New Class of CRISPR-Cas Enzymes for Precise Genome Editing

Recent research published in the journal Science Bulletin detailed the creation of a tool called ChCas12b that can accurately edit genomes and identify single-base mutations. The research team of Professor Yongming Wang from Fudan University is in charge of this study, working with the Southeast University team under the direction of Professor Renjie Chai.

Mammalian cell gene editing has extensively used RNA-guided CRISPR-Cas systems, which have shown great promise in the treatment of genetic disorders. Class II Cas9 and Class V Cas12 are currently widely utilized as gene-editing tools.

However, both the Cas9 and Cas12 nucleases show some tolerance for guide RNA (gRNA) and target site mismatches, which may result in off-target DNA cleavage, disrupting healthy genes, and even causing cancer. Researchers have been working to find CRISPR/Cas nucleases with greater specificity to combat this off-target effect.

Five Cas12b nucleases with editing activity in the mammalian genome were found after the research team used a GFP activation expression reporting system to screen 13 Cas12b nucleases. Interestingly, one of them, Candidatus Hydrogenedentes Cas12b (ChCas12b), showed that it could identify the WTN (W = T or A) PAM sequence, greatly expanding its targeting range.

The research team then created nine single amino acid mutant variants of ChCas12b by using the crystal structure of the AacCas12b protein and amino acid information related to hydrogen bonding with target DNA-sgRNA.

The GFP reporting system was then used to test the specificity of these variants. The variant ChCas12b-D496A showed less off-target activity according to GUIDE-seq analysis, which might make it a safer editing tool for clinical gene therapy applications.

The ability of ChCas12b and its high-fidelity variant ChCas12b-D496A to specifically disrupt genes containing single nucleotide polymorphisms (SNPs) was the most important demonstration the researchers made.

They also divided ChCas12b-D496A into two pieces using trans-splicing intein technology, which they then packaged into AAV viruses. In vivo experiments conducted on mice showed that ChCas12b-D496A had a higher editing efficiency and fewer off-target effects.

In conclusion, the research effectively created ChCas12b-D496A, a highly specific Cas12b nuclease that can differentiate between single-base mutations. In addition to broadening the scope of gene editing's targeting, this technology shows promise for a wide range of gene therapy applications, offering a valuable new tool for the treatment of genetic diseases in the future.