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本帖最后由 zwb96 于 2013-5-9 23:41 编辑
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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。
d/ l; f( {4 Y, R7 QMice carrying mutations in multiple genes are traditionally0 H- e2 J9 i) W% A+ k) {
generated by sequential recombination in9 _, q* e. P3 _- @
embryonic stem cells and/or time-consuming intercrossing ~1 |/ ]( F6 y! ]) i9 o
of mice with a single mutation. The
: b* u; s( B9 v% d: ~+ `CRISPR/Cas system has been adapted as an efficient2 D, B4 o$ O. P V7 O% s5 A
gene-targeting technology with the potential
# h+ J. G2 q6 a) m: v$ afor multiplexed genome editing. We demonstrate0 d0 ~9 l8 F9 ]$ v
that CRISPR/Cas-mediated gene editing allows the
0 P0 O3 s6 C9 }: P7 i! Rsimultaneous disruption of five genes (Tet1, 2, 3,
1 p0 |8 t G; C) X! q' g2 LSry, Uty - 8 alleles) in mouse embryonic stem (ES)& P0 ]# M+ H/ e; r5 j8 j0 U
cells with high efficiency. Coinjection of Cas9; u- i4 E+ W$ f5 N% n
mRNA and single-guide RNAs (sgRNAs) targeting" q8 H& a* ?* p- }& M
Tet1 and Tet2 into zygotes generated mice with biallelic
( Y8 U, Y+ ?$ Q4 E! y; rmutations in both genes with an efficiency of- s) b3 j3 y7 y- c5 o2 D0 ^- W2 I1 i
80%. Finally, we show that coinjection of Cas9
9 Q5 ]% q9 a( }. Z9 R' S, rmRNA/sgRNAs with mutant oligos generated precise2 m6 I2 j7 A3 {( i( P
point mutations simultaneously in two target
' e) p+ [' O* p" U2 U4 f8 x5 ugenes. Thus, the CRISPR/Cas system allows the
# C* z1 L) Q0 p- rone-step generation of animals carrying mutations
, {( x7 q. p* `; e$ r) g7 @, ^in multiple genes, an approach that will greatly accelerate
% R {" g. ?: [5 O% y+ g" vthe in vivo study of functionally redundant# p( p6 I) v0 Q2 x. G
genes 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
