Scientists at the Universities of Bristol, Münster, and the Lithuanian Institute of Biotechnology say they have observed and monitored the process by which CRISPRs bind to and alter the structure of DNA. They believe their finding will greatly increase the understanding of how enzymes such as CRISPRs edit genes, which may accelerate the development of novel gene therapy techniques.

The results (“Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes”), which are published in the Proceedings of the National Academy of Sciences (PNAS), provide a vital piece of the puzzle if these genome editing tools are ultimately going to be used to correct genetic diseases in humans, according to the researchers.

CRISPRs have been tailored to accurately target a single combination of letters within the three billion base pairs of the DNA molecule. This is the equivalent of correcting a single misspelt word in a 23-volume encyclopedia, note the researchers.

To find this needle in a haystack, CRISPRs rely on an RNA molecule. The targeting process requires the CRISPRs to pull apart the DNA strands and insert the RNA to form a sequence-specific structure called an R-loop. The research team tested the R-loop model using specially modified microscopes in which single DNA molecules are stretched in a magnetic field. By altering the twisting force on the DNA, the researchers could directly monitor R-loop formation events by individual CRISPRs. This allowed them to reveal previously hidden steps in the process and to probe the influence of the sequence of DNA bases.

“We use single molecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the R-loop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability,” wrote the investigators. “Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.”

“An important challenge in exploiting these exciting genome editing tools is ensuring that only one specific location in a genome is targeted,” said Mark Szczelkun, Ph.D., from the school of biochemistry at Bristol University. “Our single molecule assays have led to a greater understanding of the influence of DNA sequence on R-loop formation. In the future this will help in the rational re-engineering of CRISPR enzymes to increase their accuracy and minimize off-target effects. This will be vital if we are to ultimately apply these tools to correct genetic diseases in patients.”

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