| First Author | Zhao D | Year | 2023 |
| Journal | BMC Biol | Volume | 21 |
| Issue | 1 | Pages | 250 |
| PubMed ID | 37946200 | Mgi Jnum | J:342335 |
| Mgi Id | MGI:7547583 | Doi | 10.1186/s12915-023-01754-4 |
| Citation | Zhao D, et al. (2023) Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope. BMC Biol 21(1):250 |
| abstractText | BACKGROUND: Nme2ABE8e has been constructed and characterized as a compact, accurate adenine base editor with a less restrictive dinucleotide protospacer-adjacent motif (PAM: N4CC) but low editing efficiency at challenging loci in human cells. Here, we engineered a subset of domain-inlaid Nme2Cas9 base editors to bring the deaminase domain closer to the nontarget strand to improve editing efficiency. RESULTS: Our results demonstrated that Nme2ABE8e-797 with adenine deaminase inserted between amino acids 797 and 798 has a significantly increased editing efficiency with a wide editing window ranging from 4 to 18 bases in mammalian cells, especially at the sites that were difficult to edit by Nme2ABE8e. In addition, by swapping the PAM-interacting domain of Nme2ABE8e-797 with that of SmuCas9 or introducing point mutations of eNme2-C in Nme2ABE8e-797, we created Nme2ABE8e-797(Smu) and Nme2ABE8e-797(-C), respectively, which exhibited robust activities at a wide range of sites with N4CN PAMs in human cells. Moreover, the modified domain-inlaid Nme2ABE8e can efficiently restore or install disease-related loci in Neuro-2a cells and mice. CONCLUSIONS: These novel Nme2ABE8es with increased on-target DNA editing and expanded PAM compatibility will expand the base editing toolset for efficient gene modification and therapeutic applications. |
