DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity

Gong, Shanzhong; Yu, Helen; Johnson, Kenneth A.; Taylor, David W.

doi:10.2139/ssrn.3155775

Cited by 20 publications

(44 citation statements)

References 35 publications

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“…Stopped flow experiment was performed as previously described. 15 Briefly, cas9-gRNA complex (1:1 ratio) was mixed with tC o -labeled 55/55 nt DNA substrate at 37℃ using AutoSF-120 stoppedflow instrument (KinTek Corporation, Austin, TX). Then, the fluorophore was excited at 367 nm, and monitored at 445 nm using a single band-pass filter with 20 nm bandwidth (Semrock).…”

Section: Stopped-flow Kinetic Assaymentioning

confidence: 99%

“…However, the mechanism of target discrimination and the rules for further improving fidelity remain unclear [10][11][12][13][14] . Recently, we showed that DNA cleavage is fast and DNA unwinding is the rate-limiting and specificity-determining step for on-target cleavage by SpCas9 15 . Later single molecule studies confirmed that DNA unwinding is the rate-limiting step for SpCas9 and suggested that DNA unwinding is the determinant for specificity between wildtype and high-fidelity Cas9 variants, Cas9-HF1 and eCas9, for 3 bp PAM-distal mismatches 16 .…”

mentioning

confidence: 99%

“…Rather, because R-loop formation is fast, discrimination is a function of the kinetic partitioning between the rates of DNA release versus cleavage. In order to quantify the kinetic partitioning, we incubated enzyme and labeled DNA in the absence of Mg 2+ , which allows for R-loop formation, but prevents catalysis 15 and then added Mg 2+ and an excess of unlabeled DNA to serve as a trap. Comparison between parallel experiments performed in the presence and absence of the DNA trap provides an estimate for the fractional kinetic partitioning for dissociation versus cleavage of bound DNA.…”

mentioning

confidence: 99%

“…We propose that Cas9 uses an induced-fit mechanism analogous to DNA polymerases, where a conformational change after initial substrate binding is an important determinant of enzyme specificity. For SpCas9, the conformational change is rate-limiting and determines specificity because R-loop formation is largely irreversible and is followed by fast chemistry 15 . The higher-fidelity variants have extraordinarily slow chemistry, allowing for release of the substrate from the enzyme before the irreversible cleavage reaction even though the dissociation rate is slow.…”

mentioning

confidence: 99%

“…In vitro sgRNA transcription and refolding were performed as described previously 15 . The primers used for the templates of sgRNA transcription are listed (Table S2).…”

mentioning

confidence: 99%

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Basis for discrimination by engineered CRISPR/Cas9 enzymes

Liu

Gong

et al. 2019

Preprint

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CRISPR/Cas9 is a programmable genome editing tool that has been widely used for biological applications. While engineered Cas9s have been reported to increase discrimination against off-target cleavage compared with wild type Streptococcus pyogenes (SpCas9) in vivo, the mechanism for enhanced specificity has not been extensively characterized. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. While DNA unwinding is the rate-limiting step for on-target cleavage by SpCas9, we show that chemistry is seriously impaired by more than 100-fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the rate of chemistry without increasing the rate of DNA rewinding-the kinetic partitioning favors release rather than cleavage of a bound off-target substrate because chemistry is slow. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

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Section: Stopped-flow Kinetic Assaymentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…In vitro sgRNA transcription and refolding were performed as described previously 15 . The primers used for the templates of sgRNA transcription are listed (Table S2).…”

mentioning

confidence: 99%

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Basis for discrimination by engineered CRISPR/Cas9 enzymes

Liu

Gong

et al. 2019

Preprint

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Explicit Treatment of Non‐Michaelis‐Menten and Atypical Kinetics in Early Drug Discovery**

Srinivasan

2020

ChemMedChem

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Biological systems are highly regulated. They are also highly resistant to sudden perturbations enabling them to maintain the dynamic equilibrium essential to sustain life. This robustness is conferred by regulatory mechanisms that influence the activity of enzymes/proteins within their cellular context to adapt to changing environmental conditions. However, the initial rules governing the study of enzyme kinetics were mostly tested and implemented for cytosolic enzyme systems that were easy to isolate and/or recombinantly express. Moreover, these enzymes lacked complex regulatory modalities. Now, with academic labs and pharmaceutical companies turning their attention to more‐complex systems (for instance, multiprotein complexes, oligomeric assemblies, membrane proteins and post‐translationally modified proteins), the initial axioms defined by Michaelis‐Menten (MM) kinetics are rendered inadequate, and the development of a new kind of kinetic analysis to study these systems is required. This review strives to present an overview of enzyme kinetic mechanisms that are atypical and, oftentimes, do not conform to the classical MM kinetics. Further, it presents initial ideas on the design and analysis of experiments in early drug‐discovery for such systems, to enable effective screening and characterisation of small‐molecule inhibitors with desirable physiological outcomes.

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Two-Metal Ion Mechanism of DNA Cleavage in CRISPR-Cas9

Palermo

Casalino

Jínek

2020

Biophysical Journal

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CRISPR-Cas9 is a cutting-edge genome-editing technology, which employs the endonuclease Cas9 to cleave DNA sequences of interest. However, the catalytic mechanism of DNA cleavage and the critical role of the Mg 2+ ions have remained elusive. Here, quantum-classical QM(Car-Parrinello)/MM simulations are used to disclose the two-Mg 2+ aided mechanism of phosphodiester bond cleavage in the RuvC domain. We reveal that the catalysis proceeds through an associative pathway activated by H983 and fundamentally assisted by the joint dynamics of the two Mg 2+ ions, which cooperatively act to properly orient the reactants and lead the chemical step to completion. Cross-validation of this mechanism is achieved by evaluating alternative reaction pathways and in light of experimental data, delivering fundamental insights on how CRISPR-Cas9 cleaves nucleic acids. This knowledge is critical for improving the Cas9 catalytic efficiency and its metal-dependent function, helping also the development of novel Cas9-based genome-editing tools.

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DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity

Cited by 20 publications

References 35 publications

Basis for discrimination by engineered CRISPR/Cas9 enzymes

Basis for discrimination by engineered CRISPR/Cas9 enzymes

Explicit Treatment of Non‐Michaelis‐Menten and Atypical Kinetics in Early Drug Discovery**

Two-Metal Ion Mechanism of DNA Cleavage in CRISPR-Cas9

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