The CRISPR-Cas9 gene editing system has been widely studied because of its potential therapeutic applications, but limitations in the number of locations on the genome it can target remain a major drawback. Now scientists at the Massachusetts Institute of Technology have identified a new Cas9 enzyme that they say can help CRISPR reach more gene mutations.
CRISPR needs a "protospacer adjacent motif (PAM) sequence" to recognize a particular target on DNA for cutting. Without that PAM, no editing can occur. However, the widely used Cas9 enzyme, Streptococcus pyogenes Cas 9 (SpCas9), only recognizes nucleotides that are followed by two G nucleotides as its PAM. That significantly limits the number of locations it can target to about 9.9% of genomic sites, according to the MIT researchers.
Cas9s have been engineered to identify different PAMs, but the MIT team wanted to identify one tool that could cover a wider range of loci by itself. They found one in Streptococcus canis (ScCas9), which can target almost half of the locations on the genome, they reported in the Science Advances.
Team leader Joseph Jacobson compared CRISPR to a postal system. Previous Cas9 systems only allowed researchers to go to a ZIP code that ends in a zero, he explained. “It is very accurate and specific, but it limits you greatly in the number of locations you can go to,” he said in a statement.
Using computational algorithms, the researchers scanned bacterial sequences to find enzymes with less restrictive Pam requirements and then built synthetic versions of the candidate CRISPRs. They went on to evaluate the performance of all the candidates to narrow down the choices.
ScCas9 stood out as the most successful enzyme. It's almost identical to SpCas9 but with the ability to target more DNA sequences. In fact, rather than requiring two G nucleobases as its PAM, the new enzyme needs only one.
The search for better enzymes to perfect CRISPR's precision is a priority in the gene-editing world. A study by scientists at the University of Texas at Austin recently proposed replacing Cas9 with Cas12a to avoid toxic off-target effects, for example. And researchers at the Institute of Bioorganic Chemistry in Poland suggested using a Cas9 “nickase” enzyme that cuts only one strand of DNA instead of two.
In addition to reaching more disease-specific locations, the MIT scientists hope their new CRISPR tool can boost an even more precise variety of gene editing called base editing. While some diseases can be treated by simply knocking out the entire gene of around 1,000 bases, others, such as sickle cell anemia, are caused by the mutation of a single base, Jacobson explained. Therefore, instead of looking at hitting any one of various locations on a gene, “[y]ou need to be able to go to that very exact location, put your piece of CRISPR machinery right next to it, and then with a base editor […] go in and repair or change the base,” he said.
Jacobson and his teammates are now using their technology to identify other enzymes that could expand CRISPR’s targeting range even further.