Ultra-precise CRISPR-Cas9
Electron microscopy helped to create a new enzyme for DNA editing
Elena Kleshchenko, PCR.news
The University of Texas investigated the conformations of the Cas9 nuclease during interaction with non-target DNA sequences similar to the target. The results suggested which amino acid residues should be replaced to obtain the modified enzyme SuperFi-Cas9. It cuts the target DNA as actively as unmodified, and non-targeted cutting is dramatically reduced compared to conventional CRISPR-Cas.
A well-known problem of genomic editing using CRISPR—Cas9 is non-targeted editing, that is, incisions in areas similar to targets. More accurate variants of Cas9, constructed artificially, are usually inferior to the original version of the enzyme in efficiency. Researchers from the University of Texas (Austin) have created a new version of Cas9 for genomic editing, which cuts the target sequence as successfully as the "wild type", and at the same time much more accurately. The new enzyme was named SuperFi-Cas9. One of the leading authors of the work Kenneth Johnson believes that this development can change the rules of the game in gene editing.
It is known that three mismatched nucleotides at positions 18-20, counting from the motif adjacent to the protospacer (PAM), reduce the efficiency of cutting by about 40 times, whereas mismatches at positions 6-8, 9-11 and 15-17 — by 2000 times, so that only 20% of DNA is cleaved in 2 hours of incubation.
The authors used kinetic-guided cryo-electron microscopy (cryo-guided cryo-electron microscopy) to determine which structural elements of Cas9 make non-targeted activity possible. First, they investigated how the enzyme interacts with DNA, in which the inconsistencies are at positions 12-14 from PAM. It turned out that Cas9 in such samples demonstrates two conformations: with a linear and with a curved ("canonical") RNA-DNA duplex, and the second is observed in the samples after a longer incubation. Therefore, the researchers assumed that the linear duplex is an early intermediate that occurs before the HNH endonuclease domain takes the desired position.
When the authors studied the interactions of Cas9 with DNA with inconsistencies at position 18-20, where non-targeted editing is relatively active, they again observed linear and curved conformations. They drew attention to two domains that connect HNH to the rest of Cas9 and create a bend. This bend facilitates targeted splitting, but it also makes non-targeted splitting possible. The reorganization of the loop in the RuvC domain especially contributed to the activation of Cas9 with inappropriate activity.
"It's as if you had a chair, one of the legs of which broke, and you just taped it up," said Jack Bravo, one of the two first authors. "It can still work like a chair, but it's a little wobbly."
In order to "fix the stool"— not to allow Cas9 to cut DNA if the sequence and guide RNA match is incomplete— all seven amino acid residues stabilizing the non-target interaction were replaced with aspartic acid. The result is a design that cuts the target site almost as efficiently as the original Cas9, but at the same time the cutting of molecules with a discrepancy in positions 18-20 decreased by 500 times.
The authors of the work noted that so far they have tested SuperFi-Cas9 only on DNA in vitro, but now, together with collaborators, they will test the effectiveness on living cells.
Article by Bravo et al. The structural basis for mismatch surveillance by CRISPR–Cas9 is published in the journal Nature.
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