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; Zev Bryant; Jennifer A Doudna

doi:10.1016/j.molcel.2025.03.024

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

Mol Cell. 2025 May 1;85(9):1730-1742.e9. doi: 10.1016/j.molcel.2025.03.024. Epub 2025 Apr 23.

Authors

Affiliations

¹ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
² Department of Bioengineering, Stanford University, Stanford, CA 94305, USA; Department of Physics, Stanford University, Stanford, CA 94305, USA.
³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Innovative 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.
⁵ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ Innovative 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.
⁷ Department 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.
⁸ Innovative 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.

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.

Keywords: CRISPR-Cas9; DNA recognition; DNA unwinding; biochemistry; biophysics; genome editing; kinetic models; protospacer-adjacent motifs; single-molecule manipulation; target search.

MeSH terms

Bacterial Proteins* / genetics
Bacterial Proteins* / metabolism
CRISPR-Associated Protein 9* / genetics
CRISPR-Associated Protein 9* / metabolism
CRISPR-Cas Systems*
DNA* / genetics
DNA* / metabolism
Gene Editing* / methods
HEK293 Cells
Humans
Nucleotide Motifs
RNA, Guide, CRISPR-Cas Systems* / genetics
RNA, Guide, CRISPR-Cas Systems* / metabolism
Streptococcus pyogenes* / enzymology
Streptococcus pyogenes* / genetics

Substances

RNA, Guide, CRISPR-Cas Systems
CRISPR-Associated Protein 9
Cas9 endonuclease Streptococcus pyogenes
Bacterial Proteins
DNA

Abstract

MeSH terms

Substances

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