The next step was to examine how the CRISPR complex interacted with them, using the AlphaFold AI-based protein-folding software. Updated versions were designed to handle interactions between proteins and nucleic acids, as well as complexes of multiple proteins. So the team fed AlphaFold versions of a target DNA sequence, along with a guide RNA, the Cas9 sequence, and an enzyme that chemically modifies bases and can stick to Cas9.

Unfortunately, it choked, placing one of the proteins in what was clearly the wrong location.

Undeterred, the team simplified things and fed AlphaFold only the DNA, RNA, and Cas9 protein, since the latter is the primary factor determining its sequence specificity. This worked much better, producing a structure that agreed with ones determined by experiments with actual nucleic acids and proteins.

By comparing the structures AlphaFold generated when fed different on- and off-target sites, the researchers found a general pattern. Many (about two-thirds) of the off-target sites caused the Cas9 protein to adopt a slightly different structure. But nearly all (over 95 percent) of them altered which amino acids contacted the RNA. So there are clearly some cases where Cas9 maintains its normal structure but amino acids within it flex around in ways that accommodate the mispaired bases of off-target sites.

Conveniently, AlphaFold was already set up to identify what is termed the “contact probability,” namely, the chance that any two items, such as amino acids or nucleotides, are within a very small distance (eight Angstroms). The researchers could take the output of the contact probability analysis for on- and off-target sites and compare them, identifying exactly which amino acids in Cas9 have altered contacts when there’s a mismatch between the guide RNA and the DNA. They termed this computerized analysis setup “ContactSeek.”

Better targeting

On its own, ContactSeek tended to produce a large list of amino acids that shift around when bound to an off-target site. So the researchers focused on regions of the Cas9 protein where these amino acids clustered, viewing this as a sign that these areas were adapting to the differences caused by mismatched bases. They then began to test versions of Cas9 with different amino acids at these sites.