Schematic of a dimeric tru-RFN binding to DNA. Two FokI-dCas9 proteins are targeted to DNA half-site sequences by two tru-gRNAs (red) enabling FokI dimerization and cleavage of the intervening spacer sequence.[With permission from Human Gene Therapy]
Schematic of a dimeric tru-RFN binding to DNA. Two FokI-dCas9 proteins are targeted to DNA half-site sequences by two tru-gRNAs (red) enabling FokI dimerization and cleavage of the intervening spacer sequence.[With permission from Human Gene Therapy]

The field of molecular biology was set ablaze several years ago with the discovery of the CRISPR/Cas9 genome editing enzymes from Staphylococcus pyogenes.  In the three-year stretch since the original publication describing CRISPR’s activity and uses, over 1,300 papers have since gone on to further expound the numerous applications of this molecular editing tool.

However, like any new discovery, CRISPR/Cas9 does come with its own unique set of limitations that researchers have been diligently trying to overcome. As accurate as CRISPRs can be, with respect to the genomic sequence that they intend to edit, many researchers have noticed that the enzymes will cut off-target sites a small percentage of the time. This is particularly problematic when using CRISPR as an instrument for gene therapy, as off target genomic alterations can have large deleterious effects to the patient. 

Yet, researchers from the Massachusetts General Hospital and Harvard Medical School believe they have developed a highly specific platform for performing genome editing by combining two recently described novel strategies designed to improve Cas9 cleavage specificity.

Described in the current issue of Human Gene Therapy, which focuses on genome editing, the findings from this study were published online today through an article entitled “Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing.”

Specifically, the investigators combined truncated guide RNAs (tru-gRNAs) with a fused, dimerization-dependent non-specific FokI cleavage domain to a catalytically inactive Cas9 protein (RFN). Both of these approaches have been shown separately to enhance the specificity of CRISPR/Cas9 nucleases, but the researchers questioned if the fidelity could be amplified by combining the two technologies together into what they have named tru-RFN.

“Here we show that the on-target activities of RFNs can be directed by tru-gRNAs and that the activities of these tru-RFNs can be comparable to the activities of RFNs directed by full-length gRNAs,” the scientists explain. “Additionally, the use of tru-gRNAs can reduce the residual undesired mutagenic activity of monomeric RFNs. Our findings suggest the utility of tru-RFNs for applications requiring the highest possible genome editing specificity.”

The Boston-based researchers were able to identify the gRNA truncation length for highly specific genome editing within human cells. Interestingly, they found that while Cas9 previously showed full activity with most gRNAs that are truncated by up to three nucleotides, truRFNs induce efficient mutagenesis with the gRNAs truncated by only one nucleotide.

Additionally the tru-RFN system was able to minimize the off-target mutation rates to levels significantly lower than what has been observed for CRISPR/Cas9 previously. This led the researchers to the conclusion that “our data show that tru-RFNs provide a useful and further improved tool for high-precision genome editing applications in human cells.”

A comprehensive review of the CRISPR/Cas9 technology can also be found in the current issue of Human Gene Therapy in an article entitled “The Bacterial Origins of the CRISPR Genome-Editing Revolution.”








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