The compact high-fidelity EbCas12a enables all-in-one AAV delivery system
we characterized a novel compact Cas12a ortholog, EbCas12a, from the Erysipelotrichia bacterium with activities in mammalian cells. It is with the PAM sequence of 5'-TTTV-3' (V=A, G, C) and the smallest size of ~3.47kb among reported Cas12a orthologs so far. Moreover, enhanced EbCas12a (enEbCas12a) was also developed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells. With the help of the compact enEbCas12a, all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo. Altogether, with the help of the novel smallest high fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research. Overall design: Cells were collected after 48h post-transfection for genomic DNA extraction using FastPure Cell/Tissue DNA Isolation Mini Kit(Vazyme), Libraries were prepared according to GUIDE-seq methods. Briefly, Genomic DNA was sheared to length of about 250 bp, added Y adapter, and amplified by two rounds of nested anchored PCR. The last PCR products were GUIDE-seq libraries and sent to the company for sequencing using an Illumina sequencer