A new paper from the Doudna lab on engineering better Cas proteins
In a new paper in Cell, co-first authors Amy Eggers, Kai Chen and colleagues in the Doudna lab made advances in rational engineering of Cas proteins to increase editing efficiency. Their experiments focused on iGeoCas9, a Cas protein from the Geobacillus stearothermophilus bacterium that was evolved in the lab for increased editing efficiency. iGeoCas9 edits 100x more cells in laboratory experiments than conventional Cas9 — but what makes it so much more efficient?
Through careful experiments, the team uncovered three key mutations in a portion of the Cas protein known as the WED-domain. These mutations appear to increase contact between the WED-domain and the target DNA, increasing the speed at which the DNA double helix unwinds, ultimately allowing the Cas protein to cut the DNA. These changes also broaden the range of areas of a genome that can be targeted by reducing the need for an exact match between the place where the CRISPR complex binds to the DNA, known as the PAM sequence, and the guide RNA sequence.
Eggers, Chen, and the team created similar mutations in a related protein, Nme2Cas9, intended to increase contact between the WED-domain and the target DNA, resulting in similar dramatic increases in editing efficiency — in other words, using rational engineering to improve a Cas protein. WED-domains are present in Cas9, Cas12, as well as other related proteins.
“Scientists have used engineering strategies through intensive mutational scanning to improve genome editors, which can take significant time and resources,” says Eggers. “Now we can easily design beneficial mutations, streamlining the process for creating better genome editors. We demonstrate this by creating iNme2Cas9, a rationally engineered genome editor with substantially improved activity. Engineering the WED domain is a straightforward method to simplify and advance genome editing.”
Read the paper here.
