Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Gladstone Institutes, San Francisco, CA 94158, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.

PMID: 38781968
PMCID: PMC11658890
DOI: 10.1016/j.cell.2024.04.031

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Amy R Eggers et al. Cell. 2024.

. 2024 Jun 20;187(13):3249-3261.e14.

doi: 10.1016/j.cell.2024.04.031. Epub 2024 May 22.

Authors

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Gladstone Institutes, San Francisco, CA 94158, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.

PMID: 38781968
PMCID: PMC11658890
DOI: 10.1016/j.cell.2024.04.031

Abstract

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

Keywords: CRISPR-Cas; Cas9 engineering; DNA unwinding; GeoCas9; R-loop formation; WED domain; cryo-EM; genome editing; iGeoCas9; magnesium.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The Regents of the University of California have patents issued and pending for CRISPR technologies on which the authors are inventors. J.A.D. is a cofounder of Azalea Therapuetics, Caribou Biosciences, Editas Medicine, Evercrisp, Scribe Therapeutics, Intellia Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member at Evercrisp, Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, The Column Group, and Inari. J.A.D. is Chief Science Advisor to Sixth Street; is a Director at Johnson & Johnson, Altos, and Tempus; and has a research project sponsored by Apple Tree Partners.

Figures

**Figure 1.. Cryo-EM iGeoCas9-sgRNA-DNA ternary complex**
(A) Domain organization of GeoCas9. iGeoCas9 mutations indicated with red arrows. Deactivating mutations indicated with gray arrows. BH, bridge helix; WED, wedge; PI, PAM interacting; PLL, phosphate lock loop. (B) CRISPR-Cas9 dsDNA targeting pathway for active enzymes. Deactivated Cas9 concludes at the R-loop formation step. (C) Surface (left) and ribbon (right) representation of iGeoCas9-sgRNA-DNA ternary complex. Domains are colored as in (A). Nucleic acid is colored as in (D). BH, bridge helix; WED, wedge; PI, PAM interacting; TS, target strand; NTS, non-target strand; PAM, protospacer adjacent motif; sgRNA, single guide RNA. (D) Cartoon representation of iGeoCas9 sgRNA-dsDNA complex (left). Schematic representation of iGeoCas9 sgRNA:target DNA complex (right). PAM, protospacer adjacent motif. See also Figures S1–S3 and Table S1.

**Figure 2.. Structural comparison of wild-type GeoCas9 and iGeoCas9 WED domain**
(A) Ribbon representation of iGeoCas9 WED domain boxed with solid line. Amino acid mutation positions are boxed with a dashed line. Domains are colored as in Figure 1A. Nucleic acid is colored as in Figure 1D. TS, target strand; PAM, protospacer adjacent motif; sgRNA, single guide RNA. (B) Comparison of wild-type GeoCas9 and iGeoCas9 at WED domain amino acid positions 908, 843, and 884. Positions 908 and 843 are represented by an EM density map (gray) and model (sticks and ribbons). PAM nucleotide positions on the non-target strand are labeled in descending from −1 to −8 in the 5′ to 3′ direction. Nucleotides on the target strand are assigned the same number as their complementary nucleotides on the non-target strand. Potential DNA-protein interactions are indicated with a dashed line and atomic distance. Amino acid position 884 is represented by a Coulombic electrostatic surface potential map and encircled with a dashed line (red, negative; blue, positive; white, non-polar). NTS, non-target strand; TS, target strand; PAM, protospacer adjacent motif. See also Figures S1–S3 and Table S1.

**Figure 3.. iGeoCas9 demonstrates enhanced activity targeting wild-type GeoCas9 non-native PAMs**
(A) Schematic of the Cas9 cleavage reaction using 60 nucleotide (nt) 5′ 6-FAM labeled double-stranded (ds) DNA substrates with different PAM sequences. Nucleic acid is colored as in Figure 1D. (B) *In vitro* dsDNA cleavage activity of wild-type (WT-)GeoCas9 and iGeoCas9 determined by denaturing PAGE (n = 3, data are represented as mean ± SD). PAM contained in each substrate indicated above the graph. Fractions were collected at 0 s, 30 s, 1 min, 2.5 min, 5 min, 10 min, 30 min, 1 h, and 2 h. Fraction “0” is represented by the substrate only. The k_obs for each Cas9 are listed in the sample legend. See also Figure S4A. (C) Logo for sequences depleted from the PAM library by wild-type (WT-)GeoCas9 (left) and iGeoCas9 (right). Consensus PAM sequence located above the logo. PAM position on x axis and is numbered in the 5′ to 3′ direction in descending order from −5 to −8. C and T, blue; and A and G, green. (D) PAM nucleobase-interacting amino acids (N961, N1020, D1017, and R1035) of wild-type GeoCas9 and iGeoCas9. PAM sequence position indicated adjacent to the nucleotide. H-bond prediction was performed in ChimeraX v1.6.1 with a distance tolerance of 0.400Å and angle tolerance of 20°. Alternate rotamer conformations were observed for N1020 and R1035 in wild-type GeoCas9 and iGeoCas9. See also Figure S4A.

**Figure 4.. Thermodynamically unstable substrate mimics WED-domain mutation effects on DNA melting**
(A) Schematic of the Cas9 cleavage pathway for linear dsDNA substrates versus two base pair mismatch (2 bp mm) dsDNA substrate. The 60 nt DNA substrate is 5′ 6-FAM labeled (green). Nucleic acid is colored as in Figure 1D. (B) The order in which mutations were introduced to create GeoCas9(R1) and iGeoCas9. Mutations are listed below the arrow, and domains in which they are located are above the arrow. WED, wedge; PLL, phosphate lock loop. (C) *In vitro* dsDNA cleavage activity of wild-type (WT-)GeoCas9, iGeoCas9, GeoCas9(R1), and GeoCas9(KGR) determined by denaturing PAGE (n = 3, data are represented as mean ± SD). Substrate and PAM sequences are indicated above the graph. Fractions were collected at 0 s, 30 s, 1 min, 2.5 min, 5 min, 10 min, 30 min, 1 h, and 2 h. Fraction “0” is represented by the substrate only. The k_obs for each Cas9 are listed in the sample legend. See also Figure S4B.

**Figure 5.. Reduced concentration of MgCl₂ impacts wild-type GeoCas9 R-loop formation**
(A) *In vitro* dsDNA cleavage activity of wild-type (WT-)GeoCas9 and iGeoCas9, determined by denaturing PAGE (n = 3, data are represented as mean ± SD). MgCl₂ concentration indicated above the graph. Fractions were collected at 0 s, 30 s, 1 min, 2.5 min, 5 min, 10 min, 30 min, 1 h, and 2 h. Fraction “0” is represented by the substrate only. The rate constants k_obs are listed in the sample legend. See also Figure S4C. (B) Cartoon and chemical structure of 2-aminopurine (2AP) in the quenched and fluorescent states. Fluorescence indicated with yellow. T, thymine; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA. (C) Diagram of substrates (partial sequence) indicating positions of 2AP nucleotides measuring 5′ from the PAM (top). Drawing of the different stages of GeoCas9 R-loop formation with the three different substrates (bottom). Fluorescent 2APs are indicated in yellow. (D) 2AP fluorescence assays comparing catalytically inactivated wild-type (WT-)GeoCas9 and iGeoCas9 R-loop kinetics (n = 3, data are represented as the mean ± SD). Substrate 1, early R-loop formation. Substrate 2, mid R-loop formation. Substrate 3, late R-loop formation. MgCl₂ concentrations indicated above the graph. The rate constant k_obs are listed in the sample legend. See also Figure S4C.

**Figure 6.. WED-domain mutations greatly enhance genome-editing activities of Nme2Cas9**
(A) Model of Nme2Cas9 (PDB: 6JE3) in which all seven rationally engineered mutations are represented in the model as red sticks. Rotamers were chosen to demonstrate potential DNA interactions. (B) Workflow for EGFP-knockdown assay in HEK293T cells. Successful editing indicated by a loss of EGFP signal. (C) HEK293T cell editing by wild-type (WT-) Nme2Cas9 and iNme2Cas9 and 6 different guides (n = 4, data are represented as the mean ± SD). Neg, no treatment control; NT, non-targeting guide control. See also Figures S5 and S6.

See this image and copyright information in PMC

Update of

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.
Eggers AR, Chen K, Soczek KM, Tuck OT, Doherty EE, Thornton BW, Xu B, Trinidad MI, Doudna JA. Eggers AR, et al. bioRxiv [Preprint]. 2023 Dec 15:2023.12.14.571777. doi: 10.1101/2023.12.14.571777. bioRxiv. 2023. Update in: Cell. 2024 Jun 20;187(13):3249-3261.e14. doi: 10.1016/j.cell.2024.04.031. PMID: 38168238 Free PMC article. Updated. Preprint.

References

1. Sorek R, Lawrence CM, and Wiedenheft B (2013). CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem 82, 237–266. 10.1146/annurev-biochem-072911-172315. - DOI - PubMed
1. Jiang F, and Doudna JA (2017). CRISPR–Cas9 structures and mechanisms. Annu. Rev. Biophys 46, 505–529. 10.1146/annurev-biophys-062215-010822. - DOI - PubMed
1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, and Charpentier E (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821. 10.1126/science.1225829. - DOI - PMC - PubMed
1. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, and Zhang F (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. 10.1126/science.1231143. - DOI - PMC - PubMed
1. Wang JY, and Doudna JA (2023). CRISPR technology: A decade of genome editing is only the beginning. Science 379, eadd8643. 10.1126/science.add8643. - DOI - PubMed

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Affiliations

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Update of

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials