The gene targeting experiment was conducted on cynomolgus monkeys, which served as the experimental subjects. These monkeys were housed at Guangdong LANDAU Biotechnology Co. Ltd and TOPGENE Biotechnology Co. Ltd, where they received unrestricted access to water and were provided with a standard diet in accordance with standard care practices for monkeys. The experimental protocol adhered to the Guidelines for the Care and Use of Laboratory Animals established by Jinan University. Approval for this experiment was granted by the Animal Care and Use Committee of LANDAU (approval number: LDACU20201224-01).
The SgRNA sequences targeting the RAG1 and IL2RG genes were designed using online software (see detailed sequences in Fig. 1b). These oligonucleotides were then annealed and inserted into pBluescriptSKII-U6-sgRNA expression vectors for cloning purposes.
mRNA and RNA preparation
The CBE4max plasmids were acquired from Addgene and subsequently linearized using Not I. In order to synthesize mRNA, in vitro RNA transcription was performed using the HiScribe™ T7 ARCA mRNA Kit (tailed) from NEB. The SgRNAs were amplified and transcribed in vitro utilizing the MAXIscript T7 kit (Ambion). Following transcription, purification of the SgRNAs was carried out using the miRNeasy Mini Kit (Qiagen), following the instructions provided by the manufacturer.
Monkey zygote microinjection and animal breeding
A combination of CBE4max mRNA (200 ng/μL) and sgRNA (50 ng/μL) was injected into the zygotes of monkeys using microinjection techniques. The reconstructed embryos were subsequently placed in a culture medium for further development. Once they reached the 4–8 embryonic stage, the embryos were surgically transferred into the fallopian tubes of surrogate monkeys. Following birth, genotyping was conducted on the offspring to validate the successful breeding of animals.
PCR analysis to sequence
Tissue samples were collected from the mutant monkeys, and DNA extraction was carried out to obtain genomic DNA. For PCR analysis, rTaq polymerase (Takara, Kyoto, Japan) was used with the following cycling parameters: initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 5 min. A final extension step was performed at 72°C for 4 min. The genotyping primers used were as follows: IL2RG (forward 5′-GGCTCTGGATGACTGCGGTACC-3′; reverse 5′-GTCAGTCCGGTACTGCACCAAGTG-3′); RAG1 (forward 5′-TCAGCCAGCATGGCGTCCTCTTT-3′; reverse 5′-GAACTGGATATCTCCTGTTGTGCTCA-3′). Subsequently, Sanger sequencing analysis was conducted on the PCR products to examine the genetic sequences.
To identify specific DNA mutations in the PCR products, we employed the T7E1 assay. The PCR products were denatured and then re-annealed in NEBuffer 2 (NEB) using a thermal cycler. After re-annealing, the PCR products underwent digestion using T7 endonuclease 1 (NEB, M0302L) and were subsequently separated through agarose gel electrophoresis for analysis.
GuangZhou HeQin Bio Tec conducted the preparation of monkey DNA libraries and performed whole-genome sequencing (WGS) using the Illumina NovaSeq 6000 platform. The WGS achieved an average coverage of 30×. To process the raw sequencing data, fastp (v0.20.1) was utilized to eliminate low-quality reads and trim adapter sequences. The remaining high-quality sequencing reads were then aligned to the reference genome (Macaca_fascicularis_6.0) using BWA (v0.7.15-r1140). The resulting BAM files underwent additional processing, including read sorting, duplicate removal, and BAM file indexing, using sambamba (v0.6.6). For germline variant detection, variant calling was performed using strelka (v2.9.10). The raw variants generated by strelka, which had a filter tag of “PASS” were considered high-confidence variants for subsequent analysis. To annotate single-nucleotide variants (SNVs) and insertions/deletions (indels), ANNOVAR (v2017Jul17) was employed for genomic annotation. Custom R scripts were used for downstream analysis and visualization purposes.
Estimated edit frequency
To assess the efficiency of base editing using Sanger sequencing, we employed the EditR online software (https://moriaritylab.shinyapps.io/editr_v10/) as a tool for estimating the editing frequency.43
Histopathological analysis and immunohistochemistry
The excised tissue was immersed in 4% paraformaldehyde for 3 days to initiate fixation. Subsequently, the tissues were embedded in paraffin and sectioned at a thickness of 3 μm for both H&E staining and immunofluorescence (IF) analysis. For IF staining, antigen retrieval solution (Sigma-Aldrich, C9999) was used for a 10-min period, followed by natural cooling. The sections were then incubated in a blocking buffer (3% bovine serum albumin and 0.3% Triton X-100 in PBS) for 1 h at room temperature. Primary antibodies, including KI67 (Abcam, ab16667), GFP (Invitrogen, A11122), CD133 (Abcam, ab222782), CD56 (Abcam, ab237708), CD16 (Abcam, ab246222), IgM (Abcam, ab134159), and CD3 (Abcam, ab21703) were diluted in blocking solution and incubated overnight at 4 °C. After extensive washing with TBST (0.5% Tween 20 in TBS), the sections were incubated for 1 h with secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555, followed by the addition of DAPI. Microscopic images were captured using the TissueGnostics panoramic tissue and cell quantitative analysis system. Immunofluorescence staining was analyzed using a confocal imaging system (Olympus FV3000 microscope).
Western blot analysis
The tissues were homogenized using a grinder from Luca Sequencing Instrument Co., Ltd, Guangzhou, China. The homogenized tissues were then placed in a protein lysis buffer prepared by combining RIPA lysis buffer, protease inhibitor, and phosphatase inhibitor in a ratio of 98:1:1. After incubating the lysates on ice for 30 min, they were sonicated and centrifuged at maximum speed for 10 min. Total protein was extracted from the supernatant and quantified using the BCA protein quantification kit (Solarbio, Beijing, China). The proteins were subsequently separated by electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat dry milk for 1 h and then incubated with primary antibodies overnight at 4 °C. Antibodies specific to RAG1 (Cell Signaling Technology, 3968 S), Vinculin (Sigma, MAB3574), and IL2RG (Invitrogen, PA5-80730) were used as primary antibodies. Finally, secondary antibodies labeled with horseradish peroxidase were applied, and the protein bands were detected using an electrochemiluminescence kit from CLINX. The results were analyzed using ImageJ software.
T cells and B cells in the blood were examined using flow cytometry. EDTA-K3 tubes were used to collect blood samples for determining immunotypes and cell profiles. The BD FACS Verse flow cytometer system was employed for flow cytometry analysis to identify cell populations in the whole blood. A four-color immunofluorescence staining panel, consisting of CD4, CD3, IgM, and CD8 markers, was utilized in a single tube to detect different lymphocyte subsets. The antibodies used for detecting specific T cell subpopulations were employed at concentrations recommended by the manufacturer (refer to Supplementary Table 1 for antibody information). Data analysis was performed using FlowJo V10 software from FlowJo, Ashland, OR, USA.
Monkey tumor formation experiment
MDA-MB-231-CMV-EGFP-Luc-Puro cells of human origin were cultivated in L-15 medium supplemented with 10% fetal bovine serum, 2 μg/mL puromycin, and 1% penicillin-streptomycin (all from Gibco) at a temperature of 37 °C in a humidified environment with 100% air. When the cells reached a confluence of 80–100%, the cell suspension was harvested and subjected to centrifugation at 300×g and 4 °C for 5 min. The cells were then resuspended in Matrigel at a concentration of 1 × 107 cells/50 μL. Using an insulin syringe kept on ice, the mixture of cells and Matrigel was loaded. To conduct the procedure, the monkeys were anesthetized with isoflurane and placed in a supine position. The injection site in the axillary region was sterilized, and the cell suspension was slowly injected subcutaneously. Following the injection, the needle was held in place for 5 s. After complete recovery from anesthesia, the growth of the transplanted tumors was monitored. In accordance with approved protocols, the animals were euthanized, and the tumors were collected for in vitro assays.
In vivo imaging systems
The study comprised both wild-type (WT) monkeys and monkeys subjected to base editing. To induce deep anesthesia, Zoletil®50 (4 mg/kg, Virbac, France) was administered to the animals through intramuscular injection. The AniView100 Multimode Live Animal Imaging System (Boluteng Biological Technology Co., Ltd, Guangzhou, China) was utilized to capture images of the monkeys. Following image acquisition, the data obtained were analyzed using the AniView software (Guangzhou, China), which is specifically designed for live monkey imaging.
To obtain total RNA, tissue samples underwent RNA extraction utilizing the Trizol reagent (Thermo Fisher Scientific, MA, USA). Sequencing libraries were generated using the SEQUMED® MustSeq® 3’mRNA DEG kit (Sequmed, China). The RNA-seq analysis was conducted at SequMed Bio Technology (Guangzhou, China) using the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA). Data alignment and quantification were performed using the STAR aligner, enabling further analysis of the RNA-sequencing data.44
Tissue samples were subjected to total RNA extraction using the Trizol method. The purity and integrity of the RNA were assessed, followed by reverse transcription using the PrimeScript™ RT Kit with gDNA Eraser (Takara, Kyoto, Japan). PCR amplification was subsequently performed using the TB Green Premix Ex Taq II (Takara, Kyoto, Japan). The resulting PCR products were detected using the CFX Connect Real-Time PCR Detection system (Bio-Rad, California, USA), with triplicate detection for each group. The relative expression of genes was determined using the cycle threshold (2−ΔΔCt) method.45 Please refer to Supplementary Table 2 for the primer sequences utilized in the RT-qPCR analysis.
Potential off-target sites
To assess the possibility of off-target effects, we utilized Cas-OFFinder (http://www.rgenome.net/cas-offinder/)46 to predict and analyze potential off-target sites for each sgRNA. To validate the presence of off-target effects, PCR amplification of all predicted off-target sites was performed, followed by either Sanger sequencing or targeted deep sequencing. For a comprehensive list of the potential off-target sites and the corresponding primers used in the off-target assays, please refer to Supplementary Tables 3 and 4.
Targeted deep sequence
To sequence the genomic loci of interest, we initiated by amplifying them from genomic DNA samples using amplification primers (Supplementary Tables 5 and 6) that contained Illumina forward and reverse adapters. The specific genomic region was amplified in the first round of PCR (PCR 1) using these primers. In a subsequent PCR reaction (PCR 2), unique Illumina barcoded primer pairs were incorporated into each sample. The PCR2 products, obtained by pooling the common amplicons, were purified using a gel extraction kit (Magen, Guangzhou) after 1.5% agarose gel electrophoresis and eluted with 40 μL of water. The concentration of DNA was measured through fluorescence quantification or qPCR, and sequencing was performed on an Illumina NovaSeq instrument following the manufacturer’s protocol.43,47 Annoroad Gene Technology Co., Ltd. (Beijing) conducted targeted deep sequencing, and the resulting data were analyzed using CRISPResso2 and custom R scripts.
Group comparisons were assessed for statistical significance using a two-tailed Student’s t-test. The data are presented as mean ± standard error of the mean (SEM). All calculations were conducted using GraphPad Prism 9 software. A significance level of P < 0.05 was deemed statistically significant.