding

Structural Dynamics and Mechanochemical Coupling in Nucleoprotein MachinesR01GM106159 · NIGMS · STANFORD UNIVERSITY · PI Zev Bryant · 2014 to 2026
$3.2M
Expanding CRISPR-Cas editing technology through exploration of novel Cas proteins and DNA repair systemsU01AI142817 · NIAID · UNIVERSITY OF CALIFORNIA BERKELEY · PI BANFIELD, JILLIAN, DOUDNA, JENNIFER A · 2018 to 2022
$2.0M
Identification and Characterization of Functional lncRNAs in human cellsK99GM151484 · NIGMS · UNIVERSITY OF CALIFORNIA BERKELEY · PI COLOGNORI, DAVID · 2024 to 2025
$236k
Precision Deaminases to Expand the Scope of Diseases Amenable to CRISPR-mediated CorrectionF32GM153031 · NIGMS · UNIVERSITY OF CALIFORNIA BERKELEY · PI Erin Doherty Blackett · 2024 to 2026
$229k
Howard Hughes Medical InstituteNIAID NIH HHS U01 AI142817NIGMS NIH HHS F32 GM153031NIGMS NIH HHS K99 GM151484NIGMS NIH HHS R01 GM106159
6 · The paper itself

Abstract

RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.

Indexed as

Bacterial ProteinsCRISPR-Associated Protein 9CRISPR-Cas SystemsDNAGene EditingRNA, Guide, CRISPR-Cas SystemsStreptococcus pyogenesHEK293 CellsHumansNucleotide MotifsBacterial ProteinsCas9 endonuclease Streptococcus pyogenesCRISPR-Associated Protein 9DNARNA, Guide, CRISPR-Cas SystemsbiochemistrybiophysicsCRISPR-Cas9DNA recognitionDNA unwindinggenome editingkinetic modelsprotospacer-adjacent motifssingle-molecule manipulationtarget search

Identifiers

PMID40273916
PMCPMC12258621

What OpenQuestion holds

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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.