Rice Team Evolves CRISPR Enzyme to Unlock New DNA Targets

Rice University chemist Han Xiao has used an AI-powered method called Sequence Display to expand what CRISPR/Cas9 gene editing can target, according to a paper published in Nature Communications. Xiao’s team evolved several new versions of a compact Cas9 enzyme capable of efficiently editing stretches of DNA that had previously been out of reach.

As a proof of concept, the researchers used the evolved Cas9 enzymes to edit two genes linked to coronary heart disease.

“My team used Sequence Display technology to evolve a compact Cas9 that can efficiently target priority areas of DNA. This shows the power of our approach, which rapidly generates large datasets that AI can use to identify optimal protein sequences for a given function. In this case, it allowed us to evolve several new Cas9 variants with better activities,” explained Xiao.

Cas9, the enzyme used in CRISPR editing, works by first attaching to a short stretch of DNA called a protospacer adjacent motif, or PAM, which is typically two to six base pairs long and functions somewhat like a docking station. Once Cas9 recognizes a suitable PAM, it can edit the DNA as directed by a guide, altering an adjacent target sequence that matches the guide RNA.

Because of this, CRISPR edits must occur within a certain distance of a PAM. Many Cas enzymes can recognize several different PAMs but edit far more efficiently at some than others, meaning genes with only “slippery,” weakly bound PAMs can be difficult to edit, especially when the work calls for a specific type of Cas enzyme.

First author Linqi Cheng said: “We cannot easily or precisely change the PAMs in a given gene. But with Sequence Display technology, we could quickly evolve a Cas9 enzyme that would better recognize the PAMs already present in the gene.” 

The researchers worked with a compact, widely used Cas9 enzyme called SlugCas9, which could bind many PAMs but struggled with four of particular interest, often slipping off or failing to bind them. Other scientists had tried evolving Cas9 variants to dock on these elusive PAMs with little success, since directing evolution toward such specific binding proved difficult. Using Sequence Display, Xiao’s team found promising candidates within a week, then used the evolved SlugCas9 to edit coronary heart disease genes that had previously been hard to access.

Cheng said: “The specific sites we wanted to edit in APOC3 and PCSK9 had the PAMs that prior versions of SlugCas9 couldn’t reliably bind to. With our evolved SlugCas9, we were able to easily and precisely edit these sites in cultured cells, introducing potentially protective genetic changes linked to a lower risk of coronary heart disease, unlocking the power and possibility of CRISPR.”

According to Xiao, “CRISPR/Cas9 is one of the most promising technologies of the modern age in both research and medicine. It relies on Cas9 enzymes that are optimized and evolved for each application. This finding shows that sequence display can be key to rapidly evolve Cas9, and other proteins, to address complex and pressing technical requirements essential for successful biomedical engineering.”