Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects

Zuo, Zhicheng; Babu, Kesavan; Ganguly, Chhandosee; Zolekar, Ashwini; Newsom, Sydney; Rajan, Rakhi; Wang, Yu-Chieh; Liu, Jin

doi:10.1089/crispr.2021.0076

Cited by 14 publications

(12 citation statements)

References 51 publications

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“…A recent study by Liu and co-workers proposed two SpCas9 variants (HSC 1.1 and HSC 1.2) with enhanced specificity, also using a structure-guided engineering method. 134 The K1246 residue found from our EDA method was also seen in the HSC1.1 variant. The R691A (HiFi Cas9), 135 K526E, R661Q (evoCas9), 136 , and K890N (sniper Cas9) 137 are some of the other residues mentioned in previous studies, which are also aligned with our candidates (Table S5).…”

Section: Figures 5a and 5cmentioning

confidence: 62%

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate off-Target Effects

Maghsoud

Jayasinghe‐Arachchige

Kumari

et al. 2023

Preprint

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The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an RNA-guided targeted genome-editing tool that uses the Cas family proteins. Two magnesium-dependent nuclease domains of this enzyme termed HNH and RuvC are responsible for the cleavage of the target DNA (t-DNA) and non-target DNA strand (nt-DNA), respectively. It is believed that the HNH domain determines the DNA cleavage activity of both endonuclease domains and is sensitive to RNA-DNA base pairing complementary. However, the underlying molecular mechanisms of CRISPR-Cas9, by which it rebukes or accepts mismatches, are poorly understood. Thus, investigation of the structure and dynamics of the catalytic state of Cas9 with either matched or mismatched t-DNA can provide insights for improving its specificity in off-target cleavage. Here we focus on a recently discovered catalytic-active form of the Streptococcus pyogenes Cas9 (SpCas9) and employ classical molecular dynamics (MD) and hybrid QM/MM to study two possible reaction mechanisms of t-DNA cleavage reaction catalyzed by the HNH domain. Moreover, by designing a mismatched t-DNA structure called MM5 (C to G in the fifth position of the PAM region), the impact of single-guide RNA (sgRNA) and t-DNA complementarity on the catalysis process was investigated. Our calculated binding affinities, minimum energy paths, and analysis of catalytically important residues based on these simulations provide atomic-level details of the differences between matched and mismatched cleavage reactions. In addition, several residues exhibit significant differences in their catalytic role for the two considered systems, including K896, R820, K253, K263, K268, and R400, which will be used for further experimental investigations.

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Section: Figures 5a and 5cmentioning

confidence: 62%

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate off-Target Effects

Maghsoud

Jayasinghe‐Arachchige

Kumari

et al. 2023

Preprint

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“…In clustering, the simulation trajectories were prealigned to the starting structure, involving the backbone atoms of the PI and WED domains and those of the PAM duplex (here the four base pairs that are immediately adjacent to the protospacer). The binding energy of the above four-base pair PAM duplex to the PI and WED domains was estimated by the molecular mechanics-generalized Born surface area (MM-GBSA) approach, following the protocol as described in our previous studies. ,− The total binding energy was also decomposed on a per-residue basis. The PI and WED domains, PAM-containing full duplex, and the +1 nucleotide on the target DNA strand were subjected to further correlation network analysis, using the linear mutual information metric and a correlation cutoff of 0.4.…”

Section: Methodsmentioning

confidence: 99%

“…Several strategies have been reported to optimize the targeting specificity of SpCas9, ,, including truncation of gRNA at its 5′ end, using paired SpCas9 nickases, and dimeric fusions of catalytically dead SpCas9 to a FokI nuclease domain . In particular, recent years have seen the development of numerous high-fidelity SpCas9 variants. − Yet these versions of SpCas9-gRNA are generally incapable of distinguishing between the canonical NGG PAM and noncanonical PAMs (e.g., NGA and NAG), as they were originally engineered with the aim to improve the sensitivity of SpCas9-gRNA to imperfectly complementary DNA targets. The pioneering study by Kleinstiver et al shed light on increasing SpCas9 PAM recognition specificity.…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanism of D1135E-Induced Discriminated CRISPR-Cas9 PAM Recognition

Kang

Zuo

Yin

et al. 2022

J. Chem. Inf. Model.

Self Cite

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The off-target effects of Streptococcus pyogenes Cas9 (SpCas9) pose a significant challenge to harness it as a therapeutical approach. Two major factors can result in SpCas9 off-targeting: tolerance to target DNA−guide RNA (gRNA) mismatch and less stringent recognition of protospacer adjacent motif (PAM) flanking the target DNA. Despite the abundance of engineered SpCas9-gRNA variants with improved sensitivity to target DNA−gRNA mismatch, studies focusing on enhancing SpCas9 PAM recognition stringency are quite few. A recent pioneering study identified a D1135E variant of SpCas9 that exhibits much-reduced editing activity at the noncanonical NAG/NGA PAM sites while preserving robust on-target activity at the canonical NGG-flanking sites (N is any nucleobase). Herein, we aim to clarify the molecular mechanism by which this single D1135E mutation confers on SpCas9 enhanced specificity for PAM recognition by molecular dynamics simulations. The results suggest that the variant maintains the base-specific recognition for the canonical NGG PAM via four hydrogen bonds, akin to that in the wild type (WT) SpCas9. While the noncanonical NAG PAM is engaged to the two PAM-interacting arginine residues (i.e., R1333 and R1335) in WT SpCas9 via two to three hydrogen bonds, the D1135E variant prefers to establish two hydrogen bonds with the PAM bases, accounting for its minimal editing activity on the off-target sites with an NAG PAM. The impaired NAG recognition by D1135E SpCas9 results from the PAM duplex displacement such that the hydrogen bond of R1333 to the second PAM base is disfavored. We further propose a mechanistic model to delineate how the mutation perturbs the noncanonical PAM recognition. We anticipate that the mechanistic knowledge could be leveraged for continuous optimization of SpCas9 PAM recognition specificity toward high-precision demanding applications.

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“… SpCas9-HF1 SpCas9 Reduce the interaction between Cas9 and nontarget DNA sites HNH and REC3 domains 342 eSpCas9 SpCas9 Neutralize the positive charges of Cas9 and DNA links and sites. HNH and PAM-interacting domains 343 Sniper-Cas9 SpCas9 Sniper screen, an E. coli -based selection method 101 HypaCas9 SpCas9 REC3 and DNA complementation control HNH domain activation REC3 domain 103 evoCas9 SpCas9 Screening method using a yeast reporter strain REC3 domain 102 Cas9TX SpCas9 Prevent the perfect repair of DNA Carry optimized TREX2 110 HscCas9-v1.2 SpCas9 Substitution of amino acid residues Multiple domains 105 superFi-Cas9 SpCas9 When mismatched, sgRNA, and DNA chains form RuvC loop RuvC loop 104 efSaCas9 SaCas9 Construction of an SaCas9 variant library and directional screening system REC3 domain 106 SaCas9-HF SaCas9 Modify that residues where the distal region of PAM is linked to the target DNA Recognition lobe domain and RuvC domain 108 …”

Section: Crispr-based Gene-editing Toolsmentioning

confidence: 99%

“…[98][99][100] Due to the limitations of the PAM, the CRISPR/Cas9 gene-editing system often fails to target the proper sites. Therefore, the modification of Cas9 focuses on two goals: enhancing the security of Cas9 [101][102][103][104][105][106][107][108][109][110] and freeing it from the limitations of PAM [111][112][113][114][115][116][117] (Tables 1, 2). Method for CRISPR delivery Plasmid DNA (pDNA) is an ideal vector for loading the CRISPR system because it is not easily degradable, can be amplified in large quantities, and can be easily modified.…”

Section: Composition Of Crispr/cas9mentioning

confidence: 99%

CRISPR/Cas9 therapeutics: progress and prospects

Yang

et al. 2023

Sig Transduct Target Ther

163

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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. A variety of efficient Cas9 variants and derivatives have been developed to cope with the complex genomic changes that occur during diseases. However, strategies to effectively deliver the CRISPR system to diseased cells in vivo are currently lacking, and nonviral vectors with target recognition functions may be the focus of future research. Pathological and physiological changes resulting from disease onset are expected to serve as identifying factors for targeted delivery or targets for gene editing. Diseases are both varied and complex, and the choice of appropriate gene-editing methods and delivery vectors for different diseases is important. Meanwhile, there are still many potential challenges identified when targeting delivery of CRISPR/Cas9 technology for disease treatment. This paper reviews the current developments in three aspects, namely, gene-editing type, delivery vector, and disease characteristics. Additionally, this paper summarizes successful examples of clinical trials and finally describes possible problems associated with current CRISPR applications.

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Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects

Cited by 14 publications

References 51 publications

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate off-Target Effects

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate off-Target Effects

Molecular Mechanism of D1135E-Induced Discriminated CRISPR-Cas9 PAM Recognition

CRISPR/Cas9 therapeutics: progress and prospects

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