Dear Editor, Tay–Sachs disease (TSD) is a progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of β-hexosaminidase A (HexA) 1. The four-bases (TATC) insertion in exon 11 of the HEXA (HEXA ins TATC) accounts for 80% of Tay–Sachs disease from the Ashkenazi Jewish population 2. However, no typical clinical phenotypes, such as neurological abnormalities, the restricted pattern of distribution of GM2-ganglioside and membranous cytoplasmic bodies in the brain, were observed in HEXA−/− mouse models, due to the difference in the ganglioside degradation pathways in mice and human 3. Thus, it is desired to generate an ideal animal model to accurately mimic HEXA ins TATC in TSD patients. CRISPR–Cas9 systemmediated HDR 4 has been used to generate the mutation of HEXA ins TATC, however, low efficiency and high indels impede its application. Recently Anzalone et al. 5 described a “search-andreplace” genome editing technology named prime editing (PE) that mediates 12 possible base-to-base conversions, without requiring DSBs or donor DNA templates in human cells. In addition, a previous study showed that, compared to mice, the late onset of TSD in adult rabbits 6 shared more similarities with human regarding physiology, anatomy, and genetics 7. Thus, we generated a novel TSD rabbit model using the PE system, and characterized the typical phenotype of muscle weakness, ataxia, and mental disorders in the HEXA ins TATC rabbit model. We first validated the editing efficiencies of PEs (PE2, PE3, PE3b) in HEK293FT cells at fifteen loci: five loci for base insertion, eight loci for base substitutions, and two loci for base deletion (Supplementary Table S1). Sanger sequencing results showed that the base insertion at a frequency from 4% to 22%(Fig. 1 a and Supplementary Fig. S2), the base substitutions at a frequency from 4% to 36%, and the base deletion at a frequency from 7% to 12% were determined using PEs (Supplementary Figs. S1 and S2), respectively. These results indicate that PEs were effective in generating base insertion, substitution, and deletion in HEK293FT cells.

Next, we tested the efficiency of the PE system in rabbit embryos at three gene loci of HEXA, HBB, and TYR, which are associated with clinical diseases in ClinVar data 8 (Supplementary Table S2). Sanger sequencing results showed that 9 of 20 desired HEXA ins TATC were determined using PE2 with the efficiency of 4.1%–15.4%, while the efficiency is 8%–37.5% using PE3. In addition, 1 of 14 desired HBB with an efficiency of 10% and 1 of 10 desired TYR with an efficiency of 14% were generated using PE3, while there is no desired mutation was detected for these two sites using PE2 (Fig. 1 b and Supplementary Fig. S3).