Compact Cas9d Ultra was packaged in a single AAV vector and used to edit a gene in mouse liver
Compact Cas9d Ultra was packaged in a single AAV vector and used to edit a gene in mouse liver
On August 19, the authors published a study of Cas9d Ultra, a compact genome editor. One version, 9dCBE, converts C to T in DNA. The researchers packaged it with a guide RNA in a single AAV9 vector and administered it to newborn mice. Five weeks later, the editor had modified an average of 15.7% of the target site in the liver gene Pcsk9, and serum LDL cholesterol was lower than in mice injected with PBS.
A base editor changes a single DNA letter at a specific site. The guide RNA specifies the genomic target, the CRISPR protein holds the editor at that site, and an attached enzyme chemically converts the base. AAV is an adeno-associated virus used to deliver genetic cargo into cells. It usually accommodates about 4.7 thousand nucleotides, so large editors often have to be divided between two vectors. In this study, a single AAV9 delivered both the editor and its guide RNA.
The researchers selected Cas9d MG34-1 because of its size. The protein contains 747 amino acids and uses a guide RNA of the length commonly used in CRISPR systems. In a 2022 study, an earlier version achieved up to 22% base editing at three targets in human cells. The authors of the current study changed four amino acids in the protein and modified the guide RNA scaffold. In a cell-based assay in which DNA cleavage activates a fluorescent signal, activity increased from 20.44% to 63.32%. They then used Cas9d Ultra to construct two base editors: 9dABE converts A to G, while 9dCBE converts C to T. Across 12 and 17 tested genomic sites in human cells, their mean editing efficiencies were 54.08% and 56.12%, respectively.
Two experiments in mice tested different parts of this process. After 9dCBE was introduced into zygotes, 50 of 56 offspring carried a premature stop codon in the pigmentation gene Tyr. In the experiment targeting Pcsk9, a gene associated with LDL cholesterol levels, the authors tested whether a single AAV9 could deliver the complete editor to the liver. After five weeks, the proportion of edited cellular sequences ranged from 8.3% to 28.9%. A single vector therefore delivered the entire system, including the protein, guide RNA, and enzyme that performs the targeted DNA base conversion, and allowed the researchers to measure its activity in a living mouse.