CRISPR/Cas9系统介导果蝇miR-2基因簇的定点敲除
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摘要
microRNAs可作为肿瘤抑制因子或促进因子发挥作用,其表达与肿瘤的发生发展密切相关.miR-2基因家族成簇表达,是果蝇基因组中编码microRNA的最大的基因家族,但其功能不确定.为了研究miR-2基因家族的功能,本研究利用CRISPR/Cas9系统,成功敲除了miR-2家族位于2号染色体左臂(2L)的基因簇(ΔmiR-2),并初步确定了ΔmiR-2可以抑制促癌因子Yki活性,为研究miR-2家族的功能奠定了基础.
MicroRNAs function as tumor suppressors or promoters during tumor progression. The largest Drosophila microRNA gene family miR-2 consists of 8 members and 6 of them form two gene clusters on different chromosomes. However, the function of this gene family is unknown. In this study, we generated a specific genome deletion of miR-2 cluster located on chromosome 2 L using the CRISPR/Cas9 system in Drosophila. This knockout strain(ΔmiR-2) genetically suppress the activity of oncogenic factor Yki, indicating ΔmiR-2 will facilitate the functional study of miR-2 in the future.
MicroRNAs function as tumor suppressors or promoters during tumor progression. The largest Drosophila microRNA gene family miR-2 consists of 8 members and 6 of them form two gene clusters on different chromosomes. However, the function of this gene family is unknown. In this study, we generated a specific genome deletion of miR-2 cluster located on chromosome 2 L using the CRISPR/Cas9 system in Drosophila. This knockout strain(ΔmiR-2) genetically suppress the activity of oncogenic factor Yki, indicating ΔmiR-2 will facilitate the functional study of miR-2 in the future.
引文
1 Karginov F V, Hannon G J. The CRISPR system:small RNA-guided defense in bacteria and archaea[J]. Molecular Cell, 2010, 37(1):7-19.
2 Garneau J E, Dupuis Mè, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J]. Nature, 2010, 468(7 320):67-71.
3 Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea.[J]. Science, 2010, 327(5 962):167-170.
4 Bolotin A, Quinquis B, Sorokin A, et al. Clustered regularly interspaced short palindrome repeats(CRISPRs)have spacers of extrachromosomal origin.[J]. Microbiology, 2005, 151(Pt8):2 551-2 561.
5 Barendregt A, Zhou K. Structural basis for CRISPR RNA-guided DNA recognition by Cascade.[J]. Nature Structural&Molecular Biology, 2011, 18(5):529-536.
6 Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(39):2 579-2 586.
7 Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6 096):816-821.
8 Djuranovic S, Nahvi A, Green R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay[J]. Science, 2012, 336(6 078):237-240.
9 Shenouda S K, Alahari S K. MicroRNA function in cancer:oncogene or a tumor suppressor?[J]. Cancer Metastasis Rev, 2009, 28(3/4):369-378.
10 Dinami R, Ercolani C, Petti E, et al. miR-155 drives telomere fragility in human breast cancer by targeting TRF1[J]. Cancer Res, 2014, 74(15):4 145-4 156.
11 Li L, Li C, Wang S, et al. Exosomes derived from hypoxic oral squamous cell carcinoma cells deliver miR-21to normoxic cells to elicit a prometastatic phenotype[J]. Cancer Res, 2016, 76(7):1 770-1 780.
12 Lagosquintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs[J]. Science, 2001, 294(5 543):853-858.
13 Lau N C, Lim L P, Weinstein E G, et al. An abundant class of tiny RNAs with probable regulatory roles in caenorhabditis elegans[J]. Science, 2001, 294(5 543):858-862.
14 Lee R C, Ambros V. An extensive class of small RNAs in caenorhabditis elegans.[J]. Science, 2001, 294(5 543):862-864.
15 Ruby J G, Stark A, Johnston W K, et al. Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs[J]. Genome Research, 2007, 17(12):1 850-1 864.
16 Liu C, Kelnar K, Vlassov A V, et al. Distinct microRNA expression profiles in prostate cancer stem/progenitor cells and tumor-suppressive functions of let-7[J]. Cancer Res, 2012, 72(13):3 393-3 404.
17 Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs[J]. Science, 2001, 294(5 543):853-858.
18 Lau N C, Lim L P, Weinstein E G, et al. An abundant class of tiny RNAs with probable regulatory roles in caenorhabditis elegans[J]. Science, 2001, 294(5 543):858-862.
19 Lee R C, Ambros V. An extensive class of small RNAs in caenorhabditis elegans[J]. Science, 2001, 294(5 543):862-864.
20 Mondal T, Lavanya A V S, Mallick Akash. Novel Triazole linked 2-phenyl benzoxazole derivatives induce apoptosis by inhibiting miR-2, miR-13 and miR-14 function in drosophila melanogaster[J]. Apoptosis, 2017, 22(6):786-799.
21 Lozano J, Montanez R, Belles X. MiR-2 family regulates insect metamorphosis by controlling the juvenile hormone signaling pathway[J]. Proc Natl Acad Sci U S A, 2015, 112(12):3 740-3 745.
22 Lin L,Xie G,Li Z Q. MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori[J].RNA Biol, 2015, 12(7):742-748.
23 Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6 096):816-821.
24 Andrew R Bassett, Tibbit C, Chris P Ponting, et al. Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system[J]. Cell Reports, 2013, 4(1):220-228.
25 Ren X, Sun J, Housden B E, et al. Optimized gene editing technology for drosophila melanogaster using germ line-specific Cas9[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013,110(47):19 012-19 017.
26 李珊珊,林晓惠,吴世安. P因子介导mir-2基因簇原位过表达调节Yki的活性分析[J].南开大学学报:自然科学版,2017,50(3):54-59.
27 Cong L, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013,339(6 121):819-823.
28 Mali P, Yang L, Esvelt K M, et al. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339(6 121):823-826.
29 Auer T O, Duroure K, De C A, et al. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair[J]. Genome Research, 2014, 24(1):142-153.
30 Yin L, Maddison L A, Li M, et al. Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs[J]. Genetics, 2015, 200(2):431-441.
31 Yu Z, Ren M, Wang Z, et al. Highly efficient genome modifications mediated by CRISPR/Cas9 in drosophila[J]. Genetics, 2013, 195(1):289-291.
32 Ren X, Sun J, Housden B E, et al. Optimized gene editing technology for drosophila melanogaster using germ line-specific Cas9[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013,110(47):19 012-19 017.
33 周金伟,徐绮嫔,姚婧,等. CRISPR/Cas9基因组编辑技术及其在动物基因组定点修饰中的应用[J].遗传,2015,37(10):1 011-1 020.
2 Garneau J E, Dupuis Mè, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J]. Nature, 2010, 468(7 320):67-71.
3 Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea.[J]. Science, 2010, 327(5 962):167-170.
4 Bolotin A, Quinquis B, Sorokin A, et al. Clustered regularly interspaced short palindrome repeats(CRISPRs)have spacers of extrachromosomal origin.[J]. Microbiology, 2005, 151(Pt8):2 551-2 561.
5 Barendregt A, Zhou K. Structural basis for CRISPR RNA-guided DNA recognition by Cascade.[J]. Nature Structural&Molecular Biology, 2011, 18(5):529-536.
6 Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(39):2 579-2 586.
7 Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6 096):816-821.
8 Djuranovic S, Nahvi A, Green R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay[J]. Science, 2012, 336(6 078):237-240.
9 Shenouda S K, Alahari S K. MicroRNA function in cancer:oncogene or a tumor suppressor?[J]. Cancer Metastasis Rev, 2009, 28(3/4):369-378.
10 Dinami R, Ercolani C, Petti E, et al. miR-155 drives telomere fragility in human breast cancer by targeting TRF1[J]. Cancer Res, 2014, 74(15):4 145-4 156.
11 Li L, Li C, Wang S, et al. Exosomes derived from hypoxic oral squamous cell carcinoma cells deliver miR-21to normoxic cells to elicit a prometastatic phenotype[J]. Cancer Res, 2016, 76(7):1 770-1 780.
12 Lagosquintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs[J]. Science, 2001, 294(5 543):853-858.
13 Lau N C, Lim L P, Weinstein E G, et al. An abundant class of tiny RNAs with probable regulatory roles in caenorhabditis elegans[J]. Science, 2001, 294(5 543):858-862.
14 Lee R C, Ambros V. An extensive class of small RNAs in caenorhabditis elegans.[J]. Science, 2001, 294(5 543):862-864.
15 Ruby J G, Stark A, Johnston W K, et al. Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs[J]. Genome Research, 2007, 17(12):1 850-1 864.
16 Liu C, Kelnar K, Vlassov A V, et al. Distinct microRNA expression profiles in prostate cancer stem/progenitor cells and tumor-suppressive functions of let-7[J]. Cancer Res, 2012, 72(13):3 393-3 404.
17 Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs[J]. Science, 2001, 294(5 543):853-858.
18 Lau N C, Lim L P, Weinstein E G, et al. An abundant class of tiny RNAs with probable regulatory roles in caenorhabditis elegans[J]. Science, 2001, 294(5 543):858-862.
19 Lee R C, Ambros V. An extensive class of small RNAs in caenorhabditis elegans[J]. Science, 2001, 294(5 543):862-864.
20 Mondal T, Lavanya A V S, Mallick Akash. Novel Triazole linked 2-phenyl benzoxazole derivatives induce apoptosis by inhibiting miR-2, miR-13 and miR-14 function in drosophila melanogaster[J]. Apoptosis, 2017, 22(6):786-799.
21 Lozano J, Montanez R, Belles X. MiR-2 family regulates insect metamorphosis by controlling the juvenile hormone signaling pathway[J]. Proc Natl Acad Sci U S A, 2015, 112(12):3 740-3 745.
22 Lin L,Xie G,Li Z Q. MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori[J].RNA Biol, 2015, 12(7):742-748.
23 Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6 096):816-821.
24 Andrew R Bassett, Tibbit C, Chris P Ponting, et al. Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system[J]. Cell Reports, 2013, 4(1):220-228.
25 Ren X, Sun J, Housden B E, et al. Optimized gene editing technology for drosophila melanogaster using germ line-specific Cas9[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013,110(47):19 012-19 017.
26 李珊珊,林晓惠,吴世安. P因子介导mir-2基因簇原位过表达调节Yki的活性分析[J].南开大学学报:自然科学版,2017,50(3):54-59.
27 Cong L, Ran F A, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013,339(6 121):819-823.
28 Mali P, Yang L, Esvelt K M, et al. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339(6 121):823-826.
29 Auer T O, Duroure K, De C A, et al. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair[J]. Genome Research, 2014, 24(1):142-153.
30 Yin L, Maddison L A, Li M, et al. Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs[J]. Genetics, 2015, 200(2):431-441.
31 Yu Z, Ren M, Wang Z, et al. Highly efficient genome modifications mediated by CRISPR/Cas9 in drosophila[J]. Genetics, 2013, 195(1):289-291.
32 Ren X, Sun J, Housden B E, et al. Optimized gene editing technology for drosophila melanogaster using germ line-specific Cas9[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013,110(47):19 012-19 017.
33 周金伟,徐绮嫔,姚婧,等. CRISPR/Cas9基因组编辑技术及其在动物基因组定点修饰中的应用[J].遗传,2015,37(10):1 011-1 020.
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