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本帖最后由 zwb96 于 2013-5-9 23:41 编辑 Q, |, O, j2 z
! }7 C; u! t+ H h1 ]刚刚在Cell发表的文章（2013.5.9），转基因小鼠技术的鼻祖Rudolf Jaenisch领导的小组采用继ZFN,TALEN技术后最新的可修饰哺乳动物基因组的技术CRISPR以不可思议的高效率在小鼠胚胎干细胞一步敲除多个基因，更在短时间内通过合子内注射CRISPR系统（Cas9蛋白和sg-RNAs)建立了双基因敲除的小鼠模型，理论上只需4周时间就可完成，对基因敲除的技术来说是一次很大的飞跃，因为采用传统的同源重组的方法一般要9-15个月才能得到单个基因敲除的小鼠，敲除两个基因需要两年甚至更长的时间。以下是文章摘要。- V, T* Z1 x* H! H4 d; S" u8 W- `
Mice carrying mutations in multiple genes are traditionally% u6 X+ R1 D4 f& F9 b* g% j2 e
generated by sequential recombination in& @8 _* ?! v3 P% H
embryonic stem cells and/or time-consuming intercrossing
: r5 ~& e! P2 E2 }of mice with a single mutation. The
5 g1 ], u0 Z, S$ n; L4 P' [# a7 ACRISPR/Cas system has been adapted as an efficient
: ]& i& U+ @7 B' C* X5 hgene-targeting technology with the potential5 G6 u5 Z' Y3 x& u
for multiplexed genome editing. We demonstrate
9 x/ f% p2 ?: W7 o! Zthat CRISPR/Cas-mediated gene editing allows the
; G+ }/ y. Q z& Bsimultaneous disruption of five genes (Tet1, 2, 3," F; ?# e8 ~0 d
Sry, Uty - 8 alleles) in mouse embryonic stem (ES)5 {: ]# j4 S7 s0 Z* Q9 i
cells with high efficiency. Coinjection of Cas9; @4 l( c! o7 f* i- K/ K0 q* p
mRNA and single-guide RNAs (sgRNAs) targeting
7 ^# P! s. I: y* D$ jTet1 and Tet2 into zygotes generated mice with biallelic- `# e8 Z+ M* V7 U5 J
mutations in both genes with an efficiency of9 j9 w+ L5 i" G( y1 [+ T
80%. Finally, we show that coinjection of Cas9
& e/ I# A# r9 kmRNA/sgRNAs with mutant oligos generated precise
0 Y( x/ W' v& s- }: ~$ }point mutations simultaneously in two target1 e, H" J9 K i( I: [$ f
genes. Thus, the CRISPR/Cas system allows the
4 [. h' M( J. _7 K4 f) I0 {one-step generation of animals carrying mutations
/ g' F( L- d win multiple genes, an approach that will greatly accelerate5 t) s+ T" w$ H5 H I1 x
the in vivo study of functionally redundant
3 e# [$ X+ @3 ^1 ggenes and of epistatic gene interactions. |
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发表于 2013-5-9 23:38
发表于 2013-5-10 10:26
