利用CRISPR/Cas9对基因组中高度同源DNA片段编辑多样性的遗传学研究
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摘要
在基因组中,编码区存在许多高度相似的基因簇或基因群(多拷贝基因),非编码区也存在大量的重复序列。这些重复序列能通过改变染色体的三维结构调控基因的转录,对于生物体的遗传与进化起到了重要的作用。其高度同源的特征使得利用CRISPR/Cas9技术进行基因组编辑时面临更加复杂的状况。如果编辑的片段是二倍体或多倍体,还会产生各条染色单体上的编辑情况不相同的现象。为此本文选择了2个位于同一染色体相距11 kb的高度同源300 bp片段(L1和L2)进行CRISPR介导的DNA片段编辑。采用一对sgRNA(分别共同靶向两片段的上、下游位点)引导Cas9对HepG2细胞两个高度相似的DNA片段进行切割。片段编辑的细胞进一步单克隆化后,对获得的22个L1/L2编辑的CRISPR单克隆细胞株进行详细的基因型鉴定。结果发现除了这两个DNA片段本身被删除外,它们之间的大片段也存在被删除的现象,三个片段的各种反转组合也很频繁。该研究结果对于采用CRISPR/Cas9系统编辑多拷贝基因或重复序列,尤其是对二倍体或多倍体生物进行基因组编辑时具有重要的借鉴和参考价值。
In complex genomes, there are a large number of duplicated genes in the coding regions and many more repetitive sequences in the non-coding regions. Repetitive sequences can exert great impacts on the heredity and evolution of the organisms, as well as their genome 3D architecture and transcriptional regulation. The high homology nature of repetitive sequences renders their editing by CRISPR/Cas9 very complex. At diploid or polyploid situations,such repetitive sequences could be edited differently on each chromosome or chromatid. To explore such possibilities,we had studied the editing of two highly homologous DNA fragments(L1 and L2), each about 300 bp in size and 11 kb apart on the same chromosome. We designed a pair of sgRNAs targeting the upstream and downstream of the two DNA fragments to guide the Cas9 cleavage of the two fragments in the human HepG2 cells. We further established single-cell CRISPR clones for DNA-fragment-edited cells. A total of 22 CRISPR cell clones were characterized for their DNA fragment editing patterns. In addition to the deletion of L1/L2 fragments, we had also identified the deletion of the large internal fragment between L1 and L2 fragments, and the various combinations of inversions and deletions of the three DNA fragments. Our results have demonstrated the potential issues with important implications for using CRISPR/Cas9 to edit duplicated genes or repetitive sequences in diploid or polyploid species or cell lines.
In complex genomes, there are a large number of duplicated genes in the coding regions and many more repetitive sequences in the non-coding regions. Repetitive sequences can exert great impacts on the heredity and evolution of the organisms, as well as their genome 3D architecture and transcriptional regulation. The high homology nature of repetitive sequences renders their editing by CRISPR/Cas9 very complex. At diploid or polyploid situations,such repetitive sequences could be edited differently on each chromosome or chromatid. To explore such possibilities,we had studied the editing of two highly homologous DNA fragments(L1 and L2), each about 300 bp in size and 11 kb apart on the same chromosome. We designed a pair of sgRNAs targeting the upstream and downstream of the two DNA fragments to guide the Cas9 cleavage of the two fragments in the human HepG2 cells. We further established single-cell CRISPR clones for DNA-fragment-edited cells. A total of 22 CRISPR cell clones were characterized for their DNA fragment editing patterns. In addition to the deletion of L1/L2 fragments, we had also identified the deletion of the large internal fragment between L1 and L2 fragments, and the various combinations of inversions and deletions of the three DNA fragments. Our results have demonstrated the potential issues with important implications for using CRISPR/Cas9 to edit duplicated genes or repetitive sequences in diploid or polyploid species or cell lines.
引文
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[8]Coufal NG,Garcia-Perez JL,Peng GE,Yeo GW,Mu YL,Lovci MT,Morell M,O'Shea KS,Moran JV,Gage FH.L1retrotransposition in human neural progenitor cells.Nature,2009,460(7259):1127-1131.
[9]Gerstein MB,Bruce C,Rozowsky JS,Zheng DY,Du J,Korbel JO,Emanuelsson O,Zhang ZD,Weissman S,Snyder M.What is a gene,post-ENCODE?History and updated definition.Genome Res,2007,17(6):669-681.
[10]Shapiro JA,Von Sternberg R.Why repetitive DNA is essential to genome function.Biol Rev Camb Philos Soc,2005,80(2):227-250.
[11]Cournac A,Koszul R,Mozziconacci J.The 3D folding of metazoan genomes correlates with the association of similar repetitive elements.Nucleic Acids Res,2016,44(1):245-255.
[12]Huang HY,Wu Q.CRISPR double cutting through the labyrinthine architecture of 3D genomes.J Genet Genomics,2016,43(5):273-288.
[13]Ong CT,Corces VG.CTCF:an architectural protein bridging genome topology and function.Nat Rev Genet,2014,15(4):234-246.
[14]Guo Y,Xu Q,Canzio D,Shou J,Li JH,Gorkin DU,Jung I,Wu HY,Zhai YN,Tang YX,Lu YC,Wu YH,Jia ZL,Li W,Zhang MQ,Ren B,Krainer AR,Maniatis T,Wu Q.CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function.Cell,2015,162(4):900-910.
[15]Kim TH,Abdullaev ZK,Smith AD,Ching KA,Loukinov DI,Green RD,Zhang MQ,Lobanenkov VV,Ren B.Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome.Cell,2007,128(6):1231-1245.
[16]Schmidt D,Schwalie PC,Wilson MD,Ballester B,Gon?alves?,Kutter C,Brown GD,Marshall A,Flicek P,Odom DT.Waves of retrotransposon expansion remodel genome organization and CTCF binding in multiple mammalian lineages.Cell,2012,148(1-2):335-348.
[17]Monahan K,Rudnick ND,Kehayova PD,Pauli F,Newberry K M,Myers RM,Maniatis T.Role of CCCTC binding factor(CTCF)and cohesin in the generation of single-cell diversity of protocadherin-αgene expression.Proc Natl Acad Sci USA,2012,109(23):9125-9130.
[18]Guo Y,Monahan K,Wu HY,Gertz J,Varley KE,Li W,Myers RM,Maniatis T,Wu Q.CTCF/cohesin-mediated DNA looping is required for protocadherinαpromoter choice.Proc Natl Acad Sci USA,2012,109(51):21081-21086.
[19]Zhai YN,Xu Q,Guo Y,Wu Q.Characterization of a cluster of CTCF-binding sites in a protocadherin regulatory region.Hereditas(Beijing),2016,38(4):323-336.翟亚男,许泉,郭亚,吴强.原钙粘蛋白基因簇调控区域中成簇的CTCF结合位点分析.遗传,2016,38(4):323-336.
[20]Li JH,Shou J,Guo Y,Tang YX,Wu YH,Jia ZL,Zhai YN,Chen ZF,Xu Q,Wu Q.Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.J Mol Cell Biol,2015,7(4):284-298.
[21]Li JH,Shou J,Wu Q.DNA fragment editing of genomes by CRISPR/Cas9.Hereditas(Beijing),2015,37(10):992-1002.李金环,寿佳,吴强.CRISPR/Cas9系统在基因组DNA片段编辑中的应用.遗传,2015,37(10):992-1002.
[22]Zhang T,Haws P,Wu Q.Multiple variable first exons:a mechanism for cell-and tissue-specific gene regulation.Genome Res,2004,14(1):79-89.
[23]Li C,Wu Q.Adaptive evolution of multiple-variable exons and structural diversity of drug-metabolizing enzymes.BMC Evol Biol,2007,7:69.
[24]Huang HY,Wu Q.Cloning and comparative analyses of the zebrafish UGT repertoire reveal its evolutionary diversity.PLo S One,2010,5(2):e9144.
[25]Wang YM,Huang HY,Wu Q.Characterization of the zebrafish UGT repertoire reveals a new class of drug-metabolizing UDP glucuronosyltransferases.Mol Pharmacol,2014,86(1):62-75.
[26]Yang J,Cai L,Huang HY,Liu B,Wu Q.Genetic variations and haplotype diversity of the UGT1 gene cluster in the Chinese population.PLo S One,2012,7(4):e33988.
[27]Deltcheva E,Chylinski K,Sharma CM,Gonzales K,Chao YJ,Pirzada ZA,Eckert MR,Vogel J,Charpentier E.CRISPR RNA maturation by trans-encoded small RNAand host factor RNase III.Nature,2011,471(7340):602-607.
[28]Garneau JE,Dupuis Mè,Villion M,Romero DA,Barrangou R,Boyaval P,Fremaux C,Horvath P,Magadán AH,Moineau S.The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.Nature,2010,468(7320):67-71.
[29]Li TM,Du B.CRISPR-Cas system and coevolution of bacteria and phages.Hereditas(Beijing),2011,33(3):213-218.李铁民,杜波.CRISPR-Cas系统与细菌和噬菌体的共进化.遗传,2011,33(3):213-218.
[30]Cong L,Ran FA,Cox D,Lin SL,Barretto R,Habib N,Hsu PD,Wu XB,Jiang WY,Marraffini LA,Zhang F.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339(6121):819-823.
[31]Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna JA,Charpentier E.A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[32]Mali P,Yang LH,Esvelt KM,Aach J,Guell M,Di Carlo JE,Norville JE,Church GM.RNA-guided human genome engineering via Cas9.Science,2013,339(6121):823-826.
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