近日，來自美國(guó)威斯康星大學(xué)的華人科學(xué)家Su-Chun Zhang在國(guó)際學(xué)術(shù)期刊Cell Stem Cell發(fā)表了一項(xiàng)最新研究進(jìn)展，他們利用CRISPR/CAS9技術(shù)實(shí)現(xiàn)了對(duì)人類干細(xì)胞系進(jìn)行可誘導(dǎo)基因敲除，這一方法的成功對(duì)于研究基因在干細(xì)胞及分化不同階段中的作用具有重要推動(dòng)作用。
對(duì)基因表達(dá)進(jìn)行精確的時(shí)間調(diào)控對(duì)于闡明一個(gè)基因在生物學(xué)系統(tǒng)中的功能至關(guān)重要，但到目前為止，在人類多能干細(xì)胞中實(shí)現(xiàn)可誘導(dǎo)基因敲除，建立可誘導(dǎo)基因敲除的人類干細(xì)胞系仍存在很大挑戰(zhàn)。
在該項(xiàng)研究中，研究人員結(jié)合CRISPR/CAS9介導(dǎo)的基因組編輯和Flp/FRT以及Cre/LoxP系統(tǒng)成功實(shí)現(xiàn)建立了可誘導(dǎo)基因敲除的人類多能干細(xì)胞系。研究人員發(fā)現(xiàn)dual-sgRNA的靶向作用對(duì)于將FRT序列進(jìn)行精確的雙等位基因敲入非常重要。除此之外，他們還開發(fā)了出一種新的策略將一個(gè)可調(diào)控活性的重組酶表達(dá)體系同時(shí)導(dǎo)入細(xì)胞，移除了藥物抗性基因，利用這種方法可以加快iKO hPSC細(xì)胞系的獲得速度。
研究人員通過這種兩步法敲除策略，對(duì)人類胚胎干細(xì)胞和誘導(dǎo)多能干細(xì)胞中的SOX2,PAX6,OTX2,AGO2等基因?qū)崿F(xiàn)了可誘導(dǎo)性基因敲除，建立了相關(guān)細(xì)胞系。
研究人員相信，利用這種方法建立iKO hPSC細(xì)胞系將會(huì)改變?nèi)藗円酝鶎?duì)人類細(xì)胞中基因功能研究的方式，對(duì)于推動(dòng)干細(xì)胞研究進(jìn)展具有重要意義。
原文內(nèi)容：
Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9
Yuejun Chen6correspondenceemail, Jingyuan Cao6, Man Xiong6, Andrew J. Petersen, Yi Dong, Yunlong Tao, Cindy Tzu-Ling Huang, Zhongwei Du, Su-Chun Zhang
Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that "dual-sgRNA targeting" is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6,OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.
原文地址：http://www.cell.com/cell-stem-cell/abstract/S1934-5909(15)00261-1