The new CRISPR tool opens more genome to the edition

The targeting system can move to different parts of the genome, guided by a short sequence of RNA, where a DNA cleavage enzyme called Cas9 then makes the desired changes.

However, despite the considerable success of the gene editing tool, CRISPR-Cas9 remains limited in the number of sites that it can visit on the genome.

Indeed, CRISPR needs a specific sequence flanking the target location of the genome, called the adjacent protospacer motif, or MAP, to allow it to recognize the site.

For example, the most widely used Cas9 enzyme, Streptococcus pyogenes Cas9 (SpCas9), requires two G nucleotides as a PAM sequence, which significantly limits the number of sites targeted to approximately 9.9% of the genome sites.

To date, there is only a handful of CRISPR enzymes with minimal requirements for PAM, which means they can target more sites.

Researchers at the MIT Media Lab, led by Joseph Jacobson, a professor of media arts and science and head of the Molecular Machines research group, have discovered a Cas9 enzyme that can target nearly half of the genome sites. significantly expands its potential. use. They report their findings in the Progress of science.

"CRISPR is like a very accurate and efficient postal system, which can reach any location very precisely, but only if the postal code ends in a zero," Jacobson said. "So it's very precise and precise, but it limits you a lot in the number of places where you can go."

To develop a more general CRISPR system, researchers implemented computer algorithms to conduct bioinformatic research on bacterial sequences to determine if similar enzymes with less stringent PAM requirements existed.

To carry out the research, the researchers developed a software tool for data analysis, which they called SPAMALOT (Target Alignment PAM Search).

This revealed a number of interesting possible enzymes, but no clear winner. The team then created synthetic versions of CRISPR in the laboratory to evaluate their performance.

They discovered that the most potent enzyme, a Streptococcus canis Cas9 (ScCas9), was strikingly similar to the already widely used Cas9 enzyme, according to co-principal author Pranam Chatterjee, a student. graduated from the Media Lab, which led the research. alongside Noah Jakimo, another graduate student.

"The enzyme seems almost identical to the one that was originally discovered … but it is able to target DNA sequences that the commonly used enzyme can not", said Chatterjee.

Rather than two G nucleotides as the PAM sequence, the new enzyme needs only one G, thus opening up many more regions on the genome.

This should allow CRISPR to target many disease-specific mutations that were previously beyond the reach of the system.

For example, a typical gene has a length of about 1,000 bases, offering researchers a number of different locations to target if they simply want to destroy the entire gene, says Jacobson.

However, many diseases, such as sickle cell disease, are caused by the mutation of a single base, making them much more difficult to target.

"The basic editing is not just about hitting this gene anywhere on 1000 bases and knocking it out; just enter and correct, very accurately, the base you want to change, "said Jacobson. .

"You have to be able to get to that very specific place, put your CRISPR equipment next, and then with a basic editor – another CRISPR-related protein – go fix or modify the base," he says.

The new CRISPR tool could be particularly useful in such applications.

"We are excited to put ScCas9 in the hands of the genome publishing community and to receive feedback for their future development," said Chatterjee.

Researchers are now hoping to use their technique to find other enzymes that can further expand the targeting range of the CRISPR system, without reducing its accuracy, according to Jacobson.

"We are confident that we can take care of every genome address," he said.

Explore further:
Biochemists discover the cause of genome editing failures with the CRISPR system in vogue

More information:
"Minimum PAM specificity of a SpCas9 orthologue very similar" Progress of science (2018). advance.sciencemag.org/content/4/10/eaau0766