Fig 2: Base-edited monkeys were generated using the CBE4max system. a Schematic illustrating the process of generating base-edited monkeys. b Specific gRNAs designed to target RAG1 and IL2RG genes. The gRNA target sites are highlighted in red, while the edited bases are indicated in green. c The status of the embryos after CBE4max systemic injection. d T7E1 assay for base-edited embryos. e Following the microinjection of 152 embryos, 35 embryos in the developmental stage were transferred to surrogate female monkeys. Subsequently, five recipient monkeys were confirmed to be pregnant, resulting in the successful acquisition of six newborn monkeys. f The editing efficiency in the peripheral blood of the edited animals was assessed using Sanger sequencing-based EditR software. g The genotype of the base-edited monkeys (mutation) was determined. Red boxes indicate the presence of stop codons after the substitution, while the asterisk (*) denotes a substituted base. The gRNA sequencing map of the RAG1 gene is derived from monkey NO.1, while the gRNA sequencing map of the IL2RG gene is from monkey NO.3. h The frequencies of base substitution in deceased base-edited monkeys determined through targeted deep sequencing

Fig 3: Phenotypic and histopathological changes in base-edited monkeys. a, b Comparison of body weights, body lengths, and head circumferences between mutant monkeys and age-matched WT monkeys while being subjected to the same feeding environment. c, d Analysis of thymus and spleen in base-edited monkeys. e H&E staining depicting the development of thymus and spleen in base-edited monkeys. Scale bars: 50 µm. f–k Evaluation of RAG1 and IL2RG protein expression in the thymus and spleen of mutant monkeys. Western blot analysis illustrating RAG1 and IL2RG protein expression levels and quantification in the thymus, spleen, liver, and lung of mutant and WT monkeys (f, g). Immunohistochemistry depicting the expression and quantification of RAG1 and IL2RG in the thymus and spleen of mutant and WT monkeys (h–k). Statistical analysis was performed using an unpaired two-tailed t-test to compare the two groups. P < 0.05 was considered statistically significant. Three replicates were performed for each analysis. Scale bars: 20 µm