CRISPR-Cas9, a popular genome-editing tool that allows researchers to make double-strand breaks to edit parts of the genome. It is simple, efficient, and customizable, which is advantageous for CRISPR-Cas9 over other gene-editing tools. However, Cas9 is an endonuclease that might cause unintended double-strand breaks in DNA, leading to an off-target effect. There have been many improvements to the CRISPR-Cas system. However, up until recently, many trials failed to insert DNA into specific locations without causing the toxic double-strand breaks in DNA. The presence of short CRISPR arrays similar to the one found in the type I-F CRISPR-Cas system has been reported in several bacterial Tn7-like transposons. Recently published studies showed that a Vibrio Cholera Tn6677 transposon consists of two main components; the Cascade (CRISPR-associated complex for antiviral defense), which consists of Cas6, six subunits of Cas7, and fused Cas8/5 (simply known as Cas8). The second component is the transposition subunits (TniQ, TnsC, TnsA, and TnsB) [see Fig.1]. The whole complex is called the INTEGRATE system (INsert Transposable Elements by Guide RNA-Assisted TargEting). This newly discovered system can accurately insert DNA sequences without causing a double-strand break in the target DNA. The INTEGRATE System is very promising and could be a very accurate gene-editing tool. DNA binding pathway of the INTEGRATE complex follows three major steps: - Assembly of the Cascade complex around CRISPR-RNA (crRNA) - Binding to a transposition protein (TniQ) - Binding to a double-stranded DNA (dsDNA) to initiate the RNA-guided DNA insertion process. It is worth noting that the published results reveal some of the on-target RNA-guided DNA transposition key factors, such as the formation of a complete R-loop structure as a checkpoint state. We hypothesize that more checkpoint states (conformations) can be discovered in atomic details using Molecular Dynamic Simulation (MD Simulation). The overall aim is that the resulting MD simulation will help to identify some of the on-target RNA-guided DNA transposition key factors and obtain molecular-level information on conformational changes that take place in the INTEGRATE system. Hopefully, the result will provide new insights into the molecular mechanisms of DNA targeting by the INTEGRATE system and improve the engineering of CRISPR-Cas systems with higher on-target accuracy.

Utilizing MD simulation to understand the molecular and structural mechanism underlying the DNA targeting by the INTEGRATE system

ACS Spring 2022

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