靶向PRRSV的FnCas9-rgRNA敲除载体构建研究
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摘要
为了检测靶向猪繁殖与呼吸综合征病毒(PRRSV)的FnCas9-rgRNA敲除载体是否构建成功,试验先构建FnCas9-rgRNA敲除载体骨架并对其进行测序,然后将黄色荧光蛋白(EYFP)、PRRSV分别与各自的FnCas9-rgRNA敲除载体转染至HEK293、Marc145细胞内,通过流式细胞仪分析HEK293细胞内的荧光强弱及Marc145细胞的病变程度,并对FnCas9-rgRNA敲除载体的初步构建、载体活性进行研究。结果表明:FnCas9-rgRNA敲除载体骨架测序正确,流式细胞仪分析显示FnCas9-rgRNA敲除载体对HEK293细胞内EYFP基因的表达产生明显抑制作用,表达荧光的细胞数量明显下降,3种针对PRRSV设计的rgRNA分别与FnCas9表达载体共转染细胞,接种病毒后Marc145细胞病变程度明显降低。说明靶向PRRSV的FnCas9-rgRNA敲除载体构建方式正确且FnCas9-rgRNA敲除载体具有活性。
To detect the construction of FnCas9-rgRNA knockout vector targeting PRRSV, the FnCas9-rgRNA vector skeleton system was set up and sequenced in this experimant. Then, the enhanced yellow fluorescent protein(EYFP) and PRRSV were transfected into HEK293 and Marc145 cells respectively with FnCas9-rgRNA knockout vector, the fluorescence intensity in HEK293 cells and the degree of lesion of Marc145 cells were analyzed by flow cytometry. The preliminary construction of FnCas9-rgRNA knockout vector and its activity was sudied. The results showed that the FnCas9-rgRNA vector skeleton system was constructed correctly.The flow cytometry analysis showed that Fncas9-rgRNA system inhibited the expression of EYFP gene in HEK293 cells significantly, and the number of cells expressing fluorescence decreased. Three rgRNAs designed for PRRSV were co-infectecl with FnCas9 expression vector, and the degree of Marc145 cell lesion decresed after inoculation. It indicats that the FnCas9-rgRNA knockout vector targeting PRRSD is constructed correctly and it is active.
To detect the construction of FnCas9-rgRNA knockout vector targeting PRRSV, the FnCas9-rgRNA vector skeleton system was set up and sequenced in this experimant. Then, the enhanced yellow fluorescent protein(EYFP) and PRRSV were transfected into HEK293 and Marc145 cells respectively with FnCas9-rgRNA knockout vector, the fluorescence intensity in HEK293 cells and the degree of lesion of Marc145 cells were analyzed by flow cytometry. The preliminary construction of FnCas9-rgRNA knockout vector and its activity was sudied. The results showed that the FnCas9-rgRNA vector skeleton system was constructed correctly.The flow cytometry analysis showed that Fncas9-rgRNA system inhibited the expression of EYFP gene in HEK293 cells significantly, and the number of cells expressing fluorescence decreased. Three rgRNAs designed for PRRSV were co-infectecl with FnCas9 expression vector, and the degree of Marc145 cell lesion decresed after inoculation. It indicats that the FnCas9-rgRNA knockout vector targeting PRRSD is constructed correctly and it is active.
引文
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[3] DELA F C, LU T K. CRISPR-Cas9 technology: applications in genome engineering, development of sequence-specific antimicrobials, and future prospects[J]. Integr Biol (Camb),2017, 9(2): 109-122.
[4] MAKAROVA K S, ZHANG F, KOONIN E V. SnapShot: Class 2 CRISPR-Cas Systems[J]. Cell,2017, 168(1/2): 328.
[5] SAMPSON T R, SAROJ S D, LLEWELLYN A C, et al. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence[J]. Nature,2013, 497(7448): 254-257.
[6] PRICE A A, SAMPSON T R, RATNER H K, et al. Cas9-mediated targeting of viral RNA in eukaryotic cells[J]. Proc Natl Acad Sci USA, 2015, 112(19): 6164-6169.
[7] HIRANO H, GOOTENBERG J S, HORII T, et al. Structure and engineering of francisella novicida Cas9[J]. Cell,2016, 164(5): 950-961.
[8] 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.
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[10] WANG M, MAO Y, LU Y, et al. Multiplex gene editing in rice using the CRISPR-Cpf1 system[J]. Mol Plant,2017, 10(7): 1011-1013.
[11] FONFARA I, RICHER H, BRATOVIC M, et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA[J]. Nature,2016, 532(7600): 517-521.
[12] KIM D, KIM J, HUR J K, et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells[J]. Nat Biotechnol,2016, 34(8): 863-868.
[13] GAO P, YANG H, RAJASHANKAR K R, et al. Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition[J]. Cell Res,2016, 26(8): 901-913.