To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9H982A) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn2+-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn2+, but not Mg2+, produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.
Get full access to this article
View all access options for this article.
References
1.
MojicaFJ, Diez-VillasenorC, Garcia-MartinezJ, et al.Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol, 2005; 60(2):174–182; doi: 10.1007/s00239-004-0046-3
2.
MakarovaKS, GrishinNV, ShabalinaSA, et al.A putative RNA-interference-based immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct, 2006; 1:7; doi: 10.1186/1745-6150-1-7
3.
BolotinA, QuinquisB, SorokinA, et al.Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology, 2005; 151(Pt 8):2551–2561; doi: 10.1099/mic.0.28048-0
4.
GleditzschD, PauschP, Müller-EsparzaH, et al.PAM identification by CRISPR-Cas effector complexes: Diversified mechanisms and structures. RNA Biol, 2019; 16(4):504–517; doi: 10.1080/15476286.2018.1504546
5.
NewsomS, ParameshwaranHP, MartinL, et al.The CRISPR-Cas mechanism for adaptive immunity and alternate bacterial functions fuels diverse biotechnologies. Front Cell Infect Microbiol, 2020; 10:619763; doi: 10.3389/fcimb.2020.619763
6.
MakarovaKS, WolfYI, IranzoJ, et al.Evolutionary classification of CRISPR-Cas systems: A burst of class 2 and derived variants. Nat Rev Microbiol, 2020; 18(2):67–83; doi: 10.1038/s41579-019-0299-x
7.
JinekM, ChylinskiK, FonfaraI, et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012; 337(6096):816–821; doi: 10.1126/science.1225829
8.
NishimasuH, RanFA, HsuPD, et al.Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 2014; 156(5):935–949; doi: 10.1016/j.cell.2014.02.001
9.
JiangF, DoudnaJA. CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys, 2017; 46:505–529; doi: 10.1146/annurev-biophys-062215-010822
10.
JinekM, JiangF, TaylorDW, et al.Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 2014; 343(6176):1247997; doi: 10.1126/science.1247997
11.
SternbergSH, ReddingS, JinekM, et al.DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 2014; 507(7490):62–67; doi: 10.1038/nature13011
12.
JiangF, TaylorDW, ChenJS, et al.Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science, 2016; 351(6275):867–871; doi: 10.1126/science.aad8282
13.
JiangF, ZhouK, MaL, et al.Structural biology. A Cas9-guide RNA complex preorganized for target DNA recognition. Science, 2015; 348(6242):1477–1481; doi: 10.1126/science.aab1452
14.
SternbergSH, LaFranceB, KaplanM, et al.Conformational control of DNA target cleavage by CRISPR-Cas9. Nature, 2015; 527(7576):110–113; doi: 10.1038/nature15544
15.
JosephsEA, KocakDD, FitzgibbonCJ, et al.Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Res, 2016; 44(5):2474; doi: 10.1093/nar/gkv1293
16.
RaperAT, StephensonAA, SuoZ. Functional insights revealed by the kinetic mechanism of CRISPR/Cas9. J Am Chem Soc, 2018; 140(8):2971–2984; doi: 10.1021/jacs.7b13047
17.
GongSZ, YuHH, JohnsonKA, et al.DNA unwinding is the primary determinant of CRISPR-Cas9 activity. Cell Rep, 2018; 22(2):359–371; doi: 10.1016/j.celrep.2017.12.041
18.
BabuK, KathiresanV, KumariP, et al.Coordinated actions of Cas9 HNH and RuvC nuclease domains are regulated by the bridge helix and the target DNA sequence. Biochemistry, 2021; 60(49):3783–3800; doi: 10.1021/acs.biochem.1c00354
19.
CasalinoL, NierzwickiL, JinekM, et al.Catalytic mechanism of non-target DNA cleavage in CRISPR-Cas9 revealed by ab initio molecular dynamics. ACS Catal, 2020; 10(22):13596–13605; doi: 10.1021/acscatal.0c03566
20.
EastKW, NewtonJC, MorzanUN, et al.Allosteric motions of the CRISPR-Cas9 HNH nuclease probed by NMR and molecular dynamics. J Am Chem Soc, 2020; 142(3):1348–1358; doi: 10.1021/jacs.9b10521
21.
NierzwickiL, ArantesPR, SahaA, et al.Establishing the allosteric mechanism in CRISPR-Cas9. Wiley Interdiscip Rev Comput Mol Sci, 2021; 11(3):e1503; doi: 10.1002/wcms.1503
22.
PalermoG. Structure and dynamics of the CRISPR-Cas9 catalytic complex. J Chem Inf Model, 2019; 59(5):2394–2406; doi: 10.1021/acs.jcim.8b00988
23.
PalermoG, RicciCG, FernandoA, et al.Protospacer adjacent motif-induced allostery activates CRISPR-Cas9. J Am Chem Soc, 2017; 139(45):16028–16031; doi: 10.1021/jacs.7b05313
24.
ZuoZ, LiuJ. Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage. Curr Opin Struct Biol, 2020; 62:166–174; doi: 10.1016/j.sbi.2020.01.013
25.
SundaresanR, ParameshwaranHP, YogeshaSD, et al.RNA-independent DNA Cleavage Activities of Cas9 and Cas12a. Cell Rep, 2017; 21(13):3728–3739; doi: 10.1016/j.celrep.2017.11.100
26.
SahaC, MohanrajuP, StubbsA, et al.Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA. Sci Adv, 2020; 6(25):eaaz4849; doi: 10.1126/sciadv.aaz4849
27.
LiB, YanJ, ZhangY, et al.CRISPR-Cas12a possesses unconventional DNase activity that can be inactivated by synthetic oligonucleotides. Mol Ther Nucleic Acids, 2020; 19:1043–1052; doi: 10.1016/j.omtn.2019.12.038
28.
SahaC, Horst-KreftD, KrossI, et al.Campylobacter jejuni Cas9 modulates the transcriptome in Caco-2 intestinal epithelial cells. Genes (Basel), 2020; 11(10):1193; doi: 10.3390/genes11101193
29.
SlaymakerIM, GaoL, ZetscheB, et al.Rationally engineered Cas9 nucleases with improved specificity. Science, 2016; 351(6268):84–88; doi: 10.1126/science.aad5227
30.
TangH, YuanH, DuW, et al.Active-site models of Streptococcus pyogenes Cas9 in DNA cleavage state. Front Mol Biosci, 2021; 8:653262; doi: 10.3389/fmolb.2021.653262
31.
BabuK, AmraniN, JiangW, et al.Bridge helix of Cas9 modulates target DNA cleavage and mismatch tolerance. Biochemistry, 2019; 58(14):1905–1917; doi: 10.1021/acs.biochem.8b01241
32.
SchneiderCA, RasbandWS, EliceiriKW. NIH Image to ImageJ: 25 Years of image analysis. Nat Methods, 2012; 9(7):671–675.
PalermoG, MiaoY, WalkerRC, et al.Striking plasticity of CRISPR-Cas9 and key role of non-target DNA, as revealed by molecular simulations. ACS Cent Sci, 2016; 2(10):756–763; doi: 10.1021/acscentsci.6b00218
35.
ZuoZ, LiuJ. Cas9-catalyzed DNA cleavage generates staggered ends: Evidence from molecular dynamics simulations. Sci Rep, 2016; 5:37584; doi: 10.1038/srep37584
36.
KuscuC, ArslanS, SinghR, et al.Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol, 2014; 32(7):677–683; doi: 10.1038/nbt.2916
37.
GrotzKK, Cruz-LeónS, SchwierzN. Optimized magnesium force field parameters for biomolecular simulations with accurate solvation, ion-binding, and water-exchange properties. J Chem Theory Comput, 2021; 17(4):2530–2540; doi: 10.1021/acs.jctc.0c01281
38.
GrotzKK, SchwierzN. Magnesium force fields for OPC water with accurate solvation, ion-binding, and water-exchange properties: Successful transfer from SPC/E. J Chem Phys, 2022; 156(11):114501; doi: 10.1063/5.0087292
39.
FuruhataY, KatoY. Asymmetric roles of two histidine residues in Streptococcus pyogenes Cas9 catalytic domains upon chemical rescue. Biochemistry, 2021; 60(3):194–200; doi: 10.1021/acs.biochem.0c00766
40.
DeweeseJE, OsheroffN. The use of divalent metal ions by type II topoisomerases. Metallomics, 2010; 2(7):450–459; doi: 10.1039/c003759a
41.
IrvingH, WilliamsR. Order of stability of metal complexes. Nature, 1948; 162:746–747; doi: 10.1038/162746a0
42.
DürrSL, BohuszewiczO, SuardiazR, et al.The dual role of histidine as general base and recruiter of a third metal ion in HIV-1 RNase H. ChemRxiv; 2019.
SwartsDC, JinekM. Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a. Mol Cell, 2019; 73(3):589.e4–600.e4; doi: 10.1016/j.molcel.2018.11.021
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
