Engineering CjCas9 for Efficient Base Editing and Prime Editing

The advent of CRISPR/Cas9 has brought hope for the cure of genetic diseases. Most gene editing tools are developed based on SpCas9, which poses challenges in their delivery due to its larger size. CjCas9, derived from Campylobacter jejuni, is one of the smallest Cas9 proteins. However, its application in prime editing results in an editing efficiency of less than 5%, limiting its use in gene therapy. To overcome this hurdle, we have enhanced its DNA binding capability through the introduction of mutations and fusion with DNA-binding proteins. This has led to the development of a compact and highly efficient prime editor called SsenCjPE, which has shown an average 24-fold increase in editing efficiency. SsenCjPE can also be combined with other efficient prime editing systems to further enhance efficiency or reduce size. By introducing a combination of mutations on CjCas9, we have further improved editing efficiency with the development of CjPEmax. In summary, CjCas9, as a compact Cas protein, demonstrates remarkable performance in both base editors and prime editors.