如何用cas9做敲入

Cas9JIEDAOTONGYUANZHONGZU(HDR)CHULEXUYAODUOYINRUYITIAOXIUFUMOBAN(repair template),JIBENCAOZUOHEZUOJIYINQIAOCHU(KO)JIBENYIZHI(Fig 1)。SHUNLIDEQINGKUANGXIAZHENGTAOCAOZUOXIALAIZHISHAOXUYAO4ZHOUSHIJIAN(XINJICHIBUDAOREDOUFULEI~~),QIZHONGDANKELONGDEHUOQUHEJIANDINGSHIZUIDADEXIANSUBUZHOU。

 

WTDECas9 (ZHEILIZHIspCas9)SHIYINQISHUANGLIANDUANLIE(DSB),JINERYINQINHEJ(nonhomologous end joining,YISIJIUSHIDUANLIEDEMODUANZHONGXINLIANQILAI,ZHEIYIGUOCHENGRONGYICHANSHENGindels)HUOZHEHDR(XIUFUMOBANCUNZAI);DANYOUYIGETUBIANTI(D10A)QUEZHIBAOLIULEQIEKOUMEIDEHUOXING(nickase) (spCas9n),YIBANBUHUIYINQINHEJ,DANHUIYINQIHDR(XIUFUMOBANCUNZAI)。DAJIAZHOUZHIWTDEspCas9HUIYOUXIEWEIDETUOBAXIAOYING,YINCILIYONGcas9nDAITIWTDEcas9CHANSHENGdeletion(XUYAOLIANGTIAOsgRNATONGSHIZUOYONG)HUOZHEHDRKEYIJIANGDIYIBUFENTUOBADEXIAOGUO(Fig. 2)。

 

 

 Fig 1. Cas9QIAOCHU/QIAORUJIYINSHIJIANANPAI

 

 

Fig 2. Cas9 (D10A)TUBIANTI(Cas9n)ZHIBAOLIUnickaseHUOXING

 

 

ZHEIYITUBIANTIBUHUIYINQINHJE。RUGUOTONGYOULIANGTIAOsgRNABAXIANGYIGEgeneDEBUTONGWEIDIAN,Cas9nHUIYINQIYIGEPIANDUAN(LIANGGEsgRNAZHIJIAN)DEdeletion。XIUFUMOBANCUNZAIDEQINGKUANGXIAKEYIYINQIHDR。

 

JINTIANDENEIRONGZHUYAOJIANGJIANGLIYONGCas9ZENMEZUOTONGYUANZHONGZU(XIUFU)(HDR)。QISHIJIYINDEQIAORU(KI)YESHIYIMOYIYANGDEZUOFA。

 

 

 如何利用Cas9做同源重组 

 

 

 YI、TONGYUANBEIDESHEJI

 

 HEZUOKOBIJIAO,ZUOHDRZUIDADEBUTONGJIUSHIYAOYOUTONGYUANMOBANDECUNZAI。TONGYUANMOBANYOUYIXIALIANGZHONGLEIXING(Fig. 3):

 

 

1、DANLIANMOBAN:SHOUDONGSHEJIDANCETONGYUANBEIZHANGDUBUDIYU40 nt,DANQIANGLIETUIJIANSHEJI~90 nt。ZHAOYINWUHECHENGGONGSIHECHENGJIAOZHANGDANLIANoligoJIKE(90+90+Insertion),ZHENDUIZHENGLIANHUOZHEFANLIANHECHENGDOUok。MEIYOUBIYAOSHIYONGPAGECHUNHUA。WEILEYANZHENGFANGBIAN,JIANYIYINRUMEIQIEWEIDIAN(RFLPFA)。DANGRAN,TONGYUANMOBANYEKEYISHIXIANXINGHUADESHUANGLIANDNAPIANDUAN,KEYITONGGUOXIANXINGHUAZAITIHUOZHEJIYINHECHENG/PCRHUODE。RONGJIECHENGZHONGNONGDU10 mM,-20°CFENZHUANGBAOCUN。

 

2、XIANXINGHUASHUANGLIANMOBAN:TONGYUANBEIZAI1000 ntZUOYOU;XIANXINGHUAZAITIHUOZHEPCRCHANWUZUOWEIZHUANRANMOBAN。

 

 

ER、ZHUANRAN

 

★ 12KONGBAN:2 ✕ 105 cells/well (50-70% confluency)

★ 500 ng Cas9(HUOZHECas9n)-sgRNA

★ 1 μlDANLIANTONGYUANMOBAN(10 μM)

 

 

SAN、JIANDINGFANGFA

 

TONGGUOGFPHUOZHEpuromycinFUJICHENGmixture,RANHOUZHIJIEPCRCEXUHUOZHELIYONGRestriction-fragment length polymorphism (RFLP) FENXIFANGFA。

 

 

 SI、JIELUN

 

1、WTDECas9ZHIJIEQIEGESHUANGLIAN,YINQIDSB,ZHONGZUXIAOGUOZUIHAO,DANHUIYINQITUOBA;

2、DANLIANoligoZUOWEIMOBANYOUYUXIANXINGHUAZAITIZUOWEIMOBAN; 

3、MAOSIFANYI oligoDEMOBANXIAOGUOYOUYUZHENGLIAN。 

 

 

Fig 3. Cas9介导同源重组

 

 

 

CANKAOWENXIAN:

1. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, and Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-308.

 

2. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, and Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910-8.

 

3. Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, and Li J. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13(6):659-62.

 

4. Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, and Zhang F. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33(1):102-6

 

5. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520(7546):186-91.

 

6. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403-7.