应用基于重组慢病毒的CRISPR/Cas9技术构建基因突变的鸡DF1细胞
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摘要
为证实基于慢病毒的CRISPR/Cas9基因编辑技术可用于鸡基因组编辑,以鸡的mavs基因为靶基因,通过设计sgRNA,构建表达靶向mavs基因sgRNA的重组慢病毒,并将其感染DF1-Cas9细胞,通过T7E1酶切试验以及mavs基因的靶向序列测定,分析该方法是否可以用于鸡细胞的基因编辑。扩增sgRNA靶向序列1 220 bp片段,经T7E1酶切琼脂糖凝胶分析可见约300 bp和900 bp大小的条带,表明感染sgRNA慢病毒的细胞mavs产生了突变。序列测定结果表明sgRNA靶向序列发生了多个碱基缺失的突变。以上结果表明基于慢病毒系统的CRISPR/Cas9技术可对鸡基因组进行编辑。
In order to confirm that CRISPR/Cas9 gene editing technology based on lentivirus could be applied to the editing of chicken genome, this study selected the mavs gene as the target, designed sgRNA, constructed the recombined lentivirus expressing sgRNA of mavs, and infected the DF1-Cas9 cells.T7 E1 enzyme digestion test and mavs gene targeting sequencing methods were used to analyze whether the technology can be used for gene editing of chicken cells.The targeting fragment of 1 220 bp was amplified, and two bands about 300 bp and 900 bp were observed by T7 E1 digestion and agarose gel analysis, indicating that the cells infected with the lentivirus were mutated.Sequence analysis revealed that multiple base deletion mutations were present in the targeting sequences, indicating that CRISPR/Cas9 technology based on lentivirus system could be used to edit the chicken genome.
In order to confirm that CRISPR/Cas9 gene editing technology based on lentivirus could be applied to the editing of chicken genome, this study selected the mavs gene as the target, designed sgRNA, constructed the recombined lentivirus expressing sgRNA of mavs, and infected the DF1-Cas9 cells.T7 E1 enzyme digestion test and mavs gene targeting sequencing methods were used to analyze whether the technology can be used for gene editing of chicken cells.The targeting fragment of 1 220 bp was amplified, and two bands about 300 bp and 900 bp were observed by T7 E1 digestion and agarose gel analysis, indicating that the cells infected with the lentivirus were mutated.Sequence analysis revealed that multiple base deletion mutations were present in the targeting sequences, indicating that CRISPR/Cas9 technology based on lentivirus system could be used to edit the chicken genome.
引文
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[3] JOUNG J, KONERMANN S, ABUDAYYEH O O, et al.Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening[J].Nat Protoc, 2017, 12(4): 828-863.
[4] PLATT R J, CHEN S, ZHOU Y, et al.CRISPR-Cas9 knockin mice for genome editing and cancer modeling[J]. Cell, 2014,159(2): 440-455.
[5] WANG C, JIN H J, GAO D M,et al.A CRISPR screen identifies CDK7 as a therapeutic target in hepatocellular carcinoma[J]. Cell Res, 2018,28(6): 690-692.
[6] HEATON B E, KENNEDY E M, DUMM R E, et al.A CRISPR activation screen identifies a pan-avian influenza virus inhibitory host factor[J].Cell Rep, 2017, 20(7): 1503-1512.
[7] ZUO Q, WANG Y, CHENG S, et al.Site-directed genome knockout in chicken cell line and embryos can use CRISPR/Cas gene editing technology[J].G3 (Bethesda), 2016, 6(6): 1787-1792.
[8] VéRON N, NADEGE Q, ZHENG D,et al.CRISPR mediated somatic cell genome engineering in the chicken[J].Developmental biology, 2015,407(1): 68-74.
[9] 左其生,王颖洁,赵瑞丰,等.CRISPR/Cas技术可有效介导家鸡基因敲除[J].畜牧兽医学报, 2016,47(6): 126-131.
[10] TRABALZA A, ELEFTHERIADOU I, SGOUROU A, et al.Enhanced central nervous system transduction with lentiviral vectors pseudotyped with RVG/HIV-1gp41 chimeric envelope glycoproteins[J].J Virol, 2014,88(5): 2877-2890.
[11] ALFRANCA A, CAMPANERO M, REDONDO J.New methods for disease modeling using lentiviral vectors[J].Trends Mol Med, 2018, 24(10):1-13.
[12] 褚波,黄雪峰.慢病毒载体及其应用进展[J].生物医学工程学杂志, 2008, 25(1): 224-227.
[13] KATO H, TAKEUCHI O, SATO S, et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses[J].Nature, 2006,441(7089): 101-105.
[14] BELGNAOUI S M, PAZ S,HISCOTT J.Orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (MAVS) adapter[J].Curr Opin Immunol, 2011, 23(5): 564-572.
[15] SETH R B, SUN L, EA C K, et al.Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF3[J].Cell, 2005, 122(5): 669-682.
[16] KIM H S, LEE K, BAE S, et al.CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection[J]. J Biol Chem, 2017,292(25): 10664-10671.
[17] ROOT D E, DAVID E, HACOHEN N, et al.Genome-scale loss-of-function screening with a lentiviral RNAi library[J]. Nature methods, 2006,3(9):715-719.
[18] WANG T, WEI J J, SABATINI D M, et al.Genetic screens in human cells using the CRISPR-Cas9 system[J]. Science, 2014,343(6166): 80-84.