猪CD163基因的单碱基编辑研究

doi:10.11843/j.issn.0366-6964.2022.04.005

Abstract

Abstract: The aim of this study was to use the YE1-BE3-FNLS vector to carry out a single-base mutation of C>T in the porcine CD163 gene to form the stop codon TAA, which would lead to the premature termination of the translation of the CD163 gene. The website was used to design high-efficiency sgRNA sequence at the 7th exon of porcine CD163 gene and make it conform to C5 feature, and the following two bases of C5 were AA, and the number of bases between CAA and the start codon ATG was the integer multiple of 3. The CD163-sgRNA and APOBEC-Cas9n-UGI fragment in the YE1-BE3-FNLS vector was amplified by PCR, and CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were produced by in vitro transcription. Porcine oocytes were cultured in vitro and undergone parthenogenetic activation. The CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were injected into parthenogenetic embryos by micromanipulator. After the embryo developed to the stage of morula and blastocyst, the genomic DNA of the embryo was extracted and its single-base editing region was subjected to PCR amplification and sequencing. The results showed that a CD163-sgRNA was successfully screened among 49 candidate sgRNAs. The CD163-sgRNA and APOBEC-Cas9n-UGI fragments were successfully amplified by PCR, and the porcine parthenogenetic embryos developed to the 2-4 cell stage after injection of CD163-sgRNA and APOBEC-Cas9n-UGI mRNA. After PCR amplification and product sequencing of selected injected 20 embryos at 2-4 cell stage, there were 12 embryos with the expected site C(num1479)>T of the 7th exon of the CD163 gene, and the single-base editing efficiency was about 60%. It was found that the CD163-sgRNA had 5 off-target regions in the case of a mismatch of 3 bases based on the off-target analysis. After analyzing these 5 regions, it was found that there was no C>T mutation using PCR amplification and sanger sequencing. The pig CD163 gene was suffered with base editing by YE1-BE3-FNLS tool at the embryo level in this study, and the 7th exon expected site (num1479) C was successfully changed to T, which can terminate the translation of CD163 gene in advance by making the codon CAA to TAA.

Key words: CRISPR/Cas9, base editing, CD163 gene, pig

CLC Number:

S828.2

ZHAO Weimin, WANG Huili, CAO Shaoxian, GUO Rihong, WANG Zeping, CHEN Zhe, XU Kui, FU Yanfeng, LI Bixia, REN Shouwen, CHENG Jinhua. The Study of Base Editing of Porcine CD163 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1041-1050.

References 36

[1]	BARRANGOU R,FREMAUX C,DEVEAU H,et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science, 2007,315(5819):1709-1712.
[2]	HORVATH P,BARRANGOU R.CRISPR/Cas,the immune system of bacteria and archaea[J].Science, 2010,327(5962):167-170.
[3]	JINEK M,CHYLINSKI K,FONFARA I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science, 2012,337(6096):816-821.
[4]	MALI P,YANG L H,ESVELT K M,et al.RNA-guided human genome engineering via Cas9[J].Science, 2013,339(6121):823-826.
[5]	HSU P D,LANDER E S,ZHANG F.Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014,157(6):1262-1278.
[6]	HILTON I B,GERSBACH C A.Enabling functional genomics with genome engineering[J].Genome Res, 2015,25(10):1442-1455.
[7]	JIANG W Z,ZHOU H B,BI H H,et al.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice[J].Nucleic Acids Res, 2013,41(20):e188.
[8]	RUAN J X,XU J,CHEN-TSAI R Y,et al.Genome editing in livestock:are we ready for a revolution in animal breeding industry?[J].Transgenic Res, 2017,26(6):715-726.
[9]	RAZZAQ A,SALEEM F,KANWAL M,et al.Modern trends in plant genome editing:an inclusive review of the CRISPR/ Cas9 toolbox[J].Int J Mol Sci, 2019,20(16):4045.
[10]	TRAN N T,SOMMERMANN T,GRAF R,et al.Efficient CRISPR/Cas9-mediated gene Knockin in mouse hematopoietic stem and progenitor cells[J].Cell Rep, 2019,28(13):3510-3522.e5.
[11]	ZHAO H,LI Y,HE L J,et al.In vivo AAV-CRISPR/Cas9-mediated gene editing ameliorates atherosclerosis in familial hypercholesterolemia[J].Circulation, 2020,141(1):67-79.
[12]	KOMOR A C,KIM Y B,PACKER M S,et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J].Nature, 2016,533(7603):420-424.
[13]	GAUDELLI N M,KOMOR A C,REES HA，et al.Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage[J].Nature, 2017,551(7681):464-471.
[14]	ZUO E W,SUN Y D,WEI W，et al.Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos[J].Science, 2019,364,364(6437):289-292.
[15]	JIN S A,ZONG Y,GAO Q,et al.Cytosine,but not adenine,base editors induce genome-wide off-target mutations in rice[J].Science, 2019,364(6437):292-295.
[16]	GRÜNEWALD J,ZHOU R H,GARCIA S P,et al.Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors[J].Nature, 2019,569(7756):433-437.
[17]	ZHOU C Y,SUN Y D,YAN R,et al.Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis[J].Nature, 2019,571(7764):275-278.
[18]	ZUO E W,SUN Y D,YUAN T L,et al.A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects[J].Nat Methods, 2020,17(6):600-604.
[19]	DOMAN J L,RAGURAM A,NEWBY G A,et al.Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors[J].Nat Biotechnol, 2020,38(5):620-628.
[20]	TIAN K G,YU X L,ZHAO T Z,et al.Emergence of fatal PRRSV variants:unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One, 2007,2(6):e526.
[21]	CALVERT J G,SLADE D E,SHIELDS S L,et al.CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses[J].J Virol, 2007,81(14):7371-7379.
[22]	VAN GORP H,DELPUTTE P L,NAUWYNCK H J.Scavenger receptor CD163,a Jack-of-all-trades and potential target for cell-directed therapy[J].Mol Immunol, 2010,47(7-8):1650-1660.
[23]	STOIAN A M M,ROWLAND R R R.Challenges for Porcine Reproductive and Respiratory Syndrome (PRRS) vaccine design:reviewing virus glycoprotein interactions with CD163 and targets of virus neutralization[J].Vet Sci, 2019,6(1):9.
[24]	HUANG C,BERNARD D,ZHU J Q,et al.Small molecules block the interaction between porcine reproductive and respiratory syndrome virus and CD163 receptor and the infection of pig cells[J].Virol J, 2020,17(1):116.
[25]	WANG H T,SHEN L C,CHEN J Y,et al.Deletion of CD163 exon 7 confers resistance to highly pathogenic porcine reproductive and respiratory viruses on pigs[J].Int J Biol Sci, 2019,15(9):1993-2005.
[26]	BURKARD C,LILLICO S G,REID E,et al.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[J].PLoS Pathog, 2017,13(2):e1006206.
[27]	YANG H Q,ZHANG J,ZHANG X W,et al.CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus[J].Antiviral Res, 2018,151:63-70.
[28]	BURKARD C,OPRIESSNIG T,MILEHAM A J,et al.Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection[J].J Virol, 2018,92(16):e00415-18.
[29]	GUO C H,WANG M,ZHU Z B,et al.Highly efficient generation of pigs harboring a partial deletion of the CD163 SRCR5 domain,which are fully resistant to porcine reproductive and respiratory syndrome virus 2 infection[J].Front Immunol, 2019,10:1846.
[30]	MONTAGUE T G,CRUZ J M,GAGNON J A,et al.CHOPCHOP:a CRISPR/Cas9 and TALEN web tool for genome editing[J].Nucleic Acids Res, 2014,42(W1):W401-W407.
[31]	OLADZAD A,PORCH T,ROSAS J C,et al.Single and multi-trait GWAS identify genetic factors associated with production traits in common bean under abiotic stress environments[J].G3 (Bethesda), 2019,9(6):1881-1892.
[32]	THURONYI B W,KOBLAN L W,LEVY J M,et al.Continuous evolution of base editors with expanded target compatibility and improved activity[J].Nat Biotechnol, 2019，37(9):1070-1079.
[33]	FU Y F,FODEN J A,KHAYTER C,et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J].Nat Biotechnol, 2013,31(9):822-826.
[34]	GRAVERSEN J H,MADSEN M,MOESTRUP S K.CD163:a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma[J].Int J Biochem Cell Biol, 2002,34(4):309-314.
[35]	XU K,ZHOU Y R,MU Y L,et al.CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance[J].elife, 2020,9:e57132.
[36]	姚旭东,蒙亚琦,任秀美奥,等.利用单碱基编辑系统定点编辑哈萨克羊MSTN 基因的研究[J].畜牧兽医学报,2021,52(8):2162-2170.YAO X D,MENG Y Q,REN X M A,et al.Study of Kazakh sheep MSTN gene site-directed editing based on the base editing system[J].Acta Veterinaria et Zootechnica Sinica, 2021,52(8):2162-2170.

The Study of Base Editing of Porcine CD163 Gene

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