基于CRISPR/Cas9技术的弓形虫rop16_(I/III)缺陷虫株的构建及毒力鉴定
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摘要
目的构建并鉴定弓形虫RH株rop16_(I/III)缺陷虫株。方法利用CRISPR-Cas9技术进行构建基因缺陷虫株。运用E-CRISPR数据库设计gRNA,并使用定点突变试剂盒突变pSAG1::Cas9-U6::sgUPRT质粒上的gRNA,构建pSAG1::Cas9-U6::sgrop16质粒。此外将rop16上游序列、乙胺嘧啶抗性基因、rop16下游序列3个片段连接成donorDNA,克隆于pUC19质粒上,PCR扩增donor DNA片段。pSAG1::Cas9-U6::sgrop16质粒和donor DNA片段电穿孔转染弓形虫,电转后悬液接种于HFF-1细胞中,3μmol/L乙胺嘧啶筛选电转后的虫株。PCR和Western blotting鉴定克隆化筛选虫株。吉姆萨染色分别比较RH株和RHΔrop16株对HFF-1细胞的增殖与入侵。并比较RH株和RHΔrop16株分别感染昆明小鼠后小鼠的生存和死亡率。结果经测序比对,成功构建了pSAG1::Cas9-U6::sgrop16质粒和pUC19-donorDNA质粒。PCR鉴定结果显示,DHFR编码(编码乙胺嘧啶抗性基因)序列成功插入至靶点位置,Western blotting分析结果未见RHΔrop16株有Rop16_(I/III)蛋白表达。吉姆萨染色后计数结果表明,RH株感染的细胞内每个纳虫泡内速殖子的平均数显著高于RHΔrop16虫株。毒力试验结果显示,RH株感染的小鼠在第7d即出现死亡,而rop16_(I/III)缺陷株在第9d出现死亡,但两种弓形虫株感染动物在第10d均全部死亡,两组间无统计学差异。结论利用CRISPR-Cas9技术成功构建了rop16_(I/III)缺陷的弓形虫RH虫株,rop16_(I/III)基因敲除对弓形虫RH株毒力无明显影响。
Toxoplasmagondii RHrop16_(I/III)deficient strain was constructed based on CRSPR/Cas9 technology.E-CRISP database was used to design gRNA;mutation of gRNA in pSAG1::Cas9-U6::sgUPRT plasmid was performed by using sitedirected mutagenesis to construct pSAG1::Cas9-U6::sgrop16plasmid.The pUC19-donorDNA plasmid was constructed and fragments were amplified by PCR.The plasmid pSAG1:Cas9-U6:sgrop16_(I/III)and donor DNA fragments were electroporated into T.gondii RH strain.Follow electroporation,suspension was inoculated into human foreskin fibroblast cells(HFF-1).The3μmol/L pyrimethamine was used to screen the electroporated parasite and monoclone was detected by PCR and Western blotting. The proliferation and invasion of RHΔrop16strain were observed in HFF-1cells by Giemsa staining.Twenty KM mice were infected with 200 tachyzoite of wild type or RHΔrop16 parasite,respectively.The animal survival was recorded.The results showed that pSAG1:Cas9-U6:sgrop16plasmid and pUC19-donor DNA plasmid were successfully constructed and confirmed by DNA sequencing.PCR identification proved that DHFR coding sequence was successfully inserted to the target position.Western blotting analysis revealed deficient expression of ROP16 in the RHΔrop16strain.Giemsa staining indicated that the number of parasites per parasite phorous vacuole of wild type Toxoplasma-infected cells was more than that of the RHΔrop16infected cells.Virulence examination indicated that,the wild type strain infected mice began to die on day 7post-infection where asΔrop16_(I/III)strain,on day 9.However,all animals infected with both strains died on day 10post-infection.TheΔrop16_(I/III)strain of T.gondii was successfully constructed by the CRISPR-Cas9 technology and no difference was noted between wild type and RHΔrop16strain in their virulence to mice.
Toxoplasmagondii RHrop16_(I/III)deficient strain was constructed based on CRSPR/Cas9 technology.E-CRISP database was used to design gRNA;mutation of gRNA in pSAG1::Cas9-U6::sgUPRT plasmid was performed by using sitedirected mutagenesis to construct pSAG1::Cas9-U6::sgrop16plasmid.The pUC19-donorDNA plasmid was constructed and fragments were amplified by PCR.The plasmid pSAG1:Cas9-U6:sgrop16_(I/III)and donor DNA fragments were electroporated into T.gondii RH strain.Follow electroporation,suspension was inoculated into human foreskin fibroblast cells(HFF-1).The3μmol/L pyrimethamine was used to screen the electroporated parasite and monoclone was detected by PCR and Western blotting. The proliferation and invasion of RHΔrop16strain were observed in HFF-1cells by Giemsa staining.Twenty KM mice were infected with 200 tachyzoite of wild type or RHΔrop16 parasite,respectively.The animal survival was recorded.The results showed that pSAG1:Cas9-U6:sgrop16plasmid and pUC19-donor DNA plasmid were successfully constructed and confirmed by DNA sequencing.PCR identification proved that DHFR coding sequence was successfully inserted to the target position.Western blotting analysis revealed deficient expression of ROP16 in the RHΔrop16strain.Giemsa staining indicated that the number of parasites per parasite phorous vacuole of wild type Toxoplasma-infected cells was more than that of the RHΔrop16infected cells.Virulence examination indicated that,the wild type strain infected mice began to die on day 7post-infection where asΔrop16_(I/III)strain,on day 9.However,all animals infected with both strains died on day 10post-infection.TheΔrop16_(I/III)strain of T.gondii was successfully constructed by the CRISPR-Cas9 technology and no difference was noted between wild type and RHΔrop16strain in their virulence to mice.
引文
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[2]Schmidt M,Sonneville R,Schnell D,et al.Clinical features and outcomes in patients with disseminated toxoplasmosis admitted to intensive care:a multicenter study[J].Clin Infect Dis,2013,57(11):1535-1541.DOI:10.1093/cid/cit557
[3]Yamamoto M,Standley DM,Takashima S,et al.A single polymorphic amino acid on Toxoplasma gondii kinase ROP16determines the direct and strain-specific activation of Stat3[J].J Exp Med,2009,206(12):2747-2760.DOI:10.1084/jem.20091703
[4]Lehmann T,Marcet PL,Graham DH,et al.Globalization and the population structure of Toxoplasma gondii[J].Proc Nat Acad Sci USA,2006,103(30):11423-11428.DOI:10.1073/pnas.0601438103
[5]Esvelt KM,Wang HH.Genome-scale engineering for systems and synthetic biology[J].Mol Syst Biol,2013,9:641.DOI:10.1038/msb.2012.66
[6]Urnov FD,Miller JC,Lee YL,et al.Highly efficient endogenous human gene correction using designed zinc-finger nucleases[J].Nature,2005,435(7042):646-651.DOI:10.1038/nature03556
[7]Reyon D,Tsai SQ,Khayter C,et al.FLASH assembly of TALENs enables high-throughput genome editing[J].Nat Biotechnol,2012,30(5):460-465.DOI:10.1038/nbt.2170
[8]Hsu PD,Lander ES,Zhang F.Development and applications of CRISPR-Cas9for genome engineering[J].Cell,2014,157(6):1262-1278.DOI:10.1016/j.cell.2014.05.010
[9]Mussolino C,Cathomen T.RNA guides genome engineering[J].Nat Biotechnol,2013,31(3):208-209.DOI:10.1038/nbt.2527
[10]Irion U,Krauss J,Nusslein-Volhard C.Precise and efficient genome editing in zebrafish using the CRISPR/Cas9system[J].Development,2014,141(24):4827-4830.DOI:10.1242/dev.115584
[11]Sidik SM,Hackett CG,Tran F,et al.Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9[J].PLoS One,2014,9(6):e100450.DOI:10.1371/journal.pone.0100450.eCollection 2014