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Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells

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Abstract

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3′ end of the intron 5) and splice donor site (GT sequence at the 5′ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

References

  • Anzalone AV, Koblan LW, Liu DR (2020) Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 38:824–844

    Article  CAS  PubMed  Google Scholar 

  • Burkard C, Lillico SG, Reid E, Jackson B, Mileham AJ, Ait-Ali T, Whitelaw CB, Archibald AL (2017) Precision engineering for PRRSV resistance in pigs: macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathog 13:e1006206

    Article  PubMed  PubMed Central  Google Scholar 

  • Cui D, Li F, Li Q, Li J, Zhao Y, Hu X, Zhang R, Li N (2015) Generation of a miniature pig disease model for human Laron syndrome. Sci Rep 5:15603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Doudna JA (2020) The promise and challenge of therapeutic genome editing. Nature 578:229–236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J (2018) CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 24:927–930

    Article  CAS  PubMed  Google Scholar 

  • Han X, Gao Y, Li G, Xiong Y, Zhao C, Ruan J, Ma Y, Li X, Li C, Zhao S, Xie S (2020) Enhancing the antibacterial activities of sow milk via site-specific knock-in of a lactoferrin gene in pigs using CRISPR/Cas9 technology. Cell Biosci 10:133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hinrichs A, Kessler B, Kurome M, Blutke A, Kemter E, Bernau M, Scholz AM, Rathkolb B, Renner S, Bultmann S, Leonhardt H, de Angelis MH, Nagashima H, Hoeflich A, Blum WF, Bidlingmaier M, Wanke R, Dahlhoff M, Wolf E (2018) Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver. Mol Metab 11:113–128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang S, Liao Z, Li X, Liu Z, Li G, Li J, Lu Z, Zhang Y, Li X, Ma X, Sun Q, Huang X (2019) Developing ABEmax-NG with precise targeting and expanded editing scope to model pathogenic splice site mutations in vivo. iScience 15:640–648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, Kommineni S, Chen J, Sondey M, Ye C, Randhawa R, Kulkarni T, Yang Z, McAllister G, Russ C, Reece-Hoyes J, Forrester W, Hoffman GR, Dolmetsch R, Kaykas A (2018) p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med 24:939–946

    Article  CAS  PubMed  Google Scholar 

  • Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, Liang C, Wang D, Qiu JL, Zhang F, Gao C (2019) Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364:292–295

    Article  CAS  PubMed  Google Scholar 

  • Koblan LW, Erdos MR, Wilson C, Cabral WA, Levy JM, Xiong ZM, Tavarez UL, Davison LM, Gete YG, Mao X, Newby GA, Doherty SP, Narisu N, Sheng Q, Krilow C, Lin CY, Gordon LB, Cao K, Collins FS, Brown JD, Liu DR (2021) In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice. Nature 589:608–614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Koppes EA, Redel BK, Johnson MA, Skvorak KJ, Ghaloul-Gonzalez L, Yates ME, Lewis DW, Gollin SM, Wu YL, Christ SE, Yerle M, Leshinski A, Spate LD, Benne JA, Murphy SL, Samuel MS, Walters EM, Hansen SA, Wells KD, Lichter-Konecki U, Wagner RA, Newsome JT, Dobrowolski SF, Vockley J, Prather RS, Nicholls RD (2020) A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing. JCI Insight 5:e141523

    Article  PubMed Central  Google Scholar 

  • Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36:765–771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kurtz S, Lucas-Hahn A, Schlegelberger B, Göhring G, Niemann H, Mettenleiter TC, Petersen B (2021) Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs. Proc Natl Acad Sci USA 118(2):e2008743118

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Duan X, An X, Feng T, Li P, Li L, Liu J, Wu P, Pan D, Du X, Wu S (2018) Efficient RNA-guided base editing for disease modeling in pigs. Cell Discov 4:64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Long C, Li H, Tiburcy M, Rodriguez-Caycedo C, Kyrychenko V, Zhou H, Zhang Y, Min YL, Shelton JM, Mammen PPA, Liaw NY, Zimmermann WH, Bassel-Duby R, Schneider JW, Olson EN (2018) Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing. Sci Adv 4:eaap9004

    Article  PubMed  PubMed Central  Google Scholar 

  • Min YL, Bassel-Duby R, Olson EN (2019) CRISPR correction of duchenne muscular dystrophy. Annu Rev Med 70:239–255

    Article  CAS  PubMed  Google Scholar 

  • Montes M, Sanford BL, Comiskey DF, Chandler DS (2019) RNA splicing and disease: animal models to therapies. Trends Genet 35:68–87

    Article  CAS  PubMed  Google Scholar 

  • Pan JS, Lin ZS, Wen JC, Guo JF, Wu XH, Liu YY, Lai WJ, Liang QY, Xie YS, Chen YR, Chen YH, Yan AF, Feng J, Liu L, Gong DY, Zhu XX, Lu JH, Tang DS (2021) Application of the modified cytosine base-editing in the cultured cells of bama minipig. Biotechnol Lett 43(9):1699–1714

    Article  CAS  PubMed  Google Scholar 

  • Perleberg C, Kind A, Schnieke A (2018) Genetically engineered pigs as models for human disease. Dis Model Mech 11:dmm030783

    Article  PubMed  PubMed Central  Google Scholar 

  • Petersen B (2017) Basics of genome editing technology and its application in livestock species. Reprod Domest Anim 52(Suppl 3):4–13

    Article  CAS  PubMed  Google Scholar 

  • Porto EM, Komor AC, Slaymaker IM, Yeo GW (2020) Base editing: advances and therapeutic opportunities. Nat Rev Drug Discov 19:839–859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rees HA, Wilson C, Doman JL, Li DR (2019) Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5:eaax5717

    Article  PubMed  PubMed Central  Google Scholar 

  • Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR (2020) Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol 38:883–891

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ryu J, Prather RS, Lee K (2018) Use of gene-editing technology to introduce targeted modifications in pigs. J Anim Sci Biotechnol 9:5

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang R, Zhang JY, Lu KH, Lu SS, Zhu XX (2019) Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing. In Vitro Cell Dev Biol Anim 55:784–792

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Bi D, Qin G, Song R, Yao J, Cao C, Zheng Q, Hou N, Wang Y, Zhao J (2020) Cytosine base editor (hA3A-BE3-NG)-mediated multiple gene editing for pyramid breeding in pigs. Front Genet 11:592623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wei YY, Zhan QM, Zhu XX, Yan AF, Feng J, Liu L, Li JH, Tang DS (2020) Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method. Biotechnol Lett 42:2091–2109

    Article  CAS  PubMed  Google Scholar 

  • Whitworth KM, Rowland RR, Ewen CL, Trible BR, Kerrigan MA, Cino-Ozuna AG, Samuel MS, Lightner JE, McLaren DG, Mileham AJ, Wells KD, Prather RS (2016) Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat Biotechnol 34:20–22

    Article  CAS  PubMed  Google Scholar 

  • Wilkinson ME, Charenton C, Nagai K (2020) RNA splicing by the spliceosome. Annu Rev Biochem 89:359–388

    Article  CAS  PubMed  Google Scholar 

  • Xie J, Ge W, Li N, Liu Q, Chen F, Yang X, Huang X, Ouyang Z, Zhang Q, Zhao Y, Liu Z, Gou S, Wu H, Lai C, Fan N, Jin Q, Shi H, Liang Y, Lan T, Quan L, Li X, Wang K, Lai L (2019) Efficient base editing for multiple genes and loci in pigs using base editors. Nat Commun 10:2852

    Article  PubMed  PubMed Central  Google Scholar 

  • Xie J, Huang X, Wang X, Gou S, Liang Y, Chen F, Li N, Ouyang Z, Zhang Q, Ge W, Jin Q, Shi H, Zhuang Z, Zhao X, Lian M, Wang J, Ye Y, Quan L, Wu H, Wang K, Lai L (2020) ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems. BMC Biol 18:131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu K, Zhou Y, Mu Y, Liu Z, Hou S, Xiong Y, Fang L, Ge C, Wei Y, Zhang X, Xu C, Che J, Fan Z, Xiang G, Guo J, Shang H, Li H, Xiao S, Li J, Li K (2020) CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance. Elife 9:e57132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu S, Kim J, Tang Q, Chen Q, Liu J, Xu Y, Fu X (2020b) CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway. Protein Cell 11:352–365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yan S, Tu Z, Liu Z, Fan N, Yang H, Yang S, Yang W, Zhao Y, Ouyang Z, Lai C, Yang H, Li L, Liu Q, Shi H, Xu G, Zhao H, Wei H, Pei Z, Li S, Lai L, Li XJ (2018) A Huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington’s disease. Cell 173:989–1002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu H, Long W, Zhang X, Xu K, Guo J, Zhao H, Li H, Qing Y, Pan W, Jia B, Zhao HY, Huang X, Wei HJ (2018) Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer. J Transl Med 16:41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yuan J, Ma Y, Huang T, Chen Y, Peng Y, Li B, Li J, Zhang Y, Song B, Sun X, Ding Q, Song Y, Chang X (2018) Genetic modulation of RNA splicing with a CRISPR-guided cytidine deaminase. Mol Cell 72:380–394

    Article  CAS  PubMed  Google Scholar 

  • Yuan H, Yu T, Wang L, Yang L, Zhang Y, Liu H, Li M, Tang X, Liu Z, Li Z, Lu C, Chen X, Pang D, Ouyang H (2020) Efficient base editing by RNA-guided cytidine base editors (CBEs) in pigs. Cell Mol Life Sci 77:719–733

    Article  CAS  PubMed  Google Scholar 

  • Yue Y, Xu W, Kan Y, Zhao HY, Zhou Y, Song X, Wu J, Xiong J, Goswami D, Yang M, Lamriben L, Xu M, Zhang Q, Luo Y, Guo J, Mao S, Jiao D, Nguyen TD, Li Z, Layer JV, Li M, Paragas V, Youd ME, Sun Z, Ding Y, Wang W, Dou H, Song L, Wang X, Le L, Fang X, George H, Anand R, Wang SY, Westlin WF, Guell M, Markmann J, Qin W, Gao Y, Wei HJ, Church GM, Yang L (2021) Extensive germline genome engineering in pigs. Nat Biomed Eng 5:134–143

    Article  CAS  PubMed  Google Scholar 

  • Zhao JG, Lai LX, Ji WZ, Zhou Q (2019) Genome editing in large animals: current status and future prospects. Natl Sci Rev 6:402–420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu XX, Zhong YZ, Ge YW, Lu KH, Lu SS (2018) CRISPR/Cas9-mediated generation of Guangxi Bama minipigs harboring three mutations in alpha-synuclein causing Parkinson’s disease. Sci Rep 8:12420

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhu X, Wei Y, Zhan Q, Yan A, Feng J, Liu L, Tang D (2020a) CRISPR/Cas9-mediated biallelic knockout of IRX3 reduces the production and survival of somatic cell-cloned Bama minipigs. Animals 10:501

    Article  PubMed Central  Google Scholar 

  • Zhu XX, Zhan QM, Wei YY, Yan AF, Feng J, Liu L, Lu SS, Tang DS (2020b) CRISPR/Cas9-mediated MSTN disruption accelerates the growth of Chinese Bama pigs. Reprod Domest Anim 55:1314–1327

    Article  CAS  PubMed  Google Scholar 

  • Zuccaro MV, Xu J, Mitchell C, Marin D, Zimmerman R, Rana B, Weinstein E, King RT, Palmerola KL, Smith ME, Tsang SH, Goland R, Jasin M, Lobo R, Treff N, Egli D (2020) Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell 183:1650–1664

    Article  CAS  PubMed  Google Scholar 

  • Zuo E, Sun Y, Wei W, Yuan T, Ying W, Sun H, Yuan L, Steinmetz LM, Li Y, Yang H (2019) Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364:289–292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was jointly supported by the Guangdong Provincial R&D Project in Key Areas (2018B020203003), the National Natural Science Foundation of China (82070199, 81900775), the Guangdong Basic and Applied Basic Research Fund (2019A1515110280), the Guangdong Science and Technology Innovation Strategy Fund (The Special Fund for “Climbing Plan”; pdjh2020a0616), the Special Project in Key Fields of Guangdong Provincal Universities (2021ZDZX2050) and the Free Exploration Fund for Postgraduates of Foshan University (2020ZYTS38).

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  1. Xiang-xing Zhu, Jia-sheng Pan and Tao Lin have contributed equally to this work.

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, Gene Editing Technology Center of Guangdong Province, Foshan University, Foshan, 528225, China

    Xiang-xing Zhu, Jia-sheng Pan, Ye-cheng Yang, Qiu-yan Huang, Shuai-peng Yang, Zi-xiao Qu, Zi-sheng Lin, Jian-cong Wen, Ai-fen Yan, Juan Feng, Lian Liu, Xiao-li Zhang & Dong-sheng Tang

  2. School of Life Sciences and Food Engineering, Hebei University of Engineering, Handan, 056038, China

    Tao Lin

  3. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, 9999078, China

    Jia-hong Lu

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  1. Xiang-xing Zhu
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  2. Jia-sheng Pan
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Contributions

DST, JHL and XXZ conceived and designed the experiments. XXZ, JSP, TL, YCY, QYH, SPY and ZXQ conducted the experiments and analyzed the results. ZSL and JCW participated in experiment assistant. XXZ, JSP, TL, JHL and DST drafted the manuscript. All authors participated in discussions of the results and reviewed the manuscript.

Supplementary information

The complete sequence of PX-ABEmaxAW vector is provided online with this paper.

Corresponding authors

Correspondence to Jia-hong Lu or Dong-sheng Tang.

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All authors declare no competing financial and non-financial interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All of the animal procedures used in this study were carried out in accordance with the Guide for Care and Use of Laboratory Animals (8th edition, released by the National Research Council, USA), and were approved by the Animal Care & Welfare Committee of Foshan University (Approval No. 2019020). All of the surgical procedures were performed under anesthesia by a veterinarian, and all efforts were made to minimize animal suffering.

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Zhu, Xx., Pan, Js., Lin, T. et al. Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells. Biotechnol Lett 44, 59–76 (2022). https://doi.org/10.1007/s10529-021-03214-x

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