Molecular Simulations have Boosted Knowledge of CRISPR/Cas9: A Review

Abstract

Genome editing allows scientists to change an organism's DNA. One promising genome editing protocol, already validated in living organisms, is based on clustered regularly interspaced short palindromic repeats (CRISPR)/Cas protein-nucleic acid complexes. When the CRISPR/Cas approach was first demonstrated in 2012, its advantages with respect to previously available techniques, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), immediately got attention and the method has seen a surge of experimental and computational investigations since then. However, the molecular mechanisms involved in target DNA recognition and cleavage are still not completely resolved and need further attention. The large size and complex nature of CRISPR/Cas9 complexes has been a challenge for computational studies, but some seed results exist and are illuminating on the cleavage activity. In this short review, we present recent progress in studying CRISPR/Cas9 systems by molecular dynamics simulations with coarse-grained and atomistic descriptions, including enhanced sampling.

Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations

“…Cas9 contains two lobes (Figure ), the recognition/REC lobe (residues 56–718) and the nuclease/NUC lobe (residues 1–55 and 719–1368). , The recognition lobe contains REC-I, REC-II, and REC-III domains responsible for nucleotide recognition. , The arginine-rich bridge helix serves as a linker between the RuvC-I and REC domains and is crucial for initiating cleavage activity upon binding to target DNA . Cas9 nuclease lobes have two endonuclease domains, the HNH domain (residues 766–909, rich in histidine and asparagine residues) and the RuvC domain (residues 1–55, 719–765, and 910–1099).…”

“… Additionally, the HNH domain contains two key hinge regions near its N and C terminus, linkers L1 and L2 (Figure a), which creates a cross-talk between the RuvC and HNH domains . The PAM interacting domain (PI, residues 1100–1368) confers PAM specificity and is therefore responsible for initiating binding to DNA. , Upon DNA binding, the positively charged residues present at the interface between the REC and NUC lobes, particularly at the bridge helix, stabilize the negatively charged sgRNA:DNA hybrid . However, positively charged residues present in the linker region (L1 and L2) between the RuvC and HNH domains help to stabilize the displaced ntDNA …”

Protein-Mutation-Induced Conformational Changes of the DNA and Nuclease Domain in CRISPR/Cas9 Systems by Molecular Dynamics Simulations

“…The types I and III utilize Cas endonucleases complexed with CRISPR RNAs (crRNAs) to mediate the recognition and subsequent degradation of target DNA, , whereas the type II recognizes and cleaves the target DNA via a RNA-guided endonuclease Cas protein . More classification details are reported elsewhere …”

“…Molecular dynamics (MD) simulations are a robust tool for deciphering the structure, dynamics, and energetics of biomolecules at an atomistic level . Atomistic MD studies have already revealed crucial features of CRISPR/Cas complexes, such as the role of the ntDNA strand in the process of activation of the HNH domain for tDNA strand cleavage, the recognition between RNA and Cas9, the evidence of a PAM-induced allosteric mechanism, allosteric motions of the HNH nuclease domain of CRISPR/Cas9, deciphering off-target effects, and others. , Following the hypothesis by Slaymaker and co-workers, we aim at developing an understanding of the atomic-level effects of protein mutations on the DNA-bound CRISPR/Cas9:sgRNA complex.…”

“…8 More classification details are reported elsewhere. 9 One such Class 2 type II CRISPR/Cas system is CRISPR/ Cas9, with CRISPR-associated protein 9. This complex is composed of a single guide RNA (sgRNA) and a 160 kDa DNA endonuclease enzyme, Cas9.…”