Genetically humanized pigs exclusively expressing human insulin

Journal of Molecular Cell Biology Advance Access published March 18, 2016

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Genetically humanized pigs exclusively expressing human insulin are generated through custom endonuclease-mediated seamless engineering

Yi Yang1,?, Kepin Wang1,?, Han Wu1, Qin Jin1, Degong Ruan1, Zhen Ouyang1, Bentian Zhao1, Zhaoming Liu1, Yu Zhao1, Quanjun Zhang1, Nana Fan1, Qishuai Liu1, Shimei Guo1,3, Lei Bu4, Yong Fan5, Xiaofang Sun5, Xiaoping Li1,*, and Liangxue Lai1,2,*

1

Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong

Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

2

Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of

Veterinary Medicine, Jilin University, Changchun 130062, China

3

School of Life Sciences, University of Science and Technology of China, Hefei 230027, China 4

Leon H Charney Division of Cardiology, New York University School of Medicine, 522 First

Avenue, New York, NY 10016, USA

5

Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of

Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China

*Correspondence to: Liangxue Lai, E-mail: lai_liangxue@gibh.ac.cn; Xiaoping Li, E-mail:

li_xiaoping@gibh.ac.cn

?These authors contributed equally to this work.

? The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

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Dear Editor,

Type 1 diabetes (T1D) is a lifelong (chronic) disease and a major health problem throughout the world. This disease can be treated by either insulin injection or islet transplantation. Islet transplantation is considered as a better treatment for T1D patients, because islets can produce and release insulin at the appropriate time, resulting in tight blood glucose control. However, islet transplantation is performed only for brittle T1D patients due to lack of sufficient donors: only 1

of 333 patients with insulin-dependent diabetes mellitus (IDDM) can obtain human islets (Frank et al, 2005). Porcine insulin has been applied to treat T1D. Recently, pig islets have also proved to be an attractive alternative resource for solving the donor shortage issue for islet transplantation. Many preclinical trials of porcine islet transplantation have been performed and have already achieved long-term survival of porcine islets in primate recipients (Cardona et al, 2006; Bottino et al, 2014). Previous efforts have been focusing on overcoming rejection of recipients to the donor organs (Bottino et al, 2014). Porcine insulin differs from human insulin by one amino acid (alanine in pigs and threonine in humans) at the carboxy-terminal of the B chain (i.e. position B30) (Sonnenberg and Berger, 1983) (Supplementary Figure S1). This single amino acid difference can induce human antibodies to act against porcine insulin. As a result, the effectiveness is decreased when porcine insulin is used for the long-term treatment of diabetic patients (Clark et al, 1982). For the same reason, the transferred pig islets may not function well in human patients for a long period. To address this issue, here we attempt to generate genetically humanized pigs that produce human insulin rather than porcine insulin. We edited porcine INS (pINS) gene in fibroblasts by using transcription activator-like effector nucleases (TALENs) or CRISPR/Cas9, combined with single-stranded oligonucleotides (ssODNs) as homology donors. By using somatic cell nuclear

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transfer (SCNT) technology, we successfully generated the genetically modified pigs exclusively expressing human insulin.

Gene-editing animals have been generated by injecting the mRNA or proteins of custom endonuclease (such as zinc-finger nuclease, TALEN, and Cas9/gRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. In addition, the genotypes generated by embryo microinjection are variable and unpredictable (Crispo et al, 2015). One or

two rounds of further breeding should be performed to obtain desired homozygotes with identical genotype and phenotype, which is particularly time and labor consuming for large animals with long gestation term and sex maturation age, such as pigs. To address this issue, our group has established an approach that employs gene-targeting somatic cells with SCNT, which could generate gene-editing animals with a single and identical mutation by one round of SCNT experiment. Recent reports have demonstrated that ssODNs, which are easily available, are more effective donors than traditional double-strand DNA homology for homology-directed repair (HDR), with the aid of custom endonucleases such as zinc-finger nuclease, TALENs, and CRISPR/Cas9 (Bedell et al, 2012; Yang et al, 2013). In addition, when ssODNs are used as HDR donors, selection marker gene is not necessary, which enables to create seamless site-specific mutations. The success of ssODN-mediated targeting strategy has been reported in cell lines or early stage embryos, but not yet in somatic cells, where homologous recombination with ssODNs is expected to be more difficult due to the limited proliferation competency.

The length of ssODNs may affect the HDR efficiency in somatic cells. To optimize the length of ssODNs as donors, a HEK293 cell line with a fluorescence reporter was established, in which enhanced green fluorescent protein (EGFP) gene was mutated by deleting T at 456 site (ΔEGFP),

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and thus a stop codon (TATA>TA-A) was created to prevent the EGFP expression. Seven ssODNs with different lengths were used as donors, combined with either TALENs or Cas9/gRNA, to repair ΔEGFP (Supplementary Figure S2A). Flow cytometry analysis was performed to determine the repairing efficiency (Supplementary Figures S2B and S3). We found that the repairing efficiency increased when the length of ssODNs ranged from (0+0) nt to (25+25) nt and remarkably decreased when the length of ssODNs reached (30+30) nt. However, the

repairing efficiency increased again when the length of ssODNs increased from (30+30) nt (Figure 1A, Supplementary Figure S3). It had been reported that when short ssODNs of 15?25 nt were used as donors, an alternative genome repair mechanism, i.e. microhomology-mediated end joining, would be employed by cells and resulted in sequence deletion and chromosome translocation (McVey and Lee, 2008). Therefore, ssODNs with the length < (25+25) nt are not suitable for a precise DNA repair. Conversely, when the length of ssODN is > 35 nt, classical HDR will take place, which can accurately restore DNA sequence at a double-strand break (Supplementary Figure S2C?E). Therefore, we chose ssODN with lengths of (40+40) and (45+45) nt that show an optimum repairing efficiency as HDR donors to replace GCC with ACG in the pINS gene (Figure 1A). The efficiency of HDR mediated by ssODNs with TALENs was comparable with that of ssODNs and Cas9/gRNA. Thus, both TALENs and Cas9/gRNA were applied to modify the pINS gene (Figure 1B). Two pairs of TALENs and one gRNA targeting the exon II of pINS were designed and assembled, and their activities were evaluated by single-stranded annealing (SSA) assay (Li et al, 2014). The cleavage activity of pINS-TALEN2 is higher than that of pINS-TALEN1 and comparable with that of pINS-gRNA (Supplementary Figure S4). Thus, pINS-TALEN2 and pINS-gRNA were used to target pINS in fibroblasts. ssODNGCC>ACG with

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lengths of 88 nt (for TALENs) and 99 nt (for Cas9/gRNA), containing 41 nt right arm and 44 nt left arm, respectively, were used as HDR donors (Figure 1B). We introduced GCC>ACG mutation in the ssODN, and as a result, A>T amino acid substitution was induced at position B30 of porcine insulin. This mutation also generated a MluI restriction enzyme site, which helps to identify the genotype of targeted clones. For Cas9/gRNA, one silent mutation (GCG>GCA) on the PAM site was introduced to avoid repetitive digestion (Figure 1B).

Porcine fetal fibroblast (pFF) cells isolated from E35d embryos of Bama mini-pigs were electroporated with circular plasmids of pINS-TALEN2 or Cas9/gRNA accompanied with ssODNs. For transient durg selection, a neomycin expression cassette was inserted into the TALENs or Cas9 expression vector. After selection with G418 (1 mg/ml for 10?14 days), the surviving cell colonies were expanded and initially identified through PCR and MluI digestion. Non-homologous end joining was observed in colonies generated by either pINS-TALEN2 (72/300, 24%) or Cas9/gRNA (32/90, 35.6%) (Supplementary Tables S1, S4, and S5). Among the 300 colonies generated by TALENs, six (6/300, 2%) were identified with one allele precise substitution and no colony was found with biallelic point mutations (Figure 1C). In colonies generated through the Cas9/gRNA method, a higher editing rate of 5.6% (5/90) was obtained. Interestingly, all of the 5 cell colonies were biallelically targeted (Figure 1C). Sequencing results further confirmed that all of the colonies were correctly targeted with humanized insulin sequence (Figure 1D, Supplementary Tables S4 and S5). The correctly targeted colonies with one allelic or biallelic mutation were used as donor cells for nuclear transfer. A total of 2613 cloned embryos were generated and transferred into 11 surrogate mothers (Supplementary Table S2). Five surrogates were confirmed pregnant through ultrasound detection one month post-embryo transfer.

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