蚂蚁彩票平台 pk10 pk10开奖 pk10 pk10开奖记录 pk10



      CRISPR/Cas9TALENZFNЧDNAЭDNAд (double-strand break, DSB), (Non-homologous end joining, NHEJ) (homologous recombination, HR) 鹤УNEHJв (indel) DNA (HDR) С

    CRISPR/Cas9


CRISPR/Cas9

      CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR associated, /CRISPR) еIICRISPR/Cas9crRNAs (~20nt) DNAУtrans-activating crRNA (tracrRNA) Cas9λγCas9壬жλγ


 


CRISPR/Cas9УcrRNAtracrRNARNA (chiRNAgRNA)gRNANGGNAG PAM (protospacer adjacent motif)gRNA~20 bpУCas9дγ

        CRISPR/Cas9RNA-DNACas9жgRNAλжνоDNAó


flyCRISPR

FlyCas9

CRISPR fly design

D.melanogaster genome assembly 5DRSC Find CRISPR Tool 鷶ΧCRISPRλS2еCRISPR Efficiency Predictor gRNAеЧ

пflyCRISPR Optimal Target Finder DNAgRNAλοжЩλοаD.melanogaster release 5, release 6, С


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Bassett AR, Tibbit C, Ponting CP, Liu JL (2013) Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 4(1): 220
Kondo S, Ueda R (2013) Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195(3): 715

Ren XJ, Sun J, Housden BE, Hu YH, Roesel C, Lin SL, Liu LP, Yang ZH, Mao DC, Sun LZ, Wu QJ, Ji JY, Xi JZ, Mohr SE, Xu J, Perrimon N, Ni JQ (2013) Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. PNAS 110(47): 19012

Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, and O'Connor-Giles KM (2013) Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194: 1029-1035

Gratz SJ, Wildonger J, Harrison MM, and O'Connor-Giles KM (2013) CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand. Fly (Austin) 7

Port F, Chen HM, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. PNAS 111(29): E2967

Port F, Muschalik N, Bullock SL (2015) Systematic evaluation of Drosophila CRISPR tools reveals safe and robust alternatives to autonomous gene drives in basic research. G3 5(7): 1493

Ren X, Yang Z, Xu J, Sun J, Mao D, Hu Y, Yang SJ, Qiao HH, Wang X, Hu Q, Deng P, Liu LP, Ji JY, Li JB, Ni JQ (2014) Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila. Cell Rep 9(3): 1151



CRISPR/Cas9

flyCRISPR

vas-Cas9(X) BDSC#51323 y[1] M{vas-Cas9}ZH2A w[1118] Cas9vasУλZH-2A (2A3) λCas93xP3-GFPattPλ3xP3-RFP RFP+ GFP+
vas-Cas9(X) RFP- BDSC#55821 y[1] M{vas-Cas9.RFP-}ZH-2A w[1118] 51323/51324Cas9BDSC#24480NF - M{vas-int.Dm}ZH-102DloxP-3xP3-RFP GFP+ RFP-
vas-Cas9(II) BDSC#56552 w[1118]; PBac{y[+mDint2]=vas-Cas9}VK00037/CyO, P{w[+mC]=Tb[1]}Cpr[CyO-A] Cas9vasУλVK00037 (22A3) λ Cas93xP3-GFPw+ λCyO y+ GFP+ w+ CyO
vas-Cas9(III) BDSC#51324 w[1118]; PBac{y[+mDint2]=vas-Cas9}VK00027 Cas9vasУλVK00027 (89E11) λ Cas93xP3-GFPw[1118]; PBac{y[+mDint2]=vas-Cas9}VK00027/TM6B, Tb[1] y+ GFP+

 

FlyCas9

nos-Cas9(X) NIG-FLY#CAS-0002 y2 cho2 v1 P{nos-Cas9, y+, v+}1A/FM7c, KrGAL4 UAS-GFP Cas9nosУλX y+ v+ (red eye)
nos-Cas9(II-attP40) NIG-FLY#CAS-0001 y2 cho2 v1; attP40{nos-Cas9}/CyO Cas9nosУλIIattP40 (25C6)λ y+ v+ (v+ , red eye )
yw; nos-Cas9(II-attP40) - y1 w1118; attP40{nos-Cas9}/CyO yw()Cas9nosУ λIIattP40 (25C6)λ y+ CyO (white eye)
nos-Cas9(II-2A) NIG-FLY#CAS-0004 y2 cho2 v1; Sp/CyO, P{nos-Cas9, y+, v+}2A Cas9nosУλIICyO y+ v+ Sp CyO (v+ , red eye)
yw; nos-Cas9(III-attP2) - y1 w1118; attP2{nos-Cas9}/TM6C,Sb Tb yw()Cas9nosУ λIIIattP2 (68A4)λy1 w1118; attP2{nos-Cas9}, y+ (white eye)
nos-Cas9(III-3A) NIG-FLY#CAS-0003 y2 cho2 v1; P{nos-Cas9, y+, v+}3A/TM6C, Sb Tb Cas9nosУλIII y+ v+ TM6C Sb Tb (v+ , red eye)

 

CRISPR fly design

Act5C-Cas9.P aka CFD1 BDSC#54590 y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*] Cas9Act5CУλZH-2A(2A3) w+ RFP+
nos-Cas9.P aka CFD2 BDSC#54591 y[1] M{w[+mC]=nos-Cas9.P}ZH-2A w[*] Cas9nanosСλZH-2A(2A3) w+ RFP+
UAS-Cas9.P aka CFD4 BDSC#54594 P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP40 Cas9UAShsFLPλattP40(25C6) y+ w+
UAS-Cas9.P aka CFD3 BDSC#54592 P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP2/TM6B, Tb[1] Cas9UASСhsFLPλattP2(68A4) y+ w+ TM6B Tb
nos-Gal4/UAS-Cas9.P aka CFD3-nos BDSC#54593 P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC=UAS-Cas9.P}attP2 P{w[+mC]=GAL4::VP16-nos.UTR}CG6325[MVD1] Cas9UAS СGAL4γnos-GAL4hsFLPλattP2(68A4), 86B4 y+ w+
act-Cas9(D10A) (nickase) aka CFD13 -> P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=act-Cas9D10A}attP2 Cas9Act5CСCas9D10ACas9пλattP2(68A4) y+ w+

 

Act5C-Cas9, Lig4

Act5C-Cas9, Lig4 (X) BDSC#58492 y[1] M{Act5C-Cas9.P.RFP-}ZH-2A w[1118] Lig4[169] M{Act5C-Cas9.P}ZH-2Acre/loxPгminiwhite3xP3-RFPAct5C-Cas9λlig4169X RFP-


gRNA

attP-gRNAPhiC31бvermillion+attPG0y-v-v+v+ G2v- attPPlan HPlan Iv+gRNAgRNACas9 F1Plan RG/RH/RI


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