In a paper published in Science Bulletin, a Chinese team of scientists conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates.
Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.
Additionally, the team identified nine mutations enhancing ChCas12b specificity. More importantly, they demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele-specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.
New discovery
This study is led by Professor Yongming Wang’s research team from Fudan University, in collaboration with the team led by Professor Renjie Chai from Southeast University. This study is published in a research paper titled “Discovery and engineering of ChCas12b for precise genome editing” in the Science Bulletin, unveiling the development of a tool, ChCas12b, capable of precisely editing genomes and discerning single-base mutations.
READ MORE: NICER than CRISPR: new gene editor reduces unintended mutations
RNA-guided CRISPR-Cas systems have found extensive application in mammalian cell gene editing, particularly showcasing immense potential in the treatment of genetic diseases. Currently, Class II Cas9 and Class V Cas12 have been extensively harnessed as gene editing tools. However, both Cas9 and Cas12 nucleases exhibit a certain tolerance towards mismatches between guide RNA (gRNA) and target sites, which could potentially lead to off-target DNA cleavage, disrupting normal genes, and even triggering cancer. To tackle this off-target effect, researchers have been striving to discover CRISPR/Cas nucleases with higher specificity.
Screening nucleases
In this study, the research team screened 13 Cas12b nucleases using a GFP activation expression reporting system, identifying five Cas12b nucleases with editing activity in the mammalian genome. Notably, one among them named Candidatus Hydrogenedentes Cas12b (ChCas12b) demonstrated the ability to recognize the WTN (W=T or A) PAM sequence, thereby significantly broadening its targeting scope.
Subsequently, leveraging the crystal structure of AacCas12b protein and amino acid information involved in hydrogen bonding with target DNA-sgRNA, the research team designed nine single amino acid mutant variants of ChCas12b. These variants were then tested for specificity using the GFP reporting system. Through GUIDE-seq analysis, the variant ChCas12b-D496A exhibited lower off-target activity, potentially rendering it a safer editing tool for clinical gene therapy applications.
Selective disruption
Most significantly, the researchers demonstrated the capability of both ChCas12b and its high-fidelity variant ChCas12b-D496A to selectively disrupt genes carrying single nucleotide polymorphisms (SNPs). Additionally, they employed trans-splicing intein technology to split ChCas12b-D496A into two parts, packaging them into AAV viruses. In vivo experiments in mice revealed that ChCas12b-D496A possessed higher editing efficiency and lower off-target effects.
In summary, the study successfully developed a highly specific Cas12b nuclease—ChCas12b-D496A, capable of distinguishing single-base mutations. This technology not only expands the targeting range of gene editing but also demonstrates broad prospects for gene therapy applications, providing a significant new tool for future genetic disease treatments.
No comments yet