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. 2022 Jul 22;8(29):eabo3123.
doi: 10.1126/sciadv.abo3123. Epub 2022 Jul 22.

Cloning and base editing of GFP transgenic rhesus monkey and off-target analysis

Yu Kang  1   2   3 , Shaoxing Dai  1   3 , Yuqiang Zeng  1   3 , Fang Wang  1   3 , Pengpeng Yang  1   3 , Zhaohui Yang  1   3 , Youwei Pu  1   3 , Zifan Li  1   3 , Xinglong Chen  1   3 , Baohong Tian  1   3 , Wei Si  1   3 , Weizhi Ji  1   2   3 , Yuyu Niu  1   2   3
Affiliations

Cloning and base editing of GFP transgenic rhesus monkey and off-target analysis

Yu Kang et al. Sci Adv. .

Abstract

We report the cloning of a 12-year-old transgenic green fluorescent protein (GFP) monkey by somatic cell nuclear transfer (SCNT) and base editing of the embryos, accompanied with safety evaluation of adenine base editors (ABEs). We first show the ability of ABEmax to silence GFP through A-to-G editing of the GFP sequence in 293T cells. Subsequently, using donor cells from a monkey expressing GFP, we have successfully generated 207 ABEmax-edited (SCNT-ABE) and 87 wild-type (SCNT) embryos for embryo transfer, genotyping, and genome and transcriptome analysis. SCNT-ABE and SCNT embryos are compared for off-target analysis without the interference of genetic variants using a new method named as OA-SCNT. ABEmax does not induce obvious off-target DNA mutations but induces widespread off-target RNA mutations, 35% of which are exonic, in edited monkey embryos. These results provide important references for clinical application of ABE.

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Figures

Fig. 1.
Fig. 1.. ABE-mediated GFP knockout in SCNT monkey.
(A) The scheme of experimental procedures. TG, transgenic. (B) Representative images of ABE-mediated GFP silencing in SCNT monkey embryos. Top: SCNT blastocyst generated from transgenic GFP monkey. Bottom: GFP silencing in ABE-editing SCNT blastocyst. Left: Bright field. Right: GFP. Scale bars, 100 μm. (C) On-target analysis of ABE editing (the percentage of mutant cells in total blastomere) in monkey SCNT and SCNT-ABE embryos. (D) Image of the newborn cloned rhesus monkey generated by SCNT using adult transgenic rhesus monkey fibroblast. (E) On-target sequencing of donor cells and tissues of SCNT-ABE monkey. (F) Summary of rhesus SCNT embryos used in this study.
Fig. 2.
Fig. 2.. Comparison of SNVs and indels generated in SCNT-ABE and SCNT groups.
(A) Dot plot showing numbers of SNVs (left) and small indels (right) between SCNT and SCNT-ABE groups. Both SNVs and small indels in SCNT-ABE group are similar to those of the control group (P = 0.87 and P = 0.19, respectively). (B) Heatmap showing the number (top) and proportion (bottom) of different mutation types between SCNT and SCNT-ABE groups. A gradient of gray and blue indicates low to high number or proportion of SNVs. Each value represents the average value of different mutation types in SCNT or SCNT-ABE groups. (C) Comparison of the number of different types of off-target SNVs detected in SCNT and SCNT-ABE samples. (D) The proportion of different types of off-target SNVs detected in SCNT and SCNT-ABE samples. (E to F) Comparison of the number of A>G (E) and C>T (F) off-target SNVs in different genomic regions between SCNT and SCNT-ABE samples. P values shown above the horizontal bars were calculated by two-sided Wilcoxon rank sum test. P < 0.05 was considered significant in (A) and (C) to (F). *P < 0.05. ns, not significant.
Fig. 3.
Fig. 3.. ABE induces transcriptome-wide off-target A-to-G RNA editing in monkey embryos.
(A) Comparison of the number of all types and indicated types of off-target RNA mutation detected in SCNT and SCNT-ABE blastocyst. Both numbers of the total and of A>G RNA mutations in SCNT-ABE blastocyst are significantly higher than that in SCNT blastocyst (P = 0.0317 and P = 0.0079, respectively). (B) The proportion of indicated types of off-target RNA mutation detected in SCNT and SCNT-ABE blastocyst. (C and D) The number (C) and proportion (D) of different types of off-target SNVs detected within SCNT-ABE blastocyst. (E) Jitter plots showing efficiencies of A>G RNA editing by ABE derived from RNA-seq experiments. Samples of SCNT1 to SCNT5 and SCNT-ABE1 to SCNT-ABE5 are from SCNT and SCNT-ABE blastocyst, respectively. Top: The number of A>G RNA mutation for each sample. (F) Jitter plot showing the distribution of editing rate for each off-target A>G RNA mutation on monkey chromosomes for sample SCNT-ABE1 from (E). Chromosomes are indicated with different colors. (G) Jitter plots showing the editing rate of A>G RNA editing occurred in exons for SCNT-ABE blastocyst. Each dot represents the editing rate of one editing site in (E) to (G). The editing rate was calculated as the number of mutated reads divided by the sequencing depth for each site. (H) The stacked bar chart showing proportion of different exonic edits from (G). P values shown above the horizontal bars were calculated by two-sided Wilcoxon rank sum test. *P < 0.05; **P < 0.01.
Fig. 4.
Fig. 4.. Characterization of off-target RNA mutations.
(A and B) Principal components (PC) analysis (A) and heatmap (B) showing the clustering of samples from SCNT and SCNT-ABE blastocysts based on the overall gene expression. (C) Boxplot showing the expression of genes containing off-target RNA mutations (off-target genes) and random simulated genes (random genes) in SCNT-ABE1 and all SCNT samples. TPM, transcripts per million. (D) The Venn diagram showing overlapped editing sites (left) and genes (right) among five SCNT-ABE blastocysts. (E) Gene ontology (GO) biological process enriched by the RNA off-target editing genes from (D) in five SCNT-ABE blastocysts. GTPase, guanosine triphosphatase; snRNP, small nuclear ribonucleoprotein. (F) Sequence logos derived from A>G RNA mutations in all SCNT (top) and SCNT-ABE (bottom) blastocysts. (G) Jitter plots showing the editing rate of A>G exonic edits in the key genes at different stages of embryo development for SCNT-ABE blastocyst. Each dot represents the editing rate of an editing site. TE, trophectoderm; EPI, epiblast; PrE, primitive endoderm. The editing rate was calculated as the number of mutated reads divided by the sequencing depth for each site. (H) The stacked bar chart showing proportion of different edits from (G). (I) The bar chart showing the editing rate (15 to 100%) of 32 common editing sites in all five SCNT-ABE samples (left) and the expression of the corresponding gene (right). Significantly up- or down-regulated genes are marked with an asterisk before it. FC, fold change. P values shown above the horizontal bars were calculated by two-sided Wilcoxon rank sum test. ****P < 0.0001.
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