Investigators at MilliporeSigma recently developed a set of genomic tools that makes the CRISPR-Cas9 genome editing method more specific and efficient. The findings from the new study—published recently in Nature Communications through an article entitled “Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting”—could help researchers overcome the limited targeting abilities of the original CRISPR enzyme in human cells, thus widening the range of potential druggable genomic targets.

“With more flexible and easy-to-use genome editing technologies, there is greater potential in research, bioprocessing, and novel treatment modalities,” remarked Udit Batra, Ph.D., CEO of MilliporeSigma.

The scientists dubbed the new technique “proxy-CRISPR,” which they note provides access to previously unreachable areas of the genome. Most natural CRISPR systems, found in bacteria, cannot work in human cells without significant re-engineering. However, proxy-CRISPR provides a rapid and simple method to increase their usability without the laborious need to re-engineer native CRISPR proteins.

“In our exploration of CRISPR–Cas systems, we have uncovered that the type II-B CRISPR–Cas9 system from Francisella novicida U112 (FnCas9) possesses a novel enzymatic property that cleaves the target DNA in a staggered pattern to leave 4-nt 5′-overhangs and exhibits a higher intrinsic specificity as compared with SpCas9,” the authors wrote. “However, we have also found that FnCas9 is unable to cleave a large number of the targets that are efficiently cleaved by SpCas9 in human cells, even though the two Cas9 nucleases exhibit a similar activity on purified DNA substrates.

CRISPR genome editing technology is advancing treatment options for some of the toughest medical conditions faced today, including chronic illnesses and cancers for which there are limited or no treatment options. The applications of CRISPR are far ranging—from identifying genes associated with cancer to reversing mutations that cause blindness. CRISPR enables genome editing using an enzyme called Cas9 to cut DNA, but this has limited targeting abilities. This limitation led to the investigators focus on proxy-CRISPR.

“We developed a proximal CRISPR targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrated that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome,” the authors noted.

This new finding is complimentary to current MilliporeSigma CRISPR applications and has further solidified their commitment to advancing technology in this field. For instance, the company is looking toward the future with the launch of a suite of genome editing tools for the research community that will include novel and modified versions of Cas and Cas-like proteins.

The research team concluded that the results of this new study “provide a novel strategy to enable the use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.” 

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