Highly efficient RNA-guided base editing in rabbit

ARTICLE DOI: 10.1038/s41467-018-05232-2 OPEN Highly efficient RNA-guided base editing in rabbit 1234567890():,; Zhiquan Liu1, Mao Chen1, Siyu Chen1, Jichao Deng1, Yuning Song1, Liangxue Lai1,2 & Zhanjun Li1 Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53–88% and 44–100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit. 1 Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, Jilin University, Changchun 130062, China. 2 Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China. These authors contributed equally: Zhiquan Liu, Mao Chen, Siyu Chen. Correspondence and requests for materials should be addressed to L.L.(email: ) or to Z.L.(email: ) NATURE COMMUNICATIONS | (2018)9:2717 | DOI: 10.1038/s41467-018-05232-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05232-2 T he vast majority of human genetic diseases arise from point mutations rather than insertions and deletions (indels), which has been verified through the explosive growth of human genomic data. Although the CRISPR/Cas9 system has been widely used to facilitate genome editing in a variety of organisms, including rabbits1,2, it would induce random indels through error-prone non-homologous end-joining (NHEJ) rather than the error-free homology-directed repair (HDR)3. As a result, indels are obtained much more frequently at target sites than single-nucleotide substitutions. Recently, a programmable cytidine deaminase or adenine deaminase built on the CRISPR/Cas9 platform has been shown to achieve targeted C-to-T4–6 (BE3) or A-to-G7 (ABE7.10) conversion in living cells without generating DSBs or relying on templatedonor DNA. To date, the BE3 system has undergone various improvement to increase the utility and applicability of the base editing capability8–10 and has been applied to induce singlenucleotide modifications in various plants and animals11–14. Similarly, ABE7.10 has also been reported to work in rice15,16 and mice17. However, both systems have not been assessed for efficacy and feasibility in rabbit. In the present study, the BE3 and ABE7.10 systems were used to create targeted base substitutions in rabbit. The results provide a prospective application for the generation of rabbit models which could precisely mimic human genetic diseases. Furthermore, this is the first report, to our knowledge, demonstrating the reduction of frequencies of indels and improvement of product purity of the new BE version (BE4-Gam) in blastocysts, compared with BE3. Overall, we firstly demonstrate that base editors (both CBEs and ABEs) provide a simple and highly efficient method for inducing single-nucleotide substitution in rabbit. conversion in the rabbit genome in vivo, four target loci from three genes (Mstn, Dmd, and Tia1) were selected (Fig. 1a). Base editing was conducted in rabbit zygotes by microinjection of BE3encoding mRNA and single guide RNAs (sgRNAs). Sanger sequencing and T–A cloning were performed to verify whether the targeted point mutations were successfully incorporated in each of the target site (Figs. 1b, c, d, e and Supplementary Fig. 1). It was observed that the mutation efficiency ranged from 75% to 87%, with an average successful target mutation rate of up to 70% (Table 1). Consistent with previous reports that BE3 system can still induce proximal off-targets, indels, or non-C-to-T conversions4,13,18, these undesired mutations were also observed in mutant blastocysts, albeit at low-average frequencies of 19%, 8%, or 3% (Table 1). Within these loci, missense mutations p.P362L and p.A381T in Tia1 were recently implicated in the development of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD)19. The C–T conversion in Tia1-1 induced the desired p. P362L amino acid change, precisely mimicking the p.P362L mutation observed in humans (Fig. 1f). These results indicate that BE3 system is simple and efficient in rabbit embryos, suggesting the potential of this system to improve economical traits and develop animal models for human genetic diseases in rabbits. Base conversion at Mstn to generate double-muscled rabbits. Mstn is a member of the transforming growth factor beta (TGF-β) superfamily, which acts as a negative regulator of muscle growth. A double-muscled phenotype, with the characteristics of increased muscle mass, has been reported in Mstn-knockout (KO) sheep20, pigs21, dogs22, and rabbits23. Here, a single C-to-T conversion was designed to generate a premature stop codon (p. Gln93stop) in exon 1 of rabbit Mstn (Fig. 2a). It was expected that this mutation would inactivate the gene by directly converting normal coded codons into STOP codons24. Of the seven rabbits, this mutation was attempted in six (86%) of them who carried Results BE3 can induce C-to-T base conversion in rabbit blastocysts. To explore whether the BE3 system can catalyze site-specific base a b Dmd (WT) (double-muscle) (WT) (DMD) Tia1-1 (WT) (ALS) Tia1-2 (WT) (ALS) Dmd Sequence Target site (phenotype) Mstn c Mstn WT WT Gln Gln #6 #10 Stop Stop WT Q93X WT Q807X (0/8) (8/8) Nonsense d e Tia1-1 (0/8) (8/8) Nonsense f Tia1-2 Tia1-1 WT Human WT Val Pro Ala Rabbit #5 #5 Ile Leu WT P362L (0/8) (8/8) Missense P362L gga gtg caa ccg cct G V Q P P G V Q P P ggc gtg cag ccg cct P362L Thr WT V380I/A381T (2/8) (6/8) Missense Fig. 1 The BE3 system mediates efficient C-to-T base editing in rabbit blastocysts. a Target-site sequences within the Mstn, Dmd, Tia1-1, and Tia1-2 loci. Target sequence (black), PAM region (green), target sites (red), and mutant amino acid (underlined). WT wild-type. b–e Representative sequencing chromatograms at the Mstn, Dmd, Tia1-1, and Tia1-2 targets of WT and edited rabbit blastocysts. Target base editing (red arrows), target sequence (black), PAM region (green), target sites (red), mutant amino acid (underlined) and amino acid mutation type are indicated. The relevant codon identities at the target site are presented beneath the DNA sequence. f Diagrammatic representation of the mutations of Tia1-1 associated with (...truncated)