CRISPR-Cas9 gene-editing complex from Streptococcus pyogenes. Image: iStock
CRISPR-Cas9 gene-editing complex from Streptococcus pyogenes. Image: iStock
A team of Harvard Medical School researchers at Massachusetts General Hospital has found a way to expand the use and precision of powerful gene-editing tools called CRISPR-Cas9 RNA-guided nucleases.
In their report in Nature, the investigators describe evolved versions of the DNA-cutting Cas9 enzyme that can recognize a different range of nucleic acid sequences than is now possible with the naturally occurring form of Cas9 scientists have been using.
“In our paper we show that sites in human and zebrafish genes that could not previously be modified by wild-type Cas9 can now be targeted with the new variants we have engineered,” said Benjamin Kleinstiver, HMS research fellow in pathology at Mass General and lead author of the Nature paper. “This will allow researchers to target an expanded range of sites in a variety of genomes, which will be useful for applications requiring highly precise targeting of DNA sequences.”
CRISPR-Cas9 nucleases consist of the Cas9 bacterial enzyme and a short, 20-nucleotide RNA molecule that matches the target DNA sequence. In addition to the RNA/DNA match, the Cas9 enzyme needs to recognize a specific nucleotide sequence called a protospacer adjacent motif (PAM) adjacent to the target DNA.
The most commonly used form of Cas9, derived from the bacteria Streptococcus pyogenes and known as SpCas9, recognizes PAM sequences in which any nucleotide is followed by two guanine DNA bases. This limits the DNA sequences that can be targeted using SpCas9 to only those that include two sequential guanines.
To get around this limitation, the team set up an engineering system that allowed them to rapidly evolve the ability of SpCas9 to recognize different PAM sequences. From a collection of randomly mutated SpCas9 variants, they identified combinations of mutations that enabled SpCas9 to recognize new PAM sequences.
These evolved variants essentially double the range of sites that can now be targeted for gene editing using SpCas9. They also identified an SpCas9 variant that was less likely to induce the off-target gene mutations sometimes produced by CRISPR-Cas9 nucleases, a problem originally described in a 2013 study led by J. Keith Joung, HMS professor of pathology at Mass General and senior author of the current study.
“This additional evolved variant with increased specificity should be immediately useful to all researchers who currently use wild-type SpCas9 and should reduce the frequencies of unwanted off-target mutations,” Joung said. “Perhaps more important, our findings provide the first demonstration that the activities of SpCas9 can be altered by directed protein evolution.”
The scientists showed in their paper that the forms of Cas9 found in two other bacteria—Staphylococcus aureus and Streptococcus thermophiles—can also function in their bacterial evolution system, suggesting that their functions can be modified as well, Joung said.
“This work just scratches the surface of the range of PAMs that can be targeted by Cas9,” Joung said. “We believe that other useful properties of the enzyme may be modified by a similar approach, allowing potential customization of many important features.”
The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM107427 and R01 GM088040, a Jim and Ann Orr Research Scholar Award, and a National Sciences and Engineering Research Council of Canada Postdoctoral Fellowship. The MGH has filed a patent application on the use of the SpCas9 variants described in this paper.
Adapted from a Mass General news release.
