CRISPR/Cas9技术的应用性研究

doi:10.11843/j.issn.0366-6964.2016.07.003

Abstract

Abstract:

To investigate new application of CRISPR/Cas9 technology，we established a method to study genes function in living organism by the application of CRISPR/Cas9 in genetic manipulation.By using in vivo electroporation，immunohistochemistry and PCR analyzing，we first validated the function of DCX and the results indicated that CRISPR/Cas9 could be used in down-regulated gene expression in mouse genetic manipulation.Then this method was also used to study the function of constant exons of Pcdhα cluster. To further clarify the mechanism that led to the phenotype of neuron migration defects by DCX，we designed experiments to identify the genotype of target genes.The results indicated that CRISPR/Cas9 could lead to deletion，inversion and duplication of target genes.In conclusion，CRISPR/Cas9 technology could be used in mouse genetic manipulation and edit the target genes efficiently，which might be an ideal method to research gene function in living organism.

Key words: CRISPR/Cas9, genetic manipulation, deletion, inversion, duplication

CLC Number:

S813.3

SHU Lei-lei，JIA Zhi-lian，WU Yong-hu，SUO Lun，JIA Li-ling. The Application Research of CRISPR/Cas9[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(7): 1316-1323.

References

［1］刘志国.CRISPR/Cas9系统介导基因组编辑的研究进展［J］.畜牧兽医学报，2014，45(10)：1567-1583.
LIU Z G.Research progress on CRISPR/Cas9 mediated genome editing［J］.Acta Veterinaria et Zootechnica Sinica，2014，45(10):1567-1583.（in Chinese）
［2］SAPIR T，ELBAUM M，REINER O.Reduction of microtubule catastrophe events by LIS1，platelet-activating factor acetylhydrolase subunit［J］.EMBO J，1997,16(23)：6977-6984.
［3］GLEESON J G，LIN P T，FLANAGAN L A，et al.Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons［J］.Neuron，1999,23(2)：257-271.
［4］LOTURCO J.Doublecortin and a tale of two serines［J］.Neuron，2004,41(2)：175-177.
［5］CHEN W V，ALVAREZ F J，LEFEBVRE J L，et al.Functional significance of isoform diversification in the protocadherin gamma gene cluster［J］.Neuron，2012,75(3)：402-409.
［6］WANG X，WEINER J A，LEVI S，et al.Gamma protocadherins are required for survival of spinal interneurons［J］.Neuron，2002,36(5)：843-854.
［7］PRASAD T，WANG X，GRAY P A，et al.A differential developmental pattern of spinal interneuron apoptosis during synaptogenesis：insights from genetic analyses of the protocadherin-gamma gene cluster［J］.Development，2008,135(24)：4153-4164.
［8］SUO L，LU H，YING G，et al.Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase［J］.J Mol Cell Biol，2012,4(6)：362-376.
［9］LEDDEROSE J，DIETER S，SCHWARZ M K.Maturation of postnatally generated olfactory bulb granule cells depends on functional gamma-protocadherin expression［J］.Sci Rep，2013,3：1514.
［10］WEINER J A，WANG X，TAPIA J C，et al.Gamma protocadherins are required for synaptic development in the spinal cord［J］.Proc Natl Acad Sci U S A，2005,102(1)：8-14.
［11］GARRETT A M，WEINER J A.Control of CNS synapse development by {gamma}-protocadherin-mediated astrocyte-neuron contact［J］.J Neurosci，2009,29(38)：11723-11731.
［12］JUNGHANS D，HEIDENREICH M，HACK I，et al.Postsynaptic and differential localization to neuronal subtypes of protocadherin beta16 in the mammalian central nervous system［J］.Eur J Neurosci，2008,27(3)：559-571.
［13］LEFEBVRE J L，KOSTADINOV D，CHEN W V，et al.Protocadherins mediate dendritic self-avoidance in the mammalian nervous system［J］.Nature，2012,488(7412)：517-521.
［14］KATORI S，HAMADA S，NOGUCHI Y，et al.Protocadherin-alpha family is required for serotonergic projections to appropriately innervate target brain areas［J］.J Neurosci，2009,29(29)：9137-9147.
［15］HASEGAWA S，HAMADA S，KUMODE Y，et al.The protocadherin-alpha family is involved in axonal coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb in mouse［J］.Mol Cell Neurosci，2008,38(1)：66-79.
［16］LI J，SHOU J，GUO Y，et al.Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9［J］.J Mol Cell Biol，2015,7(4)：284-298.
［17］HORESH D，SAPIR T，FRANCIS F，et al.Doublecortin，a stabilizer of microtubules［J］.Hum Mol Genet，1999,8(9)：1599-1610.
［18］BAI J，RAMOS R L，ACKMAN J B，et al.RNAi reveals doublecortin is required for radial migration in rat neocortex［J］.Nat Neurosci，2003,6(12)：1277-1283.
［19］CHEN W V，MANIATIS T.Clustered protocadherins［J］.Development，2013,140(16)：3297-3302.

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The Application Research of CRISPR/Cas9

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