Homo sapiens Raw sequence reads for targeted loci. Homo sapiens

Cytosine base editors (CBEs) guided by Cas9 and guide RNA can specifically convert singleC-G base pairs to T-A with high efficiency; however, long-term persistence of plasmid-encodedCBEs in the cell leads to unwanted off-targeting, compromising their safety for therapeuticapplications. Delivery of CBEs as ribonucleoprotein (RNP) complexes could allow temporal anddosing control of activity but it requires high levels of pure, active protein, which has provenchallenging with current CBE variants. Moreover, many mutations remain inaccessible to editingdue to the restricted editing window of CBEs. Here, we engineered new CBE variants in whichthe cytidine deaminase A3A is embedded within Cas9, bringing the active site closer to thetarget DNA and reducing off-target editing. These Cas-embedded CBEs achieved high yield andimproved solubility when produced as recombinant proteins, allowing for their delivery as RNPsin human cells. Using different linker lengths constraining the embedded deaminase, wesuccessfully shifted and expanded the C-to-T editing window to access previously inaccessiblepositions. Following a single RNP electroporation, our CBEs achieved high levels of dose-dependent on-target editing, minimized off-target editing, and demonstrated proof-of-concepttherapeutic potential for sickle cell disease and beta-thalassemia when targeted to the BCL11Aerythroid enhancer in human hematopoietic stem and progenitor cells.