The results of a metagenomic study from the University of Trento suggest that the CRISPR toolbox will need to make room for another CRISPR enzyme. The disruption should be minimal because the newly identified enzyme is unusually compact. It consists of just over 1,000 amino acids. And yet it is also strongly active and highly precise. The hope is that it can be packaged with guide RNA within the tight quarters afforded by adeno-associated virus (AAV) vectors, and thereby expand the use of in vivo gene editing in therapeutic applications.

The study was led by Anna Cereseto, PhD, and Nicola Segata, PhD, of the department of cellular, computational, and integrative biology. Cereseto leads a laboratory that develops advanced genome editing technologies and their application in the medical sector. Segata is the head of a laboratory of metagenomics, where he studies the variety and characteristics of the human microbiome and its role in health. Their collaboration has led to the identification, in a bacterium of the intestine, of new CRISPR-Cas9 molecules that could have a clinical potential to treat genetic diseases.

Detailed findings from the study recently appeared in Nature Communications, in an article titled, “CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.”

“[Through] the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci,” the article’s authors wrote. “Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1,004 amino acids) isolated from an uncultivated Collinsella species.”

The University of Trento scientists also evaluated how CoCas9 performed in clinically relevant models, namely, human hematopoietic stem/progenitor cells and murine retinas. For example, in experiments with murine retinas, the scientists co-delivered CoCas9 with its sgRNA through AAV vectors to target the RHO gene, which is mutated in a common form of autosomal dominant retinitis pigmentosa.

“We administered an all-in-one AAV8 with CoCas9 targeting hRHO to 5-week-old mice via subretinal injection,” the article’s authors reported. “Four weeks after the administration we observed up to 35.6% of editing efficiency at the hRHO target, with a mean of 10.5% in seven retinas. These results demonstrated the potential of CoCas9 for clinical development.”

Researchers all over the world are investigating genomic therapies to find new treatments for genetic disorders. Genome editing using the CRISPR-Cas9 system is based on the use of the Cas9 protein, which can be programmed to make specific modifications in the genome to cut or replace harmful DNA sequences, correcting the mutations that cause diseases. This biotechnology was discovered in 2012 in the United States and has already led to one approved therapy, a drug for sickle cell disease. Now the study conducted by the University of Trento brings genomic research one step forward.

“Compared with other CRISPR-Cas9 approaches, the one we have identified is precise and effective, and more compact,” said Cereseto, who has been involved in studies on the genomic editor since 2018 with the development of evoCas9. “This new CRISPR-Cas9 molecule, as demonstrated by our experiments in the retina, will be more easily delivered to the organs that must be treated in therapies for genetic diseases.”

Expanding the range of CRISPR-Cas tools is necessary to speed up the development of therapies for genetic diseases. This can be done by modifying natural enzymes, as was the case with evoCas9, but discovering already evolved enzymes that can work offers great advantages. The collaboration with the laboratory of computational metagenomics of Segata has allowed the laboratory of molecular virology of Cereseto to shed light on a vast natural reserve of CRISPR-Cas9 systems from which to draw new valuable tools for human genome editing.

“By interrogating a microbiome genome database that we have created over several years, we discovered a large number of Cas9 with interesting properties for genome editing,” Cereseto and Segata noted. “We have discovered a great variety of CRISPR-Cas9 in the bacteria that inhabit the intestine. In particular, we have identified the CoCas9 nuclease in Collinsella, a bacterial genus that is often found in human guts.

“The sequencing of the entire microbiome using a metagenomic approach, followed by the laboratory reconstruction of the assembled genomes, has led to the identification of a huge variety of species. The discovery of a collection of new Cas9 nucleases, including CoCas9, makes the genome editing toolkit even larger.

“The difficulty of administration still hampers the development of therapies for genetic diseases. However, CoCas9, thanks to its small size, shows potential for gene therapy applications and is therefore a potential candidate for optimization through engineering approaches, which deserve further investigation. We are already working on clinical development projects.”

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