·編者按·

基因編輯

·編者按·

基因組編輯技術(shù)（Genome Editing）是借助核酸內(nèi)切酶，對(duì)細(xì)胞內(nèi)DNA序列進(jìn)行編輯，包括核苷酸的切除、替換或者插入，使之產(chǎn)生可遺傳的改變。這種技術(shù)的原理是構(gòu)建一個(gè)人工核酸內(nèi)切酶，在預(yù)定的基因組位置對(duì)DNA剪切，斷裂的DNA會(huì)誘導(dǎo)細(xì)胞內(nèi)的DNA修復(fù)系統(tǒng)激活，細(xì)胞內(nèi)的DNA修復(fù)機(jī)制包括非同源末端鏈接（NHEJ）和同源重組修復(fù)（HR）。前者在修復(fù)過程中引入大量突變；相反，后者根據(jù)提供的同源序列進(jìn)行修復(fù)，是真正意義上的基因編輯技術(shù)的基礎(chǔ)?；蚪M編輯技術(shù)可以實(shí)現(xiàn)三種基因組改造的目的，即基因敲除，特異突變的引入和定點(diǎn)轉(zhuǎn)基因。幾種主要的基因組編輯技術(shù)包括：鋅指核酸酶（ZincFinger Nuclease，ZFN）、轉(zhuǎn)錄激活因子樣效應(yīng)物核酸酶TALEN（Transcription Activator-like Effector Nuclease）和規(guī)律性間隔的短回文序列重復(fù)簇（Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Proteins，CRISPR）。

河北科技大學(xué)的韓春雨的團(tuán)隊(duì)發(fā)明了一種新的基因編輯技術(shù)——NgAgo-gDNA，并于2016年5月2日發(fā)表在《自然·生物技術(shù)》（Nature Biotechnology）雜志上，有望成為“第四代”基因組編輯技術(shù)。

本專題得到張智英教授（西北農(nóng)林科技大學(xué)）、陳凌懿教授（南開大學(xué)）的大力支持。

·熱點(diǎn)數(shù)據(jù)排行·

截至2016年6月1日，中國知網(wǎng)（CNKI）和Web of Science（WOS）的數(shù)據(jù)報(bào)告顯示，以“基因編輯”為詞條可以檢索到的期刊文獻(xiàn)分別為368條與1225條，本專題將相關(guān)數(shù)據(jù)按照：研究機(jī)構(gòu)發(fā)文數(shù)、作者發(fā)文數(shù)、期刊發(fā)文數(shù)、被引用頻次進(jìn)行排行，結(jié)果如下。

研究機(jī)構(gòu)發(fā)文數(shù)量排名（CNKI）

研究機(jī)構(gòu)發(fā)文數(shù)量排名（WOS）

作者發(fā)文數(shù)量排名（CNKI）

作者發(fā)文數(shù)量排名（WOS）

期刊發(fā)文數(shù)量排名（CNKI）

期刊發(fā)文數(shù)量排名（WOS）

根據(jù)中國知網(wǎng)（CNKI）數(shù)據(jù)報(bào)告，以“基因編輯”為詞條可以檢索到的高被引論文排行結(jié)果如下。

國內(nèi)數(shù)據(jù)庫高被引論文排行

根據(jù)Web of Science統(tǒng)計(jì)數(shù)據(jù)，以“基因編輯”為詞條可以檢索到的高被引論文排行結(jié)果如下。

國外數(shù)據(jù)庫高被引論文排行

·經(jīng)典文獻(xiàn)推薦·

基于Web of Science檢索結(jié)果，利用Histcite軟件選取LCS（Local Citation Score，本地引用次數(shù)）TOP 30文獻(xiàn)作為節(jié)點(diǎn)進(jìn)行分析，得到本領(lǐng)域推薦的經(jīng)典文獻(xiàn)如下。

本領(lǐng)域經(jīng)典文獻(xiàn)

An improved zinc-finger nuclease architecture for highly specific genome editing

Miller, JC; Holmes, MC; Wang, JB; et al.

來源出版物：Nature Biotechnology, 2007, 25(7): 778-785

A TALE nuclease architecture for efficient genome editing

Miller, JC; Tan, SY; Qiao, GJ; et al.

來源出版物：Nature Biotechnology, 2011, 29(2): 143-148

Targeted genome editing across species using ZFNs and TALENs

Wood, AJ; Lo, TW; Zeitler, B; et al.

來源出版物：Science, 2011, 333(6040): 307-307

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

Mussolino, C; Morbitzer, R; Lutge, F; et al.

來源出版物：Nucleic Acids Research, 2011, 39(21): 9283-9293

A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity

Jinek, M; Chylinski, K; Fonfara, I; et al.

來源出版物：Science, 2012, 337(6096): 816-821

·推薦綜述·

哺乳動(dòng)物基因組靶向修飾陽性細(xì)胞富集的報(bào)告載體系統(tǒng)研究進(jìn)展

白義春，徐坤，魏澤輝，馬琤，張智英

隨著全基因組測序技術(shù)的發(fā)展和應(yīng)用，越來越多物種的基因組序列被公布。但是獲得基因組序列并不是人們的最終目的，詮釋基因的功能才是研究這些基因的意義所在。研究基因功能最直接、最可靠的方法就是對(duì)基因組進(jìn)行靶向修飾?；蚪M靶向修飾技術(shù)最初主要是依賴細(xì)胞體內(nèi)的自發(fā)同源重組（Homologous recombination，HR）機(jī)制實(shí)現(xiàn)的。但是高等動(dòng)植物機(jī)體內(nèi)的同源重組幾率很低，只有千萬分之一，因此大大限制了該項(xiàng)技術(shù)的應(yīng)用。

已有的研究表明，如果在DNA靶位點(diǎn)上引入雙鏈斷裂（Double strand breaks，DSBs），哺乳動(dòng)物細(xì)胞中同源重組的幾率會(huì)大大提高。近年來先后發(fā)展起來的人工鋅指核酸酶（Zinc-finger nuclease，ZFN）、類轉(zhuǎn)錄激活因子效應(yīng)物核酸酶（Transcription activator-like effector nuclease，TALEN）、CRISPR/Cas（Clustered，regularly interspaced，shortpalindromic repeats（CRISPR）/CRISPR-associated protein（Cas））系統(tǒng)可以定點(diǎn)切割DNA雙鏈，從不同程度上極大提高了基因靶向修飾的效率和精確性。

但是核酸酶有時(shí)活性很低，可能只有一小部分的細(xì)胞被核酸酶靶向修飾，要從大量的細(xì)胞中篩選到基因靶向修飾的陽性細(xì)胞仍然費(fèi)時(shí)費(fèi)力，因此如何富集到陽性細(xì)胞是解決這個(gè)問題的關(guān)鍵。傳統(tǒng)的方法是與核酸酶同時(shí)轉(zhuǎn)入一個(gè)表達(dá)熒光或抗性基因的報(bào)告載體，通過熒光或抗性來進(jìn)行陽性細(xì)胞的篩選。但是通過這種方法富集陽性細(xì)胞效率很低。生物細(xì)胞內(nèi)存在自發(fā)的DNA雙鏈斷裂修復(fù)機(jī)制：同源重組（Homologous recombination，HR）、單鏈退火（Single-strand annealing，SSA）和非同源末端連接（Non-homologous end joining，NHEJ）。當(dāng)生物的基因組斷裂后，細(xì)胞會(huì)通過自身的修復(fù)機(jī)制對(duì)基因組進(jìn)行修復(fù)，從而引起基因組移碼突變或是片段的缺失。根據(jù)細(xì)胞內(nèi)的不同修復(fù)機(jī)制，近兩年逐漸發(fā)展出游離于細(xì)胞染色體之外的NHEJ和SSA介導(dǎo)的報(bào)告載體富集篩選系統(tǒng)。本文主要綜述了這兩種系統(tǒng)的原理及應(yīng)用，以期為科研工作者提供參考。

1DNA雙鏈斷裂的修復(fù)方式

DNA雙鏈斷裂后，主要通過3種方式進(jìn)行修復(fù)。HR嚴(yán)格意義上是保護(hù)基因組完整性的重要機(jī)制，當(dāng)DNA雙鏈斷裂時(shí)，以姐妹染色單體或同一染色體上含有同源DNA分子模板進(jìn)行修復(fù)，該修復(fù)是無錯(cuò)誤的修復(fù)方式，只發(fā)生在細(xì)胞周期的S/G2期。在基因組編輯中，如果人為地提供供體并對(duì)供體進(jìn)行改造（如在供體的同源臂之間插入基因序列或是對(duì)一些堿基進(jìn)行突變），就能達(dá)到基因編輯的目的，如基因敲入、基因突變等。SSA是同源重組修復(fù)方式的一種特殊情況，即在DSB兩端有一段同源重復(fù)序列的情況下，兩個(gè)同源的序列可能會(huì)相互結(jié)合發(fā)生退火，進(jìn)行后續(xù)的合成和再連接，最后刪除一個(gè)重復(fù)序列及中間包含的序列從而實(shí)現(xiàn)基因修復(fù)。NHEJ是哺乳動(dòng)物細(xì)胞中DSBs修復(fù)的主要機(jī)制，即DSB兩端不通過同源臂，而通過非特異性的末端連接實(shí)現(xiàn)修復(fù)，同時(shí)一般會(huì)引入少量核苷酸堿基的插入或缺失（Insertions and deletions，Indels），進(jìn)而可能導(dǎo)致移碼甚至是基因敲除。

2報(bào)告載體系統(tǒng)

HR介導(dǎo)的報(bào)告系統(tǒng)需要提供供體，因此關(guān)于HR介導(dǎo)的報(bào)告系統(tǒng)現(xiàn)在僅限于檢測核酸酶活性的報(bào)告細(xì)胞系，還未見HR介導(dǎo)的報(bào)告載體系統(tǒng)富集基因編輯的陽性細(xì)胞的報(bào)道。因此，下文重點(diǎn)介紹NHEJ和SSA介導(dǎo)的報(bào)告載體系統(tǒng)。

2.1NHEJ介導(dǎo)的報(bào)告載體系統(tǒng)

Kim課題組發(fā)現(xiàn)人工核酸酶如果能夠有效地切割載體上含有細(xì)胞內(nèi)源靶基因序列，那么該核酸酶也能夠有效的切割內(nèi)源染色體上的靶基因序列，這啟示在細(xì)胞中轉(zhuǎn)入一個(gè)游離的含有核酸酶靶位點(diǎn)的報(bào)告載體，該報(bào)告載體可能會(huì)反映出該細(xì)胞中核酸酶的活性，從而用報(bào)告載體來富集基因編輯的陽性細(xì)胞，基于此，Kim課題組開發(fā)出NHEJ介導(dǎo)的報(bào)告載體系統(tǒng)。

2.1.1NHEJ介導(dǎo)的熒光蛋白報(bào)告載體系統(tǒng)

熒光蛋白是一類在一定波長光照射下可以發(fā)出熒光的蛋白。最早發(fā)現(xiàn)的熒光蛋白是從維多利亞發(fā)光水母中分離的綠色熒光蛋白（Green fluorescent Protein，GFP），具有穩(wěn)定、檢測簡單、靈敏度高、對(duì)活細(xì)胞無毒性、熒光反應(yīng)不需要任何外源反應(yīng)底物等優(yōu)點(diǎn)，因此作為一種報(bào)告基因受到了廣大科研工作者的青睞。之后，人們對(duì)GFP進(jìn)行了一系列的改造，大大增強(qiáng)了GFP的熒光強(qiáng)度并且產(chǎn)生了不同顏色的突變?，F(xiàn)有的熒光蛋白有藍(lán)色熒光蛋白、紅色熒光蛋白、黃色熒光蛋白等，使熒光蛋白具有更廣泛的應(yīng)用價(jià)值。

2011年，Kim課題組利用ZFN靶向人TP53基因時(shí)，除了轉(zhuǎn)入一對(duì)靶向TP53基因的ZFN外，還轉(zhuǎn)入了一個(gè)含有ZFN靶位點(diǎn)的熒光蛋白報(bào)告載體到HEK293T細(xì)胞中。該報(bào)告載體骨架是用CMV啟動(dòng)子，將紅色熒光mRFP與綠色熒光蛋白eGFP融合表達(dá)。在mRFP與eGFP之間插入ZFN 作用的靶位點(diǎn)和終止密碼子，使eGFP的開放閱讀框移碼突變，終止eGFP綠色熒光蛋白的表達(dá)。當(dāng)沒有核酸酶作用時(shí)，轉(zhuǎn)入報(bào)告載體的細(xì)胞只發(fā)紅光，當(dāng)將該報(bào)告載體與ZFN共同轉(zhuǎn)入細(xì)胞中后，ZFN切割報(bào)告載體靶位點(diǎn)，細(xì)胞通過NHEJ機(jī)制進(jìn)行修復(fù)，伴隨著幾個(gè)堿基的插入或缺失，eGFP的閱讀框進(jìn)行重新排列，會(huì)有一部分細(xì)胞表達(dá)綠色熒光。因此該系統(tǒng)中紅光表示轉(zhuǎn)染效率，綠光表示切割效率。轉(zhuǎn)染72 h后用流式細(xì)胞儀分析，16%的細(xì)胞只發(fā)紅光不發(fā)綠光（mRFP+eGFP-），5%的細(xì)胞既發(fā)紅光也發(fā)綠光（mRFP+eGFP+）。對(duì)mRFP+eGFP+細(xì)胞用流式細(xì)胞儀分選后進(jìn)行T7E1檢測，分選后mRFP+eGFP+的細(xì)胞基因突變率為37%，比不分選的細(xì)胞高出13倍；而mRFP-eGFP-細(xì)胞、mRFP+eGFP-細(xì)胞和只轉(zhuǎn)染ZFN核酸酶沒有轉(zhuǎn)染報(bào)告載體的細(xì)胞，切割效率只有2.8%～4.8%。脫靶檢測顯示這種富集細(xì)胞的方法并沒有加劇脫靶效應(yīng)，因此這種報(bào)告載體系統(tǒng)在富集基因編輯的陽性細(xì)胞中是沒有副作用的。該課題組同時(shí)又利用兩對(duì)靶向人CCR5基因的ZFN及一對(duì)TALENs和對(duì)應(yīng)的報(bào)告載體分別對(duì)基因突變的細(xì)胞進(jìn)行富集，結(jié)果富集的陽性細(xì)胞率大大提高。在小鼠誘導(dǎo)性多潛能干細(xì)胞（Induced Pluripotent Stem Cells，iPS）上的實(shí)驗(yàn)結(jié)果也顯示該富集系統(tǒng)是可用且高效的。

盡管上述NHEJ介導(dǎo)的報(bào)告載體系統(tǒng)大大提高了核酸酶作用后基因組編輯陽性細(xì)胞的效率，但是該系統(tǒng)只有1/3的概率能表達(dá)有功能的eGFP蛋白，理論上只反映核酸酶1/3的活性。因此，Kim課題組又對(duì)該報(bào)告載體做了一些改進(jìn)，將上述報(bào)告載體第一個(gè)eGFP后又加了一個(gè)移碼的eGFP，這樣當(dāng)核酸酶作用于靶位點(diǎn)時(shí)，移碼3n+1或3n+2堿基的eGFP都可以正常表達(dá)，從而用流式細(xì)胞儀富集出陽性細(xì)胞。這樣理論上可以將報(bào)告基因的富集效率提高到2/3。當(dāng)將靶向人BMP1、F13A1 和PPP1R1B的CRISPR/Cas和相應(yīng)的報(bào)告載體轉(zhuǎn)染HEK293T細(xì)胞時(shí)，T7E1檢測細(xì)胞的突變效率都能達(dá)到36%以上，突變效率大部分都高于靶位點(diǎn)后僅有一個(gè)eGFP的報(bào)告載體富集陽性細(xì)胞的效率。

NHEJ介導(dǎo)的熒光蛋白基因報(bào)告載體系統(tǒng)，只用轉(zhuǎn)入一個(gè)含有靶位點(diǎn)的報(bào)告載體就可以對(duì)基因編輯的陽性細(xì)胞進(jìn)行富集，這種方法簡單、省時(shí)省力，富集基因編輯的陽性細(xì)胞效率較高。因此，Koo等采用該報(bào)告系統(tǒng)成功得到了CMAH基因編輯的豬胎兒成纖維細(xì)胞。

2.1.2NHEJ介導(dǎo)的抗性基因報(bào)告載體系統(tǒng)

盡管用流式分選可以有效地富集到基因編輯的陽性細(xì)胞，但是流式分選需要價(jià)格昂貴的流式細(xì)胞儀，并且分選的細(xì)胞有時(shí)會(huì)由于強(qiáng)激光或液體靜壓力影響細(xì)胞的活性?？剐曰蚴蔷幋a分解抗生素相關(guān)酶的基因，如嘌呤霉素（Puromycin）抗性基因、潮霉素（Hygromycin）抗性基因、新霉素（Neomycin）抗性基因等，這些是真核表達(dá)載體常用的篩選標(biāo)志，當(dāng)把抗性基因?qū)雽?duì)該抗生素敏感的細(xì)胞中，能賦予細(xì)胞對(duì)該抗生素的抗性，從而通過抗生素篩選可以得到具有抗性基因或其他用途的細(xì)胞。用抗生素篩選細(xì)胞，不需要特殊的設(shè)備，在一般的實(shí)驗(yàn)室都可以進(jìn)行，應(yīng)用很廣泛。

Kim課題組于2013年對(duì)NHEJ介導(dǎo)的熒光基因報(bào)告載體進(jìn)行改造，改造出可以用抗生素來對(duì)陽性細(xì)胞進(jìn)行富集的系統(tǒng)。該系統(tǒng)是將HygroR和eGFP融合表達(dá)，將靶位點(diǎn)插入mRFP和HygroR之間，并且在靶位點(diǎn)后面插入終止密碼子，因此HygroR和eGFP不表達(dá)，但是當(dāng)核酸酶作用于靶位點(diǎn)時(shí)，細(xì)胞在NHEJ 的作用下終止密碼子可能移碼，從而會(huì)有一部分細(xì)胞的eGFP和HygroR處于閱讀框中，重新表達(dá)，通過Hygromycin可以富集出RFP+HygroR+GFP+細(xì)胞。當(dāng)用ZFN和該系統(tǒng)靶向人的CCR5基因時(shí)，細(xì)胞的突變率達(dá)到42%，是不富集細(xì)胞的16倍。同時(shí)用TALEN靶向BRCA1基因，用該系統(tǒng)富集突變的細(xì)胞時(shí)，結(jié)果顯示富集后的細(xì)胞突變效率是不富集的細(xì)胞突變效率的11倍。

當(dāng)用CRISPR/Cas9 靶向PPP1R1B、BMP1和F13A1基因并用該報(bào)告載體系統(tǒng)富集細(xì)胞后，富集后的細(xì)胞突變效率比不富集的至少高出5.4倍。由此可見，該報(bào)告載體系統(tǒng)對(duì)于三種核酸酶都是適應(yīng)的，并且富集基因突變的陽性細(xì)胞的效率都非常高。

2.1.3NHEJ介導(dǎo)的抗原基因報(bào)告載體系統(tǒng)

用抗性基因報(bào)告載體系統(tǒng)進(jìn)行基因編輯的陽性細(xì)胞富集不需要特殊的儀器設(shè)備，細(xì)胞也不需要特殊的處理，富集細(xì)胞的陽性率也比較高，但是用該系統(tǒng)富集細(xì)胞，實(shí)驗(yàn)周期較長，一般都要用抗生素篩選7 d或者更長的時(shí)間；并且不同的細(xì)胞在不同的條件下，對(duì)于抗生素的敏感性不同，需要摸索不同細(xì)胞對(duì)不同抗生素的最適敏感濃度。抗原基因是編碼某種抗原物質(zhì)的基因，該抗原物質(zhì)可以特異性地與包被抗體的免疫磁珠結(jié)合形成免疫復(fù)合物，在外加磁場的作用下被滯留，從而與其他成分分離，免疫磁珠分離細(xì)胞速度快、效率高、操作簡單、不需要昂貴的儀器設(shè)備。

Kim課題組的抗原基因報(bào)告系統(tǒng)是將eGFP和H-2Kk蛋白通過T2A剪切肽連接，該系統(tǒng)作用機(jī)理同3.1.2，只是將HygroR基因換成H-2Kk基因。當(dāng)核酸酶工作時(shí)，H-2Kk蛋白恢復(fù)活性從而可以通過磁珠分選富集RFP+H-2Kk+GFP+細(xì)胞。用靶向CCR5基因的ZFN和報(bào)告載體轉(zhuǎn)染HEK293細(xì)胞，3 d后用結(jié)合了抗H-2Kk標(biāo)記抗體的磁珠分選RFP+H-2Kk+GFP+細(xì)胞，結(jié)果顯示H-2Kk+細(xì)胞的CCR5 基因的突變率是46%，與不分選細(xì)胞相比高出12倍之多。同時(shí)又在HEK293T和Huh7.5細(xì)胞上進(jìn)行實(shí)驗(yàn)，用ZFN、TALEN 和CRISPR/Cas9靶向TP53、CD81、F13A1、BMP1等基因并用該報(bào)告系統(tǒng)對(duì)細(xì)胞分選，分選后的細(xì)胞突變率是不分選細(xì)胞的3.7～17倍，這說明該系統(tǒng)對(duì)于3種人工核酸酶都是適應(yīng)的，并且發(fā)現(xiàn)核酸酶活性越低，分選后細(xì)胞的突變效率增加越高。Koo等用該報(bào)告系統(tǒng)富集ZFN打靶的豬CMAH基因的胎兒成纖維細(xì)胞，效率高達(dá)65%。

綜上所述，以上3種報(bào)告載體都能有效地富集基因編輯的陽性細(xì)胞，3種方法各有優(yōu)劣之處。流式分選篩選周期短，但是需要特殊的儀器設(shè)備，并且流式分選對(duì)細(xì)胞活性有影響，磁力分選比流式分選周期短并且容易操作。Koo等對(duì)流式分選和磁力分選的豬胎兒成纖維細(xì)胞的囊胚發(fā)育率比較后，認(rèn)為磁力分選的細(xì)胞更適合進(jìn)行核移植?？股睾Y選雖然周期長，但是不需要特殊的儀器設(shè)備，在一般的實(shí)驗(yàn)室都可以操作，因此可以依據(jù)實(shí)驗(yàn)室條件根據(jù)不同的需要采用不同的細(xì)胞富集篩選系統(tǒng)。

2.2SSA介導(dǎo)的報(bào)告載體

NHEJ介導(dǎo)的報(bào)告載體修復(fù)后只有1/3～2/3的幾率使eGFP報(bào)告基因修復(fù)表達(dá)，尤其是在核酸酶活性較低時(shí)，其靈敏度受到了很大的限制。Szczepek等在檢測ZFN的活性時(shí)，構(gòu)建了一個(gè)基于SSA修復(fù)機(jī)制的熒光素酶基因報(bào)告載體，該載體將熒光素酶報(bào)告基因中間插入終止密碼子和一個(gè)ZFN的靶位點(diǎn)，分開的兩部分含有870 bp的重復(fù)序列，使該基因失活。當(dāng)ZFN和該報(bào)告載體共轉(zhuǎn)染細(xì)胞時(shí)，靶位點(diǎn)被切割，細(xì)胞可能通過SSA方式來修復(fù)DSB，使細(xì)胞熒光素酶基因恢復(fù)活性，從而通過熒光素酶的表達(dá)來檢測ZFN的活性。Kuhar等運(yùn)用構(gòu)建的SSA-TLR的報(bào)告細(xì)胞系來檢測HR、NHEJ和SSA三種細(xì)胞修復(fù)方式的效率，意外發(fā)現(xiàn)SSA的修復(fù)效率要遠(yuǎn)遠(yuǎn)高于HR和NHEJ的效率，因此推測如果在靶位點(diǎn)附近存在重復(fù)序列時(shí)，SSA修復(fù)應(yīng)該是主要的修復(fù)方式。本課題組基于以上研究結(jié)果，開發(fā)出SSA介導(dǎo)的報(bào)告載體系統(tǒng)來富集基因編輯的陽性細(xì)胞。

2.2.1SSA介導(dǎo)的雙熒光報(bào)告載體系統(tǒng)

SSA介導(dǎo)的雙熒光報(bào)告載體系統(tǒng)是本課題組在檢測核酸酶活性時(shí)構(gòu)建的一個(gè)報(bào)告載體系統(tǒng)。最初的雙熒光報(bào)告載體是用mRFP作為標(biāo)記基因，用eGFP作為報(bào)告基因，mRFP和eGFP融合表達(dá)，eGFP基因被分成兩部分，其中含有200 bp的正向重復(fù)序列，重復(fù)序列中間插入核酸酶的靶位點(diǎn)，使eGFP失活。當(dāng)核酸酶作用于靶位點(diǎn)時(shí)，細(xì)胞中由于SSA的修復(fù)方式，eGFP恢復(fù)活性，從而用來檢測核酸酶的活性。但是無論是直接融合表達(dá)還是通過T2A剪切肽或IRES內(nèi)部核糖體結(jié)合位點(diǎn)來介導(dǎo)表達(dá)，eGFP報(bào)告基因的表達(dá)效率都會(huì)受到很大的影響，因此當(dāng)本課題組后來檢測CRISRP/Cas9的活性時(shí)又構(gòu)建了DsRed-eGFP報(bào)告載體。該報(bào)告載體是將標(biāo)記基因DsRed和報(bào)告基因eGFP分別用CMV啟動(dòng)子表達(dá)，將靶位點(diǎn)插入eGFP的兩個(gè)重復(fù)序列之間，通過eGFP的表達(dá)來反映核酸酶的活性，這就解決了融合表達(dá)時(shí)eGFP報(bào)告基因修復(fù)后表達(dá)強(qiáng)度較弱的問題?；谠搱?bào)告載體的StCRISPR/Cas9哺乳動(dòng)物細(xì)胞活性驗(yàn)證系統(tǒng)中，針對(duì)SP1.sgRNA.WT和SP1.sgRNA.Opti/hStCas9的報(bào)告載體檢測活性分別為10.95%和31.66%，而NHEJ介導(dǎo)的報(bào)告載體檢測活性只有3.71%和4.66%，說明用SSA介導(dǎo)的報(bào)告載體系統(tǒng)檢測核酸酶活性要優(yōu)于NHEJ介導(dǎo)的報(bào)告載體系統(tǒng)。但由于受流式細(xì)胞儀的限制，本課題組只是用該系統(tǒng)進(jìn)行了核酸酶活性的檢測，沒有用流式細(xì)胞儀富集陽性細(xì)胞。中山大學(xué)吳金青等用自主構(gòu)建的SSA介導(dǎo)的熒光報(bào)告載體系統(tǒng)通過流式分選來富集靶向IGF2基因的豬胎兒成纖維細(xì)胞，用ZFN和CRISPR/Cas9編輯過的細(xì)胞富集后打靶效率可以提高5倍。

2.2.2SSA介導(dǎo)的抗性基因報(bào)告載體系統(tǒng)

由于雙熒光報(bào)告載體系統(tǒng)受限于流式細(xì)胞儀，因此本課題組又開發(fā)出SSA介導(dǎo)的抗性基因報(bào)告載體系統(tǒng)。該系統(tǒng)是將PuroR作為報(bào)告基因，將靶位點(diǎn)插入PuroR中，靶位點(diǎn)兩邊有300 bp的同源臂，當(dāng)核酸酶作用于靶位點(diǎn)時(shí)，通過SSA機(jī)制恢復(fù)PuroR的功能，細(xì)胞表現(xiàn)出Puromysin抗性，從而可以通過Puromysin來富集陽性細(xì)胞，用3對(duì)ZFN分別靶向奶山羊乳腺上皮細(xì)胞as1-casein基因的同一位點(diǎn)，并用該系統(tǒng)富集后進(jìn)行突變檢測，3 對(duì)ZFN的靶位點(diǎn)的突變效率分別達(dá)到9.4%、15.9%和4.1%。但是這些報(bào)告載體只是進(jìn)行了陽性細(xì)胞的富集，沒有對(duì)SSA介導(dǎo)的報(bào)告載體做進(jìn)一步的研究。

2.2.3SSA介導(dǎo)的雙報(bào)告基因載體系統(tǒng)

2014年，本實(shí)驗(yàn)室Ren等對(duì)SSA介導(dǎo)的報(bào)告載體富集篩選系統(tǒng)進(jìn)行了深入的研究，開發(fā)出DsRed-PuroR-eGFP（RPG）雙報(bào)告基因載體系統(tǒng)。在該系統(tǒng)中，用CMV啟動(dòng)子啟動(dòng)紅色熒光DsRed的表達(dá)，用CAG啟動(dòng)子啟動(dòng)通過T2A融合的PuroR和eGFP作為雙報(bào)告基因，將靶位點(diǎn)插入PuroR中，靶位點(diǎn)兩側(cè)有同向重復(fù)序列，正常情況下，該報(bào)告載體不表達(dá)PuroR和eGFP，當(dāng)核酸酶作用于靶位點(diǎn)時(shí)，由于SSA的修復(fù)機(jī)制，PuroR和eGFP恢復(fù)表達(dá)，細(xì)胞表現(xiàn)出Puromysin抗性和綠色熒光，從而可以通過Puromysin或流式細(xì)胞儀對(duì)陽性細(xì)胞進(jìn)行富集。同時(shí)對(duì)SSA同源臂從50～350 bp長度進(jìn)行了比較，發(fā)現(xiàn)CRISPR/Cas9靶向切割基因的能力隨著同源臂從50～200 bp的增加而增加，從1.1%增加到6.8%，而同源臂從200～350 bp差異不大，因此確認(rèn)了最佳的同源臂為200 bp。同時(shí)在對(duì)CRISPR/Cas9靶向CCR5.a位點(diǎn)時(shí)，對(duì)該報(bào)告系統(tǒng)與Kim的NHEJ的報(bào)告系統(tǒng)對(duì)比，發(fā)現(xiàn)在同源臂為50 bp時(shí)的報(bào)告系統(tǒng)富集突變細(xì)胞的效率比NHEJ介導(dǎo)的報(bào)告系統(tǒng)的富集細(xì)胞的效率要高出3倍。隨后用ZFN靶向CCR5基因，發(fā)現(xiàn)SSA介導(dǎo)的報(bào)告載體系統(tǒng)富集陽性細(xì)胞的效率在同源臂在200～350 bp時(shí)也比NHEJ介導(dǎo)的報(bào)告載體富集陽性細(xì)胞的效率高出3倍。在用同源臂為200 bp的報(bào)告系統(tǒng)富集陽性細(xì)胞時(shí)，CRISPR/Cas9靶向AAVS1基因也得到了同樣的結(jié)果。這與Kuhar等在報(bào)告細(xì)胞系上所發(fā)現(xiàn)的結(jié)果吻合，即在靶位點(diǎn)附近存在重復(fù)序列時(shí)，SSA修復(fù)機(jī)制占主要方式。因此隨后利用SSA介導(dǎo)的報(bào)告系統(tǒng)，本課題組用CRISPR/Cas9分別靶向CCR2，CCR5.a和CCR5.b三個(gè)位點(diǎn)時(shí)，發(fā)現(xiàn)富集細(xì)胞的切割效率比不富集的細(xì)胞提高了6.3～34.8倍，證實(shí)了這是一個(gè)有效的富集陽性細(xì)胞的系統(tǒng)。目前本課題組正在用這個(gè)報(bào)告載體系統(tǒng)對(duì)雞DF-1細(xì)胞系、豬腎原代動(dòng)物或基因功能的研究奠定基礎(chǔ)。

在SSA介導(dǎo)的報(bào)告載體系統(tǒng)中，由于在兩個(gè)重復(fù)序列之間插入的片段比較短（<40 bp），當(dāng)兩個(gè)重復(fù)序列之間的靶位點(diǎn)被打斷后，SSA的修復(fù)方式是主要方式，因此SSA介導(dǎo)的報(bào)告載體系統(tǒng)富集陽性細(xì)胞的效率要優(yōu)于NEHJ介導(dǎo)的報(bào)告載體系統(tǒng)。但是在哺乳動(dòng)物細(xì)胞中，DSB主要以NHEJ為主要的修復(fù)方式，因此當(dāng)兩個(gè)重復(fù)序列之間的靶序列片段較長，是否SSA介導(dǎo)的報(bào)告載體優(yōu)于NEHJ介導(dǎo)的報(bào)告載體還需要進(jìn)一步驗(yàn)證。

3展望

基因組編輯技術(shù)是近年來研究基因功能的主要工具，ZFN、TALEN和CRISPR/Cas等基因組編輯技術(shù)的出現(xiàn)，更是推動(dòng)了人們對(duì)基因組靶向編輯技術(shù)研究的熱潮。盡管ZFN、TALEN和CRISPR/Cas技術(shù)相比傳統(tǒng)的同源重組技術(shù)，在不同程度上大大提高了基因組靶向編輯的效率，但是如果要研究基因功能，還需要篩選出比較純正的陽性克隆，因此富集陽性細(xì)胞就成為獲得陽性克隆的關(guān)鍵。NHEJ和SSA介導(dǎo)的基因組編輯陽性細(xì)胞富集系統(tǒng)，大大提高了基因編輯細(xì)胞的陽性率，并且人們可以根據(jù)不同的條件采用不同的報(bào)告載體系統(tǒng)進(jìn)行陽性細(xì)胞的富集，為基因功能的進(jìn)一步研究和轉(zhuǎn)基因動(dòng)物的生產(chǎn)提供了重要基礎(chǔ)。?

（摘自《中國科學(xué)：生命科學(xué)》2015年9期）

·高被引論文摘要·

被引頻次：34

人工鋅指核酸酶介導(dǎo)的基因組定點(diǎn)修飾技術(shù)

肖安，胡瑩瑩，王唯曄，等

摘要：鋅指核酸酶（ZFN）由鋅指蛋白（ZFP）結(jié)構(gòu)域和Fok I核酸內(nèi)切酶的切割結(jié)構(gòu)域人工融合而成，是近年來發(fā)展起來的一種可用于基因組定點(diǎn)改造的分子工具。ZFN可識(shí)別并結(jié)合特定的DNA序列，并通過切割這一序列的特定位點(diǎn)造成DNA的雙鏈斷裂（DSB）。在此基礎(chǔ)上，人們可以對(duì)基因組的特定位點(diǎn)進(jìn)行各種遺傳操作，包括基因打靶、基因定點(diǎn)插入、基因修復(fù)等，從而能夠方便快捷地對(duì)基因組實(shí)現(xiàn)靶向遺傳修飾。這種新的基因組定點(diǎn)修飾方法的突出優(yōu)勢是適用性好，對(duì)物種沒有選擇性，并且可以在細(xì)胞和個(gè)體水平進(jìn)行遺傳操作。文章綜述了ZFN技術(shù)的研究進(jìn)展及應(yīng)用前景，重點(diǎn)介紹ZFN的結(jié)構(gòu)與作用機(jī)制、現(xiàn)有的靶點(diǎn)評(píng)估及鋅指蛋白庫的構(gòu)建與篩選方法、基因組定點(diǎn)修飾的策略，以及目前利用這一技術(shù)已成功實(shí)現(xiàn)突變的物種及內(nèi)源基因，為開展這一領(lǐng)域的研究工作提供參考。

關(guān)鍵詞：鋅指核酸酶；鋅指蛋白；基因組定點(diǎn)修飾；基因打靶；分子工具

來源出版物：遺傳, 2011, 33(7): 665-683

被引頻次：33

CRISPR/Cas9介導(dǎo)的基因組定點(diǎn)編輯技術(shù)

方銳，暢飛，孫照霖，等

摘要：Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated（Cas）9系統(tǒng)成功被改造為第三代人工核酸內(nèi)切酶，與鋅指核酸內(nèi)切酶（zinc finger endonuclease，ZFN）和類轉(zhuǎn)錄激活因子效應(yīng)物核酸酶（transcription activator-like effector nuclease，TALEN）一樣可用于各種復(fù)雜基因組的編輯。目前該技術(shù)成功應(yīng)用于人類細(xì)胞、斑馬魚和小鼠以及細(xì)菌的基因組精確修飾，修飾類型包括基因定點(diǎn)InDel突變、基因定點(diǎn)敲入、兩位點(diǎn)同時(shí)突變和小片段的缺失。由于其突變效率高、制作簡單及成本低的特點(diǎn)，被認(rèn)為是一種具有廣闊應(yīng)用前景的基因組定點(diǎn)改造分子工具。本文從CRISPR/Cas的研究歷史、分類、作用機(jī)理以及基因定點(diǎn)修飾應(yīng)用等方面進(jìn)行簡單介紹，希望能夠?yàn)樵谶@一領(lǐng)域的科研工作者提供參考。

關(guān)鍵詞：人工核酸內(nèi)切酶；基因編輯；CRISPR；基因打靶；CRISPR/Cas

來源出版物：生物化學(xué)與生物物理進(jìn)展, 2013, 40(8): 691-702

被引頻次：30

CRISPR/Cas系統(tǒng)：RNA靶向的基因組定向編輯新技術(shù)

李君，張毅，陳坤玲，等

摘要：CRISPR/Cas系統(tǒng)廣泛存在于細(xì)菌及古生菌中，是機(jī)體長期進(jìn)化形成的RNA指導(dǎo)的降解入侵病毒或噬菌體DNA的適應(yīng)性免疫系統(tǒng)。對(duì)Ⅱ型CRISPR/Cas系統(tǒng)的改造使其成為繼鋅指核酸酶（ZFNs）和TALE核酸酶（TALENs）以來的另一種對(duì)基因組進(jìn)行高效定點(diǎn)修飾的新技術(shù)，與ZFNs和TALENs相比，CRISPR/Cas系統(tǒng)更簡單，并且更容易操作。文章重點(diǎn)介紹了Ⅱ型CRISPR/Cas系統(tǒng)的基本結(jié)構(gòu)、作用原理及這一技術(shù)在基因組定點(diǎn)修飾中的應(yīng)用，剖析了該技術(shù)可能存在的問題，展望了CRISPR/Cas系統(tǒng)的應(yīng)用前景，為開展這一領(lǐng)域的研究工作提供參考。

關(guān)鍵詞：CRISPR/Cas系統(tǒng)；基因組定點(diǎn)修飾；向?qū)NA

來源出版物：遺傳, 2013, 35(11): 1265-1273

被引頻次：26

CRISPR-Cas系統(tǒng)與細(xì)菌和噬菌體的共進(jìn)化

李鐵民，杜波

摘要：細(xì)菌在適應(yīng)噬菌體攻擊的過程中，進(jìn)化了多種防御系統(tǒng)，噬菌體在細(xì)菌的選擇壓力下，也在不斷進(jìn)化反防御策略，雙方的這種進(jìn)化關(guān)系與發(fā)生機(jī)制一直尚不完全清楚。近年在細(xì)菌和古細(xì)菌中發(fā)現(xiàn)一種新的免疫防御系統(tǒng)，即CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated system) 系統(tǒng)。在對(duì)其功能和作用機(jī)制深入研究的同時(shí)，也不斷地揭示了細(xì)菌和噬菌體之間的共進(jìn)化關(guān)系。為此，文章在介紹原核細(xì)胞中CRISPR-Cas系統(tǒng)介導(dǎo)的免疫機(jī)制基礎(chǔ)上，重點(diǎn)綜述了CRISPR系統(tǒng)在細(xì)菌和噬菌體進(jìn)化中的作用。

關(guān)鍵詞：CRISPR；CRISPR-Cas系統(tǒng)；噬菌體；共進(jìn)化；免疫防御

來源出版物：遺傳, 2011, 33(3): 213-218

被引頻次：14

鋅指蛋白核酸酶的作用原理及其應(yīng)用

鐘強(qiáng)，趙書紅

摘要：鋅指蛋白核酸酶（Zinc finger nucleases，ZFN）因其能特異性識(shí)別并切割DNA序列以及可設(shè)計(jì)性，被用于基因定點(diǎn)突變和外源基因定點(diǎn)整合。目前，ZFN技術(shù)以其準(zhǔn)確的靶位點(diǎn)設(shè)計(jì)能力和誘發(fā)高效率基因打靶的優(yōu)勢，越來越受到基因改造研究者的重視，已經(jīng)成功應(yīng)用于動(dòng)植物細(xì)胞、胚胎的基因改造。隨著鑒定靶DNA高親和力的鋅指蛋白（Zinc finger protein，ZFP）實(shí)驗(yàn)技術(shù)日漸成熟，可以預(yù)見到不久的將來這項(xiàng)技術(shù)會(huì)在基因工程和育種中得到廣泛應(yīng)用。文章介紹了鋅指蛋白識(shí)別DNA靶位點(diǎn)和ZFN介導(dǎo)的基因打靶（Double strand break gene targeting，DSB-GT）的原理，同時(shí)還綜述了目前ZFN技術(shù)用于基因改造的研究進(jìn)展。

關(guān)鍵詞：鋅指結(jié)構(gòu)域；鋅指蛋白；鋅指蛋白核酸酶；同源重組；基因打靶

來源出版物：遺傳, 2011, 33(2): 123-130

被引頻次：12

鋅指核酸酶在基因組靶向修飾中的應(yīng)用

王令，張存芳，張智英

摘要：同源重組和逆轉(zhuǎn)錄病毒介導(dǎo)轉(zhuǎn)基因法是目前基因組修飾中常用的兩種主要方法。由于這些傳統(tǒng)方法效率低，特異性差等缺點(diǎn)，制約了其在研究中的應(yīng)用。鋅指核酸酶（Zinc finger nuclease，ZFN）是一種人工合成酶，含有鋅指蛋白DNA結(jié)合域和非特異性核酸酶FokI結(jié)構(gòu)域。ZFN在對(duì)基因組的靶向修飾時(shí)，表現(xiàn)出高度特異性和高效性。最新研究結(jié)果顯示，鋅指核酸酶在哺乳動(dòng)物細(xì)胞和斑馬魚基因組靶向敲除的效率高達(dá)20%。這一技術(shù)的出現(xiàn)，將給基因組靶向修飾的研究和應(yīng)用領(lǐng)域帶來革命，特別是在基因治療人類疾病方面有巨大的潛力和廣闊的前景。

關(guān)鍵詞：鋅指核酸酶；同源重組；基因組；靶向修飾；基因治療

來源出版物：中國生物化學(xué)與分子生物學(xué)報(bào), 2009, 25(7): 585-589

被引頻次：7

利用CRISPR/Cas9敲除大鼠胰島素受體底物1（Irs1）基因

馬元武，馬婧，路迎冬，等

摘要：目的為研究胰島素受體底物1（Irs1）基因與代謝病之間的關(guān)系，我們利用CRISPR/Cas9系統(tǒng)敲除大鼠Irs1基因，為研究代謝病提供基因敲除大鼠。方法針對(duì)Irs1第一外顯子，設(shè)計(jì)CRISPR/Cas9作用靶點(diǎn)，構(gòu)建sgRNA表達(dá)質(zhì)粒。利用T7 RNA聚合酶體外轉(zhuǎn)錄sgRNA 和Cas9。將Cas9 mRNA和sgRNA混合物注射入SD大鼠的受精卵中，實(shí)現(xiàn)靶基因敲除。用T7EN1實(shí)驗(yàn)初步檢測靶基因的修飾情況，再經(jīng)過測序分析確定突變。結(jié)果獲得了5個(gè)在Irs1基因突變的首建鼠，突變效率為83%。結(jié)論得到了穩(wěn)定遺傳的Irs1基因敲除大鼠。

關(guān)鍵詞：Irs1；基因敲除；大鼠；CRISPR/Cas9

來源出版物：中國比較醫(yī)學(xué)雜志, 2014, 24(3): 55-60

被引頻次：7

鋅指核酸酶技術(shù)制備肌肉生長抑制素基因敲除的五指山小型豬成纖維細(xì)胞

曹隨忠，岳成鶴，李西睿

摘要：敲除豬肌肉生長抑制素（Myostatin，MSTN）基因可能提高豬瘦肉率，MSTN基因敲除豬也可作為相關(guān)疾病的動(dòng)物模型。文章利用鋅指核酸酶（Zinc-finger nucleases，ZFNs）技術(shù)敲除五指山小型豬胎兒成纖維細(xì)胞MSTN基因，為制備MSTN基因敲除豬奠定基礎(chǔ)。ZFNs質(zhì)?；蚓幋aZFNs的mRNA均能高效敲除MSTN基因，使用ZFNs mRNA能直接得到MSTN+/-和MSTN-/-兩種基因型的細(xì)胞克隆。DNA序列測定與分析發(fā)現(xiàn)，細(xì)胞克隆的突變類型多為ZFNs作用靶位點(diǎn)處不大于10 bp的堿基插入或缺失（92.18%）；氨基酸預(yù)測發(fā)現(xiàn)，突變型MSTN基因的終止密碼子常常提前出現(xiàn)。將MSTN基因敲除的細(xì)胞進(jìn)行體細(xì)胞核移植（Somatic cell nuclear transfer，SCNT）發(fā)現(xiàn)，胚胎體外早期發(fā)育潛力與野生型無顯著差異，表明這些細(xì)胞可用于后續(xù)MSTN基因敲除豬的制備。

關(guān)鍵詞：肌肉生長抑制素；基因敲除；五指山小型豬；鋅指核酸酶；MSTN-/-成纖維細(xì)胞

來源出版物：遺傳, 2013, 35(6): 778-785

被引頻次：6

CRISPR-Cas9介導(dǎo)的基因組編輯技術(shù)的研究進(jìn)展

鄭小梅，張曉立，于建東

摘要：CRISPR-Cas（clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins）系統(tǒng)為細(xì)菌與古生菌中抵御外源病毒或質(zhì)粒DNA入侵的獲得性免疫系統(tǒng)。該系統(tǒng)在crRNA的指導(dǎo)下，使核酸酶Cas識(shí)別并降解外源DNA。其中，Ⅱ型CRISPR-Cas系統(tǒng)最為簡單，僅包括一個(gè)核酸酶Cas9與tracrRNA: crRNA二聚體便可完成其生物功能?；贑RISPR-Cas9的基因組編輯技術(shù)的核心為將tracrRNA: crRNA設(shè)計(jì)為引導(dǎo)RNA，在引導(dǎo)RNA的指導(dǎo)下Cas9定位于特定DNA序列上，進(jìn)行DNA雙鏈切割，實(shí)現(xiàn)基因組的定向編輯。CRISPR-Cas9系統(tǒng)以設(shè)計(jì)操縱簡便、編輯高效與通用性廣等優(yōu)勢成為新一代基因組編輯技術(shù)，為基因組定向改造調(diào)控與應(yīng)用等帶來突破性革命。從CRISPR-Cas9介導(dǎo)的基因組編輯技術(shù)的發(fā)展與應(yīng)用等方面綜述其最新研究進(jìn)展，并著重介紹該技術(shù)的關(guān)鍵影響因素，為相關(guān)研究者提供參考。

關(guān)鍵詞：CRISPR-Cas系統(tǒng)；Cas9；基因組編輯技術(shù)

來源出版物：生物技術(shù)進(jìn)展, 2015, 1: 1-9

被引頻次：6

CRISPR-Cas9基因編輯技術(shù)在病毒感染疾病治療中的應(yīng)用

殷利眷，胡斯奇，郭斐

摘要：CRISPR-Cas9基因編輯技術(shù)是基于細(xì)菌或古細(xì)菌CRISPR介導(dǎo)的獲得性免疫系統(tǒng)衍生而來，由一段RNA通過堿基互補(bǔ)配對(duì)識(shí)別DNA，指導(dǎo)Cas9核酸酶切割識(shí)別的雙鏈DNA，誘發(fā)同源重組或非同源末端鏈接，進(jìn)而實(shí)現(xiàn)在目的 DNA上進(jìn)行編輯。病毒通過特異的受體侵染細(xì)胞，其基因組在細(xì)胞內(nèi)發(fā)生復(fù)制、轉(zhuǎn)錄、翻譯等過程完成其生活周期，某些DNA病毒或逆轉(zhuǎn)錄病毒基因組會(huì)整合到宿主基因組中?；蛑委熓遣《靖腥炯膊≈委煹男纶厔?。因此，基因編輯技術(shù)在持續(xù)感染的病毒或潛伏感染病毒疾病治療中具有重大的潛在意義。文章主要從CRISPR-Cas9作用機(jī)制以及在病毒感染疾病治療中的應(yīng)用等方面進(jìn)行了綜述。

關(guān)鍵詞：CRISPR；同源重組；非同源末端鏈接；基因編輯；病毒

來源出版物：遺傳, 2015, 37(5): 412-418

被引頻次：1741

Multiplex genome engineering using CRISPR/Cas systems

Cong, L; Ran, FA; Cox, D; et al.

Abstract: Functional elucidation of causal genetic variants and elements requires precise genome editingtechnologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

來源出版物：Science, 2013, 339(6121): 819-823

被引頻次：1268

A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity

Jinek, M; Chylinski, K; Fonfara, I; et al.

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA: crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

來源出版物：Science, 2012, 337(6096): 816-821

被引頻次：739

One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering

Wang, HY; Yang, H; Shivalila, CS; et al.

Abstract: Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming inter-crossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty-8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

來源出版物：Cell, 2013, 153(4): 910-918

被引頻次：710

A TALE nuclease architecture for efficient genome editing

Miller, JC; Tan, SY; Qiao, GJ; et al.

Abstract: Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editingand mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

來源出版物：Nature Biotechnology, 2011, 29(2): 143-148

被引頻次：655

Efficient genome editing in zebrafish using a CRISPR-Cas system

Hwang, WY; Fu, YF; Reyon, D; et al.

Abstract: In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR) CRISPR-associated (Gas) systems. Bacterial type II CRISPR systems have been adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator like effector nucleases.

來源出版物：Nature Biotechnology, 2013, 31(3): 227-229

被引頻次：552

High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

Fu, YF; Foden, JA; Khayter, C; et al.

Abstract: Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cellbased reporter assay to characterize off-target cleavage of CRISPR-associated (Cas) 9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

來源出版物：Nature Biotechnology, 2013, 31(9): 822-826

被引頻次：550

DNA targeting specificity of RNA-guided Cas9 nucleases

Hsu, PD; Scott, DA; Weinstein, JA; et al.

Abstract: The Streptococcus pyogenes Cas9 (SpCas9)nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing (1-10). Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

來源出版物：Nature Biotechnology, 2013, 31(9): 827-832

被引頻次：488

Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity

Ran, FA; Hsu, PD; Lin, CY; et al.

Abstract: Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50 to 1500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

來源出版物：Cell, 2013, 154(6): 1380-1389

被引頻次：484

Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases

Hockemeyer, D; Soldner, F; Beard, C; et al.

Abstract: Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

來源出版物：Nature Biotechnology, 2009, 27(9): 851-857

被引頻次：482

Establishment of HIV-1 resistance in CD4+T cells by genome editing using zinc-finger nucleases

Perez, EE; Wang, JB; Miller, JC; et al.

Abstract: Homozygosity for the naturally occurring Δ32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted ~50% of CCR5 alleles in a pool of primary human CD4+T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4+T cells had lower viral loads and higher CD4+T-cell counts than mice engrafted with wild-type CD4+T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4+T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN-modified autologous CD4+T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.

來源出版物：Nature Biotechnology, 2008, 26(7): 808-816

·推薦論文摘要·

利用CRISPR-Cas9系統(tǒng)定點(diǎn)突變豬MSTN基因的研究

張冬杰，劉娣，張旭，等

摘要：為了今后能夠有效的利用CRISPR-Cas9系統(tǒng)進(jìn)行基因功能研究及轉(zhuǎn)基因動(dòng)物的培育，本研究將帶有綠色熒光蛋白（GFP）和肌肉生長抑制素基因（Myostatin，MSTN）靶序列的CRISPR-Cas9系統(tǒng)轉(zhuǎn)染入PK15細(xì)胞系，利用流式細(xì)胞儀分離并收集轉(zhuǎn)染成功的陽性細(xì)胞，使用PCR擴(kuò)增結(jié)合克隆測序的方法檢測和分析CRISPR-Cas9系統(tǒng)的定點(diǎn)突變情況，以評(píng)價(jià)CRISPR-Cas9系統(tǒng)的作用效果。結(jié)果發(fā)現(xiàn)，隨機(jī)挑選的100個(gè)陽性克隆中，38個(gè)發(fā)生了變異，突變效率為38%，其中32個(gè)為插入突變，6個(gè)為缺失突變。插入突變中突變1（Mutation1）所占比例最高，為47.4%。本研究中CRISPR-Cas9系統(tǒng)的突變效率達(dá)到了38%，是目前進(jìn)行基因組編輯的一個(gè)有效工具，推測其突變類型具有一定的偏好性。

關(guān)鍵詞：CRISPR-Cas9系統(tǒng)；基因組編輯；突變率

來源出版物：畜牧獸醫(yī)學(xué)報(bào), 2015, 47(1): 207-212

聯(lián)系郵箱：劉娣，liudi1963@163.com

基因組定點(diǎn)編輯技術(shù)的研究進(jìn)展

張智輝，董少忠，寸韡，等

摘要：基因組編輯是建立在基因靶向修飾的基礎(chǔ)上，對(duì)生物基因組進(jìn)行改造的一項(xiàng)新技術(shù)。通過利用人工核酸酶ZFn、TALEN和細(xì)菌獲得性免疫系統(tǒng)CRISPR，可在靶位點(diǎn)制造DNA雙鏈切口進(jìn)而誘導(dǎo)細(xì)胞內(nèi)源性修復(fù)機(jī)制，通過同源重組修復(fù)或非同源末端連接途徑實(shí)現(xiàn)基因敲除、替換和糾正。對(duì)目前3個(gè)主要的基因組定點(diǎn)編輯技術(shù)的應(yīng)用和發(fā)展作一綜述。

關(guān)鍵詞：定點(diǎn)基因組編輯；鋅指核酸酶；TALEN；CRISPR/Cas9

來源出版物：生命科學(xué), 2013, 25(7): 735-742

聯(lián)系郵箱：董少忠，shaozhongdong@gmail.com

應(yīng)用SSA報(bào)告載體提高ZFN和CRISPR/Cas9對(duì)豬IGF2基因的打靶效率

吳金青，梅瑰，劉志國，等

摘要：IGF2（Insulin-like growth factor 2）基因作為最復(fù)雜多樣的生長因子之一，對(duì)豬胎兒發(fā)育以及出生后生長發(fā)育和肌肉生成起著非常重要的作用。通過基因組編輯技術(shù)對(duì)我國本地豬種的IGF2基因作精確的遺傳修飾，對(duì)于提高本地豬種的瘦肉率具有重要的育種意義。文章在藍(lán)塘豬胎兒成纖維細(xì)胞（Porcine fetal fibroblasts，PEF）中檢測了鋅指核酸酶（Zinc finger nucleases，ZFN）和CRISPR/Cas9 對(duì)IGF2 基因的打靶效率，結(jié)果表明CRISPR/Cas9對(duì)IGF2基因的切割效率最高可達(dá)9.2%，顯著高于ZFN的切割效率（<1%），但兩者均未達(dá)到作為體細(xì)胞核移植（Somatic nuclear transfer，SCNT）供體細(xì)胞所需的打靶效率。應(yīng)用SSA（Single-strand annealing）報(bào)告載體篩選技術(shù)來富集IGF2基因被ZFN和CRISPR/Cas9修飾過的PEF細(xì)胞，結(jié)果表明，該技術(shù)可使CRISPR/Cas9的打靶效率提高5倍左右，對(duì)ZFN的打靶效率具有更大的增強(qiáng)作用。

關(guān)鍵詞：IGF2基因；ZFN；CRISPR/Cas9；SSA報(bào)告系統(tǒng)

來源出版物：遺傳, 2015, 37(1): 55-62

聯(lián)系郵箱：何祖勇, zuyonghe@gmail.com

靈長類動(dòng)物的基因定向修飾——記運(yùn)用CRISPR/Cas9技術(shù)獲得基因定向敲除食蟹猴

牛昱宇

摘要：早在2001年1月，美國科學(xué)家在《Science》上報(bào)道了世界上第一只轉(zhuǎn)基因恒河猴，他們利用慢病毒轉(zhuǎn)染法成功地將綠色熒光蛋白（GFP）轉(zhuǎn)入恒河猴早期胚胎，并通過胚胎移植獲得了GFP整合和表達(dá)的轉(zhuǎn)基因猴ANDi，這是轉(zhuǎn)基因技術(shù)在非人靈長類上的首次成功嘗試。2008年，Shang-Hsun Yang等人成功構(gòu)建出了亨廷頓疾?。℉D）的轉(zhuǎn)基因恒河猴模型，作者將84個(gè)CAG重復(fù)序列連接到人HTT基因第一個(gè)外顯子上，并將其包裝成高滴度的慢病毒粒子（滴度>109 PFU/mL）后，在卵母細(xì)胞帶下注射病毒后再進(jìn)行單精注射受精和胚胎移植，成功得到了第一個(gè)轉(zhuǎn)基因非人靈長類疾病模型。然而，通過慢病毒介導(dǎo)的方式產(chǎn)生的轉(zhuǎn)基因動(dòng)物，外源基因的隨機(jī)插入使得它的表達(dá)和功能的實(shí)現(xiàn)具有一定的不確定性。在靈長類動(dòng)物中，由于技術(shù)限制無法通過與大小鼠同樣的技術(shù)路線獲得精確基因修飾的動(dòng)物。以TANLENs、CRISPR/Cas9技術(shù)為代表的人工核酸酶介導(dǎo)的基因組編輯技術(shù)的誕生，使得在靈長類動(dòng)物中實(shí)現(xiàn)精確基因修飾成為可能。季維智研究員團(tuán)隊(duì)與南京醫(yī)科大學(xué)沙家豪教授和南京大學(xué)黃行許教授團(tuán)隊(duì)密切合作，成功運(yùn)用Crispr/Cas9技術(shù)獲得了世界上首例基因定向敲除食蟹猴，證實(shí)了CRISPR/Cas9系統(tǒng)可以在靈長類動(dòng)物中很好地工作，并產(chǎn)生活體動(dòng)物。這為靈長類疾病動(dòng)物模型的發(fā)展奠定了良好的基礎(chǔ)。

關(guān)鍵詞：靈長類動(dòng)物；CRISPR/Cas9；轉(zhuǎn)基因動(dòng)物；沙家豪教授；人工核酸酶；非人靈長類；疾病動(dòng)物模型；恒河猴；胚胎移植；疾病模型

來源出版物：生命科學(xué), 2014, 26(004): 325-328

聯(lián)系郵箱：牛昱宇，niuyy@kbimed.com

Rationally engineered Cas9 nucleases with improved specificity

Slaymaker, IM; Gao, LY; Zetsche, B; et al.

Abstract: The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

來源出版物：Science, 2016, 351(6268): 84-88

聯(lián)系郵箱：Zhang, F; zhang@broadinstitute.org

In vivo gene editing in dystrophic mouse muscle and muscle stem cells

Tabebordbar, M; Zhu, KX; Cheng, JKW; et al.

Abstract: Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdxmouse model of DMD. Delivery by adenoassociated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAVDmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

來源出版物：Science, 2016, 351(6271): 407-411

聯(lián)系郵箱：Wagers, AJ; amy_wagers@harvard.edu

Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy

Nelson, CE; Hakim, CH; Ousterout, DG; et al.

Abstract: CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus–9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.

來源出版物：Science, 2016, 351(6271): 400-403

聯(lián)系郵箱：Olson, EN; eric.olson@utsouthwestern.edu

In vivo genome editing using Staphylococcus aureus Cas9

Ran, FA; Cong, L; Yan, WX; et al.

Abstract: The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcuspyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

來源出版物：Nature, 2015, 520(7546): 186-191

聯(lián)系郵箱：Zhang, F; zhang@broadinstitute.org

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects

Kleinstiver, BP; Pattanayak, V; Prew, MS; et al.

Abstract: CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of offtarget mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

來源出版物：Nature, 2016, 529 (7587): 490-495

聯(lián)系郵箱：Joung, JK; jjoung@mgh.harvard.edu

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

Tsai, SQ; Zheng, Z; Nguyen, NT; et al.

Abstract: CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint ‘hotspots’. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.

來源出版物：Nature Biotechnology, 2015, 33(2): 187-197

聯(lián)系郵箱：Joung, JK; stsai4@mgh.harvard.edu

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo

Zuris, JA; Thompson, DB; Shu, Y; et al.

Abstract: Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE-and Cas9-based transcription activators, and Cas9: sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9: sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNAtransfection. This approach also mediated efficient delivery of Cre recombinase and Cas9: sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cremediated recombination and 20% Cas9-mediated genome modification in hair cells.

來源出版物：Nature Biotechnology, 2015, 33(1): 73-80

聯(lián)系郵箱：Liu, DR; drliu@fas.harvard.edu

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

Liang, PP; Xu, YW; Zhang, XY; et al.

Abstract: This paper reviews the current status and implementation of battery chargers, charging power levels, and infrastructure for plug-in electric vehicles and hybrids. Charger systems are categorized into off-board and on-board types with unidirectional or bidirectional power flow. Unidirectional charging limits hardware requirements and simplifies interconnection issues. Bidirectional charging supports battery energy injection back to the grid. Typical on-board chargers restrict power because of weight, space, and cost constraints. They can be integrated with the electric drive to avoid these problems. The availability of charging infrastructure reduces on-board energy storage requirements and costs. On-board charger systems can be conductive or inductive. An off-board charger can be designed for high charging rates and is less constrained by size and weight. Level 1 (convenience), Level 2 (primary), and Level 3 (fast) power levels are discussed. Future aspects such as roadbed charging are presented. Various power level chargers and infrastructure configurations are presented, compared, and evaluated based on amount of power, charging time and location, cost, equipment, and other factors.

來源出版物：Protein & Cell, 2015, 6: 363-372

聯(lián)系郵箱：Zhou, CQ; zhoucanquan@hotmail.com

Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Kleinstiver, BP; Prew, MS; Tsai, SQ; et al.

Abstract: Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wildtype SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

來源出版物：Nature, 2015: 481–485

聯(lián)系郵箱：Joung, JK; jjoung@mgh.harvard.edu

Genetic screens in human cells using the CRISPR-Cas9 system

Wang, T; Wei, JJ; Sabatini, DM; et al.

Abstract: The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genesidentified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

來源出版物：Science, 2014, 343(6166): 80-84

聯(lián)系郵箱：Sabatini, DM; sabatini@wi.mit.edu

Improving CRISPR-Cas nuclease specificity using truncated guide RNAs

Fu, YF; Sander, JD; Reyon, D; et al.

Abstract: Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended targe. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.

來源出版物：Nature Biotechnology, 2014, 32(3): 279-284

聯(lián)系郵箱：Joung, JK; jsander@alumni.iastate.edu

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV

Tebas, P; Stein, D; Tang, WW; et al.

Abstract: BACKGROUND: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene (“gene editing”) — in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN) — is safe. METHODS: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatmentrelated adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study.

來源出版物：New England Journal of Medicine, 2014, 370(10): 901-910

聯(lián)系郵箱：Tebas, P; pablo.tebas@uphs.upenn.edu

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

Cho, SW; Kim, S; Kim, Y; et al.

Abstract: RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects ofRGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genomeediting efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

來源出版物：Genome Research, 2014, 24(1): 132-141

聯(lián)系郵箱：Kim, JS; jskim01@snu.ac.kr

Crystal structure of Cas9 in complex with guide RNA and target DNA

Nishimasu, H; Ran, FA; Hsu, PD; et al.

Abstract: The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA: DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

來源出版物：Cell, 2014, 156(5): 935-949

聯(lián)系郵箱：Zhang, F; zhang@broadinstitute.org

DNA-guided genome editing using the Natronobacterium gregoryi Argonaute

Gao, F; Shen, XZ; Jiang, F; Wu, Y; et al.

Abstract: The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA: DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system

Zetsche, B; Gootenberg, JS; Abudayyeh, OO; et al.

Abstract: The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided effectors. Class 1 effectors utilize multi-complexes, whereas class 2 effectors rely on singlecomponent effector such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from . Cpf1 is a single RNA-guided lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidominococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-systems and advances their genome editingapplications.

來源出版物：Cell, 2015, 163(3):759-71

Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

Yang, L; Güell, M; Niu, D; et al.

Abstract: The shortage of organs for transplantation is a major barrier to the treatment of organ failure. While porcine organs are considered promising, their use has been checked by concerns about transmission of porcine endogenous retroviruses (PERVs) to humans. Here, we describe the eradication of all in a porcine kidney epithelial line (PK15). We first determined the PK15 copy number to be 62. Using CRISPR-Cas9, we disrupted all 62 copies of the pol gene and demonstrated a >1000-fold reduction in transmission to using our engineered. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that can be inactivated for clinical application to porcine-to-human xenotransplantation.

來源出版物：Science, 2015, 350(6264):1101-1104

Correction of a genetic disease in mouse via use of CRISPR-Cas9

Wu, Y; Liang, D; Wang, Y; et al.

Abstract: The CRISPR-Cas9 system has been employed to generate mutant alleles in a range of different organisms. However, so far there have not been reports of use of this system for efficient correction of a genetic disease. Here we show that mice with a dominant mutation in Crygc gene that causes cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant allele. Correction occurred via homology-directed repair (HDR) based on an exogenously supplied oligonucleotide or the endogenous WT allele, with only rare evidence of off-target modifications. The resulting mice were fertile and able to transmit the corrected allele to their progeny. Thus, our study provides proof of principle for use of the CRISPR-Cas9 system to correct genetic disease.

來源出版物：Cell Stem Cell, 2013, 13(6): 659-662

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

Abudayyeh, OO; Gootenberg, JS; Konermann, S; et al.

Abstract: The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNADNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

來源出版物：Science, 2016: aaf5573

RNA-guided human genome engineering via Cas9

Mali, P; Yang, L; Esvelt, KM; et al.

Abstract: Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

來源出版物：Science, 2013, 339(6121): 823-826

編輯：王微

【作者單位：1. 西北農(nóng)林科技大學(xué)動(dòng)物科技學(xué)院；2. 陜西省興平市畜牧局】

doi:來源出版物：Nature Biotechnology, 2016 10.1038/ nbt.3547

Abstract: Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editingand mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

Abstract: Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.

Abstract: Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with singlenucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce doublestranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA: crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

Abstract:Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (> 10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavagecompetent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a > 40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.