Figure 2.

Split-Intein CBEs Can Reconstitute and Edit the Human Genome

(A) Crystal structure of the SpCas9 protein complexed with sgRNA (red) and target DNA (blue). Inset shows the disordered linker that connects the REC lobe and the NUC lobe. Light gray indicates N-terminal SpCas9 domain; dark gray indicates C-terminal SpCas9 domain (PDB: 4OO8).⁴⁸ (B) Schematic of the engineered split-intein CBE two-plasmid system. Abbreviations are as follows: CMV, cytomegalovirus promoter; NLS, nuclear localization signal; Int-N, N-terminal intein domain; Int-C, C-terminal intein domain: V5, V5 epitope tag; 3× HA, three tandem repeats of the human influenza hemagglutinin (HA) epitope tag. (C) Western blot of HEK293T cells transfected with the N-terminal split-intein CBE domain, the C-terminal, or both. The western blots were probed with anti-HA or anti-V5 antibodies to detect reconstitution of the full-length base editors. (D) Plot showing the mean frequency change of the target cytosine in the SOD1 gene by deep sequencing in HEK293T cells 6 days after transfection with the full-length CBE, the split-intein CBE, or a non-targeted control (NTG), n = 3. (E) Mean C>T conversion frequency of editing-window cytosines in the EMX1 gene by deep sequencing in HEK293T cells 6 days after transfection with full-length CBE or split-intein CBE (n = 3). Error bars indicate SD. **p < 0.01; (D and E) two-tailed unpaired t test.