Redesigned Cas9 Protein Reduces Risk of Off-Target DNA Cleavage by 4,000%

Redesigned Cas9 Protein Reduces Risk of Off-Target DNA Cleavage by 4,000%

The CRISPR-Cas9 gene-editing tool has shown great promise for editing parts of the human genome to correct mutations, remove defective genes, or add fully functional genes; however, as accurate as the system is at editing DNA there is still the potential for off-target DNA cleavage.

Cas9 is a naturally occurring protein that is used by bacteria to remove foreign genetic material. It searches for specific sections of genetic material, and when it recognizes foreign genes, it makes the cut and relies on natural processes to perform the repair. The problem is that the system is not always precise. Mismatches can occur, where similar genetic material is recognized as a match and cuts are made, and these off-target effects need to be avoided.

Any off-target DNA cleavage by the Cas9 component of the system could have serious consequences – They could be more serious than the defects the system is used to correct. It is also possible for on-target gene editing to be accompanied by the creation of dangerous new mutations.

Since 2012, several advances have been made to improve the accuracy of the tool and new variants of Cas9 have been developed that reduce the potential for mismatching; however, the problem with these variants is they are far less efficient at on-target DNA cleavage.

Significant progress has recently been made by researchers at the University of Texas at Austin. The researchers have been studying the mechanisms by which Cas9 identifies mismatches, which until now have been poorly understood. Using kinetics-guided cryo-electron microscopy, the researchers studied the structure of the Cas9 protein at different stages of contiguous triple nucleotide mismatch cleavage at different positions along the gRNA–TS duplex.

They found that when a mismatch is identified in positions 18 through 20 on a strand of DNA, rather than failing to make the cut and moving on, a finger-like structure moves in and binds to the DNA as if it was the correct sequence of DNA being targeted. When the Cas9 enzyme binds to an incorrect section of DNA, due to the mismatch the DNA is a little floppy and the cut is not made; however, the finger-like structure stabilizes the DNA which allows the Cas9 protein to make the cut, even though there is a mismatch. Until now, this finger-like structure has never been observed.

The solution to reducing mismatching was therefore to engineer the Cas9 protein to have a different projection – one that does not stabilize the DNA when there is a mismatch but repels it. The researchers called their redesigned Cas9 protein SuperFi-Cas9, which they report is 4,000 times less likely to mismatch yet is just as fast at on-target editing as the naturally occurring Cas9.

“This really could be a game-changer in terms of a wider application of the CRISPR Cas systems in gene editing,” said Kenneth Johnson, professor of molecular biosciences at the University of Texas, Austin.

You can read more about the study in the paper – Structural basis for mismatch surveillance by CRISPR–Cas9 – which was recently published in Nature. DOI: 10.1038/s41586-022-04470-1