Exploring the Catalytic Mechanism of Cas9 Using Information Inferred from Endonuclease VII

Abstract

Elucidating the nature of the gene editing mechanism of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an important task in view of the role of this breakthrough to the advancement of human medicine. In particular, it is crucial to understand the catalytic mechanism of Cas9 (one of the CRISPR associated proteins) and its role in confirming accurate editing. Thus, we focus in this work on an attempt to analyze the catalytic mechanism of Cas9. Considering the absence of detailed structural information on the active form of Cas9, we use an empirical valence bond (EVB) which is calibrated on the closely related mechanism of T4 endonuclease VII. The calibrated EVB is then used in studying the reaction of Cas9, while trying several structural models. It is found that the catalytic activation requires a large conformational change, where K848 or other positively charged group moves from a relatively large distance toward the scissile phosphate. This conformational change leads to the change in position of the Mg 2+ ion and to a major reduction in the activation barrier for the catalytic reaction. Our finding provides an important clue on the nature of the catalytic activation of CAS9 and thus should help in elucidating a key aspect of the gene editing process. For example, the approach used here should be effective in exploring the nature of off target activation and its relationship to the energetics of the unwinding process. This strategy may offer ways to improve the selectivity of Cas9.

Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain

“…Together, our results clearly show N863, instead of D861, is the functional catalytic residue. Some studies have also suggested the possibility of N863 in forming the catalytic center of the Cas9 HNH domain (Nishimasu et al, 2014; Yoon et al, 2019). In contrast to the observation from another in silico model that was recently published (Yoon et al, 2019), our computational model indicated that D861 and N863 are unlikely to be simultaneously involved in the coordination of Mg 2+ ion for cutting the tDNA.…”

“…Despite lacking experimental evidence, it is generally believed that D861 directly participates in Mg 2+ chelation and tDNA cleavage (Huai et al, 2017; Palermo et al, 2018; Palermo et al, 2017; Zuo and Liu, 2017). An in silico model (Yoon et al, 2019) that was recently reported also suggested that D861 and N863 are potentially involved in chelating the Mg 2+ ion at the catalytic center of the HNH domain, although this discovery also remained untested in an experimental setting. In the diverse homologous structures of DNA/RNA nucleases from other species (Yang, 2008; Yang, 2011), however, the residues spatially equivalent to the D861 of SpyCas9 are conserved as an asparagine.…”

Enhancing ade novoenzyme activity by computationally-focused ultra-low-throughput screening

“…48,49,[91][92][93][94][95] In addition, whereas we and others have been able to obtain high delity with experimental values across a wide range of enzymes and enzyme variants even in the case of far more complex systems than the current Kemp eliminase. [98][99][100][104][105][106][107][108][109] This makes EVB useful as a predictive tool for systems where the changes in energy involved are not as subtle as in the case of Kemp elimination.…”

Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

“…46,[87][88][89][90][91][92] In addition, whereas we and others have been able to obtain high fidelity with experimental values across a wide range of enzymes and enzyme variants even in the case of far more complex systems than the current Kemp eliminase. [95][96][97][101][102][103][104][105][106] This makes EVB useful as a predictive tool for systems where the changes in energy involved as not as subtle as in the case of Kemp elimination.…”