An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a

Saha, Aakash; Ahsan, Mohd; Arantes, Pablo Ricardo; Schmitz, M. Lienhard; Chanez, Christelle; Jinek, Martin; Palermo, Giulia

doi:10.1101/2022.09.05.506663

Cited by 5 publications

(8 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3 and 4). A number of studies have revealed DNA conformational changes post NTS-nicking in Cas12a (24,30,31,45).…”

Section: Discussionmentioning

confidence: 99%

“…While Cas9 cleaves a DNA target using two nuclease domains in a parallel-sequential fashion (27)(28)(29), Cas12a causes double-stranded DNA breaks using a single RuvC nuclease domain following a sequential mechanism, with the NTS being cleaved first at a site located in the PAM-distal segment of the protospacer, around 14-18 nucleotides from the PAM (19). Following nicking of the NTS, the protein-RNA-DNA complex undergoes additional conformational changes, eventually enabling cleavage of the TS at a site located 1-2 nucleotides beyond the DNA/RNA hybrid within the R-loop flank (19,30,24,31). Consequently, Cas12a yields a mismatch tolerance pattern that is clearly different from that of Cas9 (32), with little to no tolerance for PAM-distal mismatches (33,34).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A DNA Unwinding Equilibrium Serves as a Checkpoint for CRISPR-Cas12a Target Discrimination

Singh

Liu

Allen

et al. 2023

Preprint

View full text Add to dashboard Cite

CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The data sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.

show abstract

“…3 and 4). A number of studies have revealed DNA conformational changes post NTS-nicking in Cas12a (24,30,31,45).…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A DNA Unwinding Equilibrium Serves as a Checkpoint for CRISPR-Cas12a Target Discrimination

Singh

Liu

Allen

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…158 However, enhanced sampling simulations have recently revealed that an α-helical lid, located within the RuvC domain, also aids in the traversal of the TS by anchoring the crRNA:TS hybrid. 159 ■ BEST PRACTICES TO MODEL PROTEIN/NUCLEIC…”

Section: ■ Gene Editing Systemsmentioning

confidence: 99%

“…Multi-μs MD simulation revealed that DNA binding induces coupled dynamics of the REC2 and RuvC that prime the conformational transition of TS toward the catalytic site . However, enhanced sampling simulations have recently revealed that an α-helical lid, located within the RuvC domain, also aids in the traversal of the TS by anchoring the crRNA:TS hybrid …”

Section: Gene Editing Systemsmentioning

confidence: 99%

Machines on Genes through the Computational Microscope

Sinha

Pindi

Ahsan

et al. 2023

J. Chem. Theory Comput.

Self Cite

View full text Add to dashboard Cite

Macromolecular machines acting on genes are at the core of life’s fundamental processes, including DNA replication and repair, gene transcription and regulation, chromatin packaging, RNA splicing, and genome editing. Here, we report the increasing role of computational biophysics in characterizing the mechanisms of “machines on genes”, focusing on innovative applications of computational methods and their integration with structural and biophysical experiments. We showcase how state-of-the-art computational methods, including classical and ab initio molecular dynamics to enhanced sampling techniques, and coarse-grained approaches are used for understanding and exploring gene machines for real-world applications. As this review unfolds, advanced computational methods describe the biophysical function that is unseen through experimental techniques, accomplishing the power of the “computational microscope”, an expression coined by Klaus Schulten to highlight the extraordinary capability of computer simulations. Pushing the frontiers of computational biophysics toward a pragmatic representation of large multimegadalton biomolecular complexes is instrumental in bridging the gap between experimentally obtained macroscopic observables and the molecular principles playing at the microscopic level. This understanding will help harness molecular machines for medical, pharmaceutical, and biotechnological purposes.

show abstract

“…The role of H983 as a nucleophilic activator is in accordance with mutation data revealing a hampered cleavage of the ntDNA strand when H983 is mutated to alanine. Further in-depth discussion can be found elsewhere ( Nierzwicki et al, 2021 ; Patel and Palermo, 2022 ; SahaAshan et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

Wang

Arantes

Ahsan

et al. 2023

Front. Mol. Biosci.

Self Cite

View full text Add to dashboard Cite

The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

show abstract

An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a

Cited by 5 publications

References 55 publications

A DNA Unwinding Equilibrium Serves as a Checkpoint for CRISPR-Cas12a Target Discrimination

A DNA Unwinding Equilibrium Serves as a Checkpoint for CRISPR-Cas12a Target Discrimination

Machines on Genes through the Computational Microscope

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes

Contact Info

Product

Resources

About