The development of SpCas9 variants with high specificity and efficiency based on the HH Theory

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1–V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

About the authors

G. H. Wang

Guangdong Industry Polytechnic

Email: lijuan860103@163.com

School of Food and Biotechnology

China, Guangzhou, 510300

C. M. Wang

Nanfang Hospital, Southern Medical University; Southern University of Science and Technology Hospital (SUS Tech Hospital)

Email: lijuan860103@163.com

Department of Pharmacy

China, Guangzhou, 510515; Shenzhen, 518000

X. J. Wu

The University of North Carolina at Chapel Hill

Email: lijuan860103@163.com

Lineberger Comprehensive Cancer Center

United States, Chapel Hill, North Carolina, 27517

T. Chu

Sangon Biotech (Shanghai) Co., Ltd.

Email: lijuan860103@163.com
China, Shanghai

D. W. Huang

Jinhua Polytechnic

Email: lijuan860103@163.com

Pharmaceutical and Material Engineering School

China, Jinhua, 321007

J. Li

Southern Medical University

Author for correspondence.
Email: lijuan860103@163.com

Department of Histology and Embryology, School of Basic Medical Sciences

China, Guangzhou, 510515

References

  1. Komor A.C., Badran A.H., Liu D.R. (2017) CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 169, 559.
  2. Doudna J.A. (2020) The promise and challenge of therapeutic genome editing. Nature. 578, 229‒236.
  3. Zhu X., Clarke R., Puppala A.K., Chittori S., Merk A., Merrill B.J., Simonovic M., Subramaniam S. (2019) Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9. Nat. Struct. Mol. Biol. 26, 679‒685.
  4. Wang G., Li J. (2021) Review, analysis, and optimization of the CRISPR Streptococcus pyogenes Cas9 system. Med. Drug Discov. 9, 100080. doi: 10.1016/j.medidd.(2021)100080
  5. Mozo-Villarías A., Querol E. (2019) A protein self-assembly model guided by electrostatic and hydrophobic dipole moments. PLoS One. 14, e0216253.
  6. Shashikala H.B.M., Chakravorty A., Alexov E. (2019) Modeling electrostatic force in protein-protein recognition. Front. Mol. Biosci. 6, 94.
  7. Grdadolnik J., Merzel F., Avbelj F. (2017) Origin of hydrophobicity and enhanced water hydrogen bond strength near purely hydrophobic solutes. Proc. Natl. Acad. Sci. USA. 114, 322‒327.
  8. Galamba N. (2013) Water’s structure around hydrophobic solutes and the iceberg model. J. Phys. Chem. B. 117, 2153‒2159.
  9. Kinoshita M. (2009) Importance of translational entropy of water in biological self-assembly processes like protein folding. Int. J. Mol. Sci. 10, 1064‒1080.
  10. Harano Y., Kinoshita M. (2004) Large gain in translational entropy of water is a major driving force in protein folding. Chem. Phys. Lett. 399, 342‒348. doi: 10.1016/j.cplett.2004.09.140
  11. Harano Y., Kinoshita M. (2005) Translational-entropy gain of solvent upon protein folding. Biophys. J. 89, 2701‒2710.
  12. Feng B., Sosa R.P., Martensson A.K.F., Jiang K., Tong A., Dorfman K.D., Takahashi M., Lincoln P., Bustamante C.J., Westerlund F., Norden B. (2019) Hydrophobic catalysis and a potential biological role of DNA unstacking induced by environment effects. Proc. Natl. Acad. Sci. USA. 116, 17169‒17174.
  13. Yakovchuk P., Protozanova E., Frank-Kamenetskii M.D. (2006) Base-stacking and base-pairing contributions into thermal stability of the DNA double helix. Nucleic Acids Res. 34, 564‒574.
  14. Vologodskii A., Frank-Kamenetskii M.D. (2018) DNA melting and energetics of the double helix. Phys. Life Rev. 25, 1‒21.
  15. Chen J.S., Dagdas Y.S., Kleinstiver B.P., Welch M.M., Sousa A.A., Harrington L.B., Sternberg S.H., Joung J.K., Yildiz A., Doudna J.A. (2017) Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature. 550, 407‒410.
  16. Kleinstiver B.P., Pattanayak V., Prew M.S., Tsai S.Q., Nguyen N.T., Zheng Z., Joung J.K. (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 529, 490‒495.
  17. Fu Y., Sander J.D., Reyon D., Cascio V.M., Joung J.K. (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279‒284.
  18. Slaymaker I.M., Gao L., Zetsche B., Scott D.A., Yan W.X., Zhang F. (2016) Rationally engineered Cas9 nucleases with improved specificity. Science. 351, 84‒88.
  19. Guo M., Ren K., Zhu Y., Tang Z., Wang Y., Zhang B., Huang Z. (2019) Structural insights into a high fidelity variant of SpCas9. Cell Res. 29, 183‒192.
  20. Wang G., Wang C., Chu T., Wu X., Anderson C.M., Huang D., Li J. (2023) Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency. J. Biotechnol. 368, 42‒52.
  21. Sykes P.J., Neoh S.H., Brisco M.J., Hughes E., Condon J., Morley A.A. (1992) Quantitation of targets for PCR by use of limiting dilution. Biotechniques. 13, 444‒449.
  22. Rose J.C., Stephany J.J., Valente W.J., Trevillian B.M., Dang H.V., Bielas J.H., Maly D.J., Fowler D.M. (2017) Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics. Nat. Methods. 14, 891‒896.
  23. Miyaoka Y., Mayerl S.J., Chan A.H., Conklin B.R. (2018) Detection and quantification of HDR and NHEJ induced by genome editing at endogenous gene loci using droplet digital PCR. Methods Mol. Biol. 1768, 349‒362.
  24. Wei C.T., Maly D.J., Fowler D.M. (2020) Temporal and rheostatic control of genome editing with a chemically-inducible Cas9. Methods Enzymol. 633, 119‒141.
  25. Dibitetto D., La Monica M., Ferrari M., Marini F., Pellicioli A. (2018) Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR. DNA Repair (Amst.). 68, 68‒74.
  26. Helfer-Hungerbuehler A.K., Shah J., Meili T., Boenzli E., Li P., Hofmann-Lehmann R. (2021) Adeno-associated vector-delivered CRISPR/SaCas9 system reduces feline leukemia virus production in vitro. Viruses. 13(8), 1636.
  27. Guschin D.Y., Waite A.J., Katibah G.E., Miller J.C., Holmes M.C., Rebar E.J. (2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247‒256.
  28. Auer B., Kumar R., Schmidt J.R., Skinner J.L. (2007) Hydrogen bonding and Raman, IR, and 2D-IR spectroscopy of dilute HOD in liquid D2O. Proc. Natl. Acad. Sci. USA. 104, 14215‒14220.
  29. Shibata M., Nishimasu H., Kodera N., Hirano S., Ando T., Uchihashi T., Nureki O. (2017) Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy. Nat. Commun. 8, 1430.
  30. Nishimasu H., Ran F.A., Hsu P.D., Konermann S., Shehata S.I., Dohmae N., Ishitani R., Zhang F., Nureki O. (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 156, 935‒949.
  31. Palecz B. (2002) Enthalpic homogeneous pair interaction coefficients of L-alpha-amino acids as a hydrophobicity parameter of amino acid side chains. J. Am. Chem. Soc. 124, 6003‒6008.
  32. Fauchère J.L., Charton M., Kier L.B., Verloop A., Pliska V. (1988) Amino acid side chain parameters for correlation studies in biology and pharmacology. Int. J. Pept. Protein Res. 32, 269‒278.
  33. Fu Y., Foden J.A., Khayter C., Maeder M.L., Reyon D., Joung J.K., Sander J.D. (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822‒826.
  34. Hsu P.D., Scott D.A., Weinstein J.A., Ran F.A., Konermann S., Agarwala V., Li Y., Fine E.J., Wu X., Shalem O., Cradick T.J., Marraffini L.A., Bao G., Zhang F. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827‒832.
  35. Vakulskas C.A., Dever D.P., Rettig G.R., Turk R., Jacobi A.M., Collingwood M.A., Bode N.M., McNeill M.S., Yan S., Camarena J., Lee C.M., Park S.H., Wiebking V., Bak R.O., Gomez-Ospina N., Pavel-Dinu M., Sun W., Bao G., Porteus M.H., Behlke M.A. (2018) A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216‒1224.
  36. Kulcsár P.I., Tálas A., Tóth E., Nyeste A., Ligeti Z., Welker Z., Welker E. (2020) Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5’G-extended sgRNAs. Nat. Commun. 11, 1223.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Supplementary 1
Download (223KB)
Indexing metadata
3. Supplementary 2
Download (6MB)
Indexing metadata

Note

#Полный текст статьи на английском языке размещен на сайте издательства Springer – https://link.springer.com/journal/11008


Copyright (c) 2024 Russian Academy of Sciences