李金環(huán)，壽佳，吳強

上海交通大學(xué)系統(tǒng)生物醫(yī)學(xué)研究院比較生物醫(yī)學(xué)研究中心，系統(tǒng)生物醫(yī)學(xué)教育部重點實驗室，上海 200240

CRISPR/Cas9系統(tǒng)在基因組DNA片段編輯中的應(yīng)用

李金環(huán)，壽佳，吳強

上海交通大學(xué)系統(tǒng)生物醫(yī)學(xué)研究院比較生物醫(yī)學(xué)研究中心，系統(tǒng)生物醫(yī)學(xué)教育部重點實驗室，上海 200240

源于細菌和古菌的Ⅱ型成簇規(guī)律間隔短回文重復(fù)系統(tǒng)[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)，CRISPR/Cas9]近年被改造成為基因組定點編輯的新技術(shù)。由于它具有設(shè)計簡單、操作方便、費用低廉等巨大優(yōu)勢，給遺傳操作領(lǐng)域帶來了一場革命性的改變。本文重點介紹了CRISPR/Cas9系統(tǒng)在基因組DNA片段靶向編輯方面的研究和應(yīng)用，主要包括DNA片段的刪除、反轉(zhuǎn)、重復(fù)、插入和易位，這一有效的DNA片段編輯方法為研究基因功能、調(diào)控元件、組織發(fā)育和疾病發(fā)生發(fā)展提供了有力手段。本文最后展望了Ⅱ型CRISPR/Cas9系統(tǒng)的應(yīng)用前景和其他類型CRISPR系統(tǒng)的應(yīng)用潛力，為開展利用基因組DNA片段靶向編輯進行基因調(diào)控和功能研究提供參考。

CRISPR/Cas9系統(tǒng)；DNA片段編輯；基因功能；調(diào)控元件；疾病發(fā)生

隨著人類基因組計劃(Human Genome Project, HGP)和 DNA元件百科全書(Encyclopedia of DNA Elements, ENCODE)項目的完成，科學(xué)家們分析和鑒定了大量的人類基因組中的DNA調(diào)節(jié)元件[1～4]。這些在基因表達調(diào)控中起重要作用的 DNA調(diào)節(jié)元件包括啟動子、增強子、絕緣子和沉默子等。然而很多調(diào)控元件由于遺傳操作方法的限制沒有得到實驗的驗證和功能的闡明[3, 5～10]。其次，人類基因組中包含很多串聯(lián)排列、高度相似和功能冗余的基因，這些基因形成復(fù)雜的基因簇，例如原鈣粘蛋白和尿苷二磷酸葡醛酸轉(zhuǎn)移酶(UGT)基因簇[6, 11～13]，研究基因簇的調(diào)控和功能面臨巨大的挑戰(zhàn)，急需開發(fā)遺傳學(xué)基因編輯的新技術(shù)和新方法。最后，人類基因組中存在很多結(jié)構(gòu)多樣性(Structural variation)，如DNA片段的刪除、反轉(zhuǎn)、重復(fù)、插入和易位[14,15]，基因組結(jié)構(gòu)的多樣性與復(fù)雜疾病的相關(guān)性研究也多見報道[16～21]。因此有效的 DNA片段編輯方法對闡明染色體重排(Chromosomal rearrangement)和基因組結(jié)構(gòu)多樣性及其如何影響復(fù)雜疾病的發(fā)生發(fā)展具有重要作用。

依賴于同源重組的方法進行 DNA片段編輯的傳統(tǒng)遺傳操作，包括DNA片段的刪除、反轉(zhuǎn)、重復(fù)、插入和易位，均已經(jīng)得到很好的發(fā)展[22～27]。近幾年新出現(xiàn)的基因組編輯核酸酶，如鋅指核酸酶(Zinc finger nucleases, ZFN)和類轉(zhuǎn)錄激活因子效應(yīng)物核酸酶(Transcription activator-like effector nucleases, TALEN)更進一步加快了基因組編輯技術(shù)的發(fā)展[28～30]。然而這些方法在操作上技術(shù)難度大，耗時費力昂貴并且效率不高[24, 30, 31]。源于細菌和古菌的Ⅱ型成簇規(guī)律間隔短回文重復(fù)系統(tǒng)[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)，CRISPR/Cas9]是新興基因組編輯技術(shù)，由于它設(shè)計非常簡單和操作方便，給遺傳操作領(lǐng)域帶來了一場革命性的改變。本文主要圍繞CRISPR/Cas9系統(tǒng)在基因組DNA片段編輯方面的應(yīng)用展開介紹。

1 CRISPR/Cas9基因組編輯系統(tǒng)

CRISPR/ Cas系統(tǒng)廣泛存在于細菌和古菌中，是它們的一種適應(yīng)性免疫系統(tǒng)，能夠識別自身和外源入侵DNA片段。CRISPR/ Cas系統(tǒng)有3種類型：Ⅰ型、Ⅱ型和Ⅲ型[32]。Ⅰ型和Ⅲ型 CRISPR/Cas系統(tǒng)較為復(fù)雜，需要多個Cas蛋白形成復(fù)合體切割DNA雙鏈，而Ⅱ型CRISPR/Cas系統(tǒng)只需要一個Cas9蛋白核酸酶來切割 DNA雙鏈,即為現(xiàn)在廣泛應(yīng)用于遺傳學(xué)基因編輯的CRISPR/Cas9系統(tǒng)。在CRISPR/Cas9系統(tǒng)中，Cas9核酸酶在兩個非編碼RNA[crRNA (CRISPR RNA)和tracrRNA (trans-activating crRNA)]的指導(dǎo)下直接對含有PAM (Protospacer adjacent motif)的DNA雙鏈上游3 bp處進行靶向雙鏈切割，形成特定位置的鈍末端 DNA 雙鏈斷裂(Double strand breaks, DSBs)[33～35]。

2012年，Jinek等[36]將 CRISPR/Cas9系統(tǒng)中crRNA和tracrRNA兩個非編碼RNA改造成一個RNA，即單導(dǎo)向 RNA (Single-guide RNA, sgRNA)，它能夠指導(dǎo)Cas9蛋白對特定的DNA序列進行靶向斷裂，為CRISPR/Cas9系統(tǒng)的廣泛應(yīng)用奠定基礎(chǔ)。隨后CRISPR/Cas9系統(tǒng)被應(yīng)用到真核生物中，引起一場遺傳操作的革命性改變。2013年，麻省理工學(xué)院張鋒團隊[37]和哈佛大學(xué) Church團隊[38]在Science雜志上同時發(fā)表了CRISPR/Cas9系統(tǒng)在哺乳動物細胞中的應(yīng)用，多個靶向 sgRNAs可以同時對基因組的多個位點進行斷裂，通過DNA修復(fù)系統(tǒng)的不精確修復(fù)在多個斷裂點周圍同時引發(fā)突變。但是，多個sgRNAs也可能引起意想不到的DNA片段編輯，包括可能的非常復(fù)雜的組合型(Combinatorial)DNA片段敲除、反轉(zhuǎn)、重復(fù)(多個sgRNAs靶點在同一條染色體上)[39]和復(fù)雜的染色體易位(多個 sgRNAs靶點在不同的染色體上)[39]。同時 Doudna實驗室[40]和Kim實驗室[41]也分別報道了CRISPR在哺乳動物細胞中的應(yīng)用。同年，張鋒和 Jaenisch團隊[42]在 Cell雜志上發(fā)表了CRISPR/Cas9系統(tǒng)在小鼠中的應(yīng)用，進一步推動了該系統(tǒng)在哺乳動物中的應(yīng)用研究。CRISPR/Cas9系統(tǒng)只需對sgRNA進行設(shè)計，具有實驗簡單、操作容易和節(jié)省時間等優(yōu)勢，已經(jīng)在不同物種中迅速發(fā)展起來并得到廣泛應(yīng)用[43～61]。

2 CRISPR/Cas9系統(tǒng)在基因組DNA片段編輯中的應(yīng)用

2.1 DNA片段靶向刪除(Targeted deletion of DNA fragments)

研究基因和調(diào)控元件的功能，可以通過刪除這一段 DNA片段來進行探索(圖 1A)。2013年，CRISPR/Cas9系統(tǒng)被應(yīng)用到哺乳動物細胞中，張鋒團隊[37]和 Church團隊[38]發(fā)現(xiàn)位于同一條染色體上的兩個sgRNAs對DNA雙鏈進行操作時，在獲得定點突變的同時，也存在DNA片段刪除的情況(圖1A)。張鋒團隊[37]在EMX1位點設(shè)計的相距119 bp的兩個靶向sgRNAs對該119 bp DNA片段進行了刪除。Church團隊[38]針對AAVS1位點設(shè)計的T1和T2靶向sgRNAs，同時轉(zhuǎn)染細胞后有效地獲得了19 bp的DNA片段的刪除。這一研究技術(shù)的出現(xiàn)，對闡明基因和調(diào)控元件的功能具有重要的意義。同年，F(xiàn)ujii 等[62]成功獲得了～10 kb的DNA片段刪除小鼠，并且成功繁育出下一代。同一時期，張博團隊[29]成功地在斑馬魚中對DNA片段進行了刪除。以上研究表明CRISPR/Cas9系統(tǒng)可以實現(xiàn)在不同物種、不同細胞系的DNA片段的刪除(圖1A)。

隨后，不同實驗室詳盡報道了DNA片段刪除的操作方法和效率[39, 63～65]。Canver等[63]和吳強團隊[39]在哺乳動物細胞中對幾十個堿基到大約一兆堿基的DNA片段進行了有效的編輯，表明此方法可以有效地對處于基因組任意位置的，任意長度的DNA片段進行操作(圖 1A)。Kraft等[64]和吳強團隊[39]在小鼠中對大約一千個堿基到一兆多堿基的不同長度的DNA 片段進行了有效操作，更加確定了CRISPR/Cas9系統(tǒng)可以在不同物種中進行任意長度的 DNA 片段操作(圖 1A)。Wang等[66]通過CRISPR/Cas9系統(tǒng)刪除了基因組中的DNA片段，鑒定了神經(jīng)系統(tǒng)中Blimp1的增強子的存在，并闡述了增強子在Blimp1基因中的重要作用。同時在神經(jīng)系統(tǒng)中還有其他的應(yīng)用，包括對 GluN1[67]、GluA2[67]和Grin1[68]等基因的刪除，表明CRISPR/Cas9系統(tǒng)在神經(jīng)相關(guān)基因研究中的重要作用。近年來越來越多的研究表明非編碼 RNA的存在可能具有重要的作用，通過 CRISPR/Cas9系統(tǒng)對非編碼 RNA (miR-21、miR-29a和 lncRNA-21A)進行基因組操作[69]，對于闡明非編碼RNA的功能具有重要作用。通過刪除15號染色體上大約30 Mb的DNA片段能夠獲得單倍體細胞系[70]，為研究基因組功能和進行遺傳篩選提供有用工具。這些研究表明CRISPR/Cas9系統(tǒng)可以實現(xiàn)從幾十堿基對到幾十兆堿基對的DNA片段的靶向刪除(圖1A)。

圖1 CRISPR/Cas9系統(tǒng)在基因組DNA片段編輯中的應(yīng)用

Cas9存在一定的脫靶效應(yīng)[71, 72]。Ran等[47]在小鼠受精卵中通過4個靶向sgRNAs在Cas9切口酶作用下實現(xiàn)DNA片段的刪除，他們在DYRK1A位點處成功地嘗試了500～6000 bp DNA片段的刪除。這種方法需要設(shè)計多一倍的靶向sgRNAs，雖然會減少脫靶率，但是多個 sgRNAs也可能引起復(fù)雜的組合型DNA片段編輯[39]?？傊?，利用CRISPR/Cas9系統(tǒng)可以實現(xiàn) DNA片段的靶向刪除(圖 1A)，盡管這一技術(shù)還有待進一步優(yōu)化發(fā)展。

2.2 DNA 片段靶向反轉(zhuǎn)(Targeted inversion of DNA fragments)

2014年，Choi等[73]將 CRISPR/Cas9系統(tǒng)應(yīng)用到模擬肺癌中的基因反轉(zhuǎn)事件(圖1B)，即EML4-ALK 和KIF5B-RET反轉(zhuǎn)事件(它們導(dǎo)致癌癥發(fā)生)。對于EML4-ALK反轉(zhuǎn)事件，在人胚腎HEK293T細胞中，他們在ALK和EML4基因里分別設(shè)計一個sgRNA，大約相距12 Mb，在Cas9作用下，形成DSB，檢測到了EML4-ALK反轉(zhuǎn)事件，同樣原理檢測到大約相距11 Mb的KIF5B-RET反轉(zhuǎn)事件。隨后Canver等[63]在研究DNA片段刪除時，也發(fā)現(xiàn)不同長度的DNA片段反轉(zhuǎn)事件的存在，包括2～1000 kb的DNA片段反轉(zhuǎn)(圖1B)，效率為12.8%～0.5%。同年，Maddalo 等[74]在小鼠肺組織中模擬了 EML4-ALK反轉(zhuǎn)事件，他們將Cas9和兩個sgRNAs一起構(gòu)建到腺病毒載體上，腺病毒可以有效地感染小鼠肺，4～7周后，在感染了腺病毒的小鼠肺上會頻繁地檢測到腫瘤。然而這種方法不能得到可以遺傳的小鼠后代，屬于體細胞的改變。

2015 年，Kraft等[64]在小鼠胚胎干細胞(Embryonic stem cells, ESCs)中，通過Cas9和兩個sgRNAs對1.1 kb～1.6 Mb之間的6個不同位點進行操作，檢測到反轉(zhuǎn)事件的存在(圖 1B)，然后將具有反轉(zhuǎn)事件的克隆培育以獲得嵌合體小鼠，該方法存在的缺陷是即使獲得了反轉(zhuǎn)事件的克隆，但有些克隆在后面培育小鼠的過程中并沒有成功，嵌合體小鼠獲得率低。吳強團隊[39]在哺乳動物細胞和小鼠中對DNA片段反轉(zhuǎn)進行了詳盡的研究(圖1B)。他們在哺乳動物細胞中對709 bp～1 Mb的7個不同長度的DNA片段進行了有效地反轉(zhuǎn)(圖 1B)；同時他們將Cas9 mRNA和兩個靶向sgRNAs注射到小鼠受精卵中，再將被注射后存活的受精卵移植到假孕鼠中獲得后代，篩選得到了反轉(zhuǎn)的嵌合小鼠，對960 bp～30 kb的不同長度的3個DNA片段進行了反轉(zhuǎn)，并且獲得了反轉(zhuǎn)事件的嵌合小鼠并實現(xiàn) DNA片段反轉(zhuǎn)的種系傳代，成功獲得了F1代反轉(zhuǎn)小鼠(圖1B)。在人類細胞系中，他們還利用 Cas9和兩個靶向sgRNAs靶向反轉(zhuǎn)了7個位于不同染色體、不同大小的DNA片段(圖1B)。這一簡單、高效、快速的DNA片段靶向反轉(zhuǎn)方法對于研究人類基因組中幾萬個潛在沉默子和啟動子、十幾萬個潛在絕緣子、幾十萬個潛在增強子和幾百萬個潛在調(diào)控序列非常有用，同時也將促進對串聯(lián)排列、高度相似和功能冗余的基因簇的調(diào)控和功能研究。例如，這種方法可以用來研究原鈣粘蛋白基因簇中方向相反的潛在絕緣子以及其與增強子、沉默子和啟動子之間的復(fù)雜關(guān)系[75]。隨后吳強團隊[76]利用此方法[39]反轉(zhuǎn)絕緣子相關(guān)元件CBS(CTCF-binding sites)來研究基因組三維高級拓?fù)浣Y(jié)構(gòu)以及增強子和啟動子的相互作用，闡述了CBS方向性在基因組三維高級結(jié)構(gòu)和基因表達調(diào)控中的重要作用。同年，Lupiá?ez等[77]根據(jù)Kraft等[64]發(fā)表的方法用小鼠建立了人類肢體發(fā)育不正常的DNA片段反轉(zhuǎn)模型。通過反轉(zhuǎn)DNA片段，能夠改變這個 DNA反轉(zhuǎn)片段所在位點周圍的染色質(zhì)相互作用，從而能夠研究DNA結(jié)構(gòu)多樣性在人類肢體發(fā)育畸形中所起的重要作用?？傊?，利用CRISPR/Cas9系統(tǒng)可以實現(xiàn)DNA片段的靶向反轉(zhuǎn)(圖1B)，進而研究基因表達調(diào)控和疾病發(fā)生機制。

2.3 DNA 片段靶向重復(fù)(Targeted duplication of DNA fragments)

在小鼠中能夠通過基因打靶和跨等位基因重組(Trans-allelic recombination)的方法獲得靶向DNA片段重復(fù)(圖1C)，但操作難度大，且效率偏低[27]。Kraft 等[64]在小鼠ESCs中，通過Cas9和兩個靶向sgRNAs對從1.1 kb～1.6 Mb之間的6個不同位點進行操作，在其中4個位點檢測到DNA重復(fù)事件的存在(圖1C)然后將具有 DNA片段重復(fù)事件的克隆進行培育以獲得嵌合體小鼠。吳強團隊[39]在哺乳動物細胞中對709 bp～1 Mb的7個不同長度的DNA片段進行操作時，其中在5個位點檢測到DNA片段靶向重復(fù)(圖1C)。在小鼠中對960 bp～30 kb的不同長度的3個DNA片段進行了操作，將Cas9 mRNA和兩個靶向sgRNAs注射到受精卵中，再將被注射后存活的受精卵移植到假孕鼠中獲得后代，在對1241 bp DNA片段操作時，從26只刪除小鼠中篩選到1只DNA片段靶向重復(fù)小鼠(圖1C)。盡管利用CRISPR/cas9系統(tǒng)進行DNA片段靶向重復(fù)研究才剛剛起步(圖1C)，效率還有待提高，但這一技術(shù)將促進 CNV(Copynumber variation)的功能研究。

2.4 DNA片段靶向插入(Targeted insertion of DNA fragments)

麻省理工學(xué)院張鋒團隊[37]在 EMX1位點利用Cas9切口酶形成的DNA切口或Cas9形成的DNA雙鏈斷裂都可以有效地介導(dǎo)同源重組(homologous recombination，HR)來插入包含兩個限制性酶切位點的 DNA片段(圖 1D)。哈佛大學(xué) Church團隊[38]在AAVS1位點設(shè)計的T1和T2靶向sgRNAs，在Cas9和供體DNA模板作用下，通過HR可以將綠色熒光蛋白基因(Green fluorescent protein gene，GFP)內(nèi)原來已經(jīng)插入的終止子以及來自于AAVS1位點68 bp的片段刪除，以得到正常的GFP，在此系統(tǒng)下他們也嘗試了另外5個靶向sgRNAs，都成功的通過HR表達了綠色熒光蛋白。所以，在哺乳動物細胞中CRISPR/Cas9系統(tǒng)可以有效地介導(dǎo)HR和實現(xiàn)DNA片段靶向插入。同年，Wang等[42]針對 Tet1設(shè)計sgRNA，并提供能夠?qū)?nèi)源的限制性酶切位點 SacⅠ改變成為EcoRⅠ的外源DNA模板，在Cas9作用下通過 HR成功地將小鼠受精卵基因組改變，同樣也針對Tet2進行操作，通過HR將內(nèi)源限制性酶切位點EcoR V改變成為EcoRⅠ。更為有趣的是，他們同時對 Tet1和 Tet2進行操作，不僅獲得了 Tet1和Tet2分別改變的受精卵，而且獲得了Tet1和Tet2同時改變的受精卵和小鼠，表明通過 HR介導(dǎo)可以同時實現(xiàn)多位點的DNA片段靶向插入。

Byrne等[78]詳盡地闡述了在人類誘導(dǎo)的多能干細胞中如何通過同源重組并結(jié)合 CRISPR技術(shù)進行靶向精確DNA片段插入。他們將小鼠的THY1基因靶向插入到人的 THY1基因位置并將其置換。對同源臂長度研究發(fā)現(xiàn)，選取同源臂長度從約100 bp～5 kb，利用CRISPR產(chǎn)生兩個DSB時，同源臂長度在～2 kb時插入效率最高，隨著同源臂長度減小同源重組效率降低，然而過長的同源臂并沒有明顯提高效率。對于存在一個DSB時，效率和左右兩側(cè)同源臂長度及 sgRNA靶向指導(dǎo)的切割位點位置有關(guān)；sgRNA切割在左側(cè)，右側(cè)同源臂長些時效率更高；sgRNA切割在右側(cè)，左側(cè)同源臂長些時效率更高。這種非對稱同源臂的設(shè)計允許一側(cè)較短的同源臂存在，可以更好地設(shè)計引物通過 PCR方法篩選克隆。Maruyama等[79]和Chu等[80]通過小分子抑制劑Scr7抑制NHEJ修復(fù)途徑中的連接酶IV，大大提高了HR效率，為 HR介導(dǎo)的基因組編輯更廣泛的應(yīng)用奠定基礎(chǔ)?？傊肅RISPR/Cas9系統(tǒng)可以實現(xiàn)DNA片段的靶向插入(圖1D)。

2.5 染色體靶向易位(Targeted translocation of Chromosomal fragments)

染色體易位常常與腫瘤發(fā)生有著密不可分的關(guān)系，因此染色體易位的研究對于人們認(rèn)識腫瘤的發(fā)病機理尤為重要[81, 82]。例如，在慢性粒細胞白血病發(fā)生發(fā)展中，染色體易位造成了費城染色體(Philadelphia chromosome)形成，它能夠編碼BCR-ABL融合蛋白產(chǎn)生白血病[83]。在不同的染色體上引入同時出現(xiàn)的 DSBs是引發(fā)染色體易位必不可少的環(huán)節(jié)之一。利用I-Sce I或鋅指核酸酶(ZFN)在染色體的特定位點引入DSBs從而引發(fā)染色體易位[84～86]。自 CRISPR技術(shù)出現(xiàn)以來，因其高效、便捷、易操作等特性，已有不少科研團隊將其應(yīng)用于染色體易位的相關(guān)研究(圖1E)。

2014年，Choi等[73]利用CRISPR/Cas9技術(shù)在人胚腎HEK293T細胞和肺的上皮細胞AALE中模擬了引發(fā)肺癌的ROS1基因和CD74基因的染色體易位，說明Cas9所誘導(dǎo)的雙鏈斷裂能夠?qū)崿F(xiàn)染色體靶向易位。同年，Brunet團隊[87]在人類細胞中利用ZFNs、TALENs和Cas9三種核酸酶，在不同的兩條染色體上引入 DSBs造成染色體易位，通過對其斷裂連接點的研究，發(fā)現(xiàn)染色體易位修復(fù)機制具有物種特異性，人類細胞和老鼠細胞利用不同的DNA修復(fù)系統(tǒng)。2015年，Lagutina等[88]在鼠的肌細胞中利用CRISPR系統(tǒng)模擬了人橫紋肌肉瘤的Pax3-Foxo1基因易位事件。以上研究表明，CRISPR系統(tǒng)有助于人們構(gòu)建與癌癥相關(guān)的染色體易位模型(圖 1E)，從而促進癌癥發(fā)病機理研究。

3 結(jié)語與展望

Ⅱ型CRISPR/Cas9系統(tǒng)自出現(xiàn)以來，得到迅速而廣泛的應(yīng)用，是生物學(xué)研究歷史上類似PCR一樣重要的技術(shù)變革。由于它具有設(shè)計簡單，容易操作和成本低廉等重要優(yōu)勢，全世界生物學(xué)實驗室都可以利用它去進行基因編輯研究。CRISPR/Cas9可以實現(xiàn)基因組定點編輯，在DNA修復(fù)系統(tǒng)的作用下實現(xiàn)特定位點突變[37, 38]，特定位點的靶向突變可以實現(xiàn)基因功能的喪失。但有時在一個靶向 sgRNA和Cas9作用后所帶來的突變并不能完全達到效果，這時設(shè)計兩個靶向sgRNAs即可以通過DNA片段的靶向刪除[29, 37～39, 62～64]，有效地實現(xiàn)基因功能缺失。通過兩個sgRNAs也可以實現(xiàn)DNA片段的反轉(zhuǎn)和重復(fù)[39, 64, 77]。如果設(shè)計外源DNA模板，也可以通過HR實現(xiàn)精確的DNA靶向插入[37, 38, 78, 79]。如果靶點在不同染色體上，還可以實現(xiàn)DNA片段易位。所以CRISPR/Cas9系統(tǒng)可以實現(xiàn)DNA片段的刪除、反轉(zhuǎn)、重復(fù)、插入和易位(圖1)，為研究基因功能、調(diào)控元件和疾病病理機制提供有效手段。

CRISPR/Cas9系統(tǒng)對靶向DNA片段的編輯效率受多種因素的影響。其中，靶向 sgRNA設(shè)計對于DNA片段的編輯效率尤為重要。靶向 sgRNA序列可以選擇 GC含量高一些的序列，盡量堿基分布均勻，還有靶點盡量選擇在DNA超敏位點，這樣可以更好地實現(xiàn)Cas9對DNA片段的切割[39]。DNA片段長度能夠影響其刪除效率[63]，隨著 DNA片段長度的增加 DNA片段刪除效率降低[63]，但片段長度也可能不影響其編輯效率[39]，具體的 DNA片段編輯效率可能與基因組三維高級構(gòu)象(Three dimensional higher-order architecture)相關(guān)。CRISPR系統(tǒng)可以實現(xiàn)多位點DNA編輯[37, 42]，但是當(dāng)多個靶向sgRNAs作用在同一條染色體上，也可能會引起非常復(fù)雜的組合型DNA片段編輯(刪除、反轉(zhuǎn)、重復(fù)可能會同時發(fā)生)[39]，因此在用多個靶向sgRNAs進行實驗時要考慮到 DNA片段編輯的復(fù)雜性。對于兩個靶向sgRNAs來說，DNA片段刪除和反轉(zhuǎn)的效率高于DNA片段重復(fù)，DNA片段刪除和反轉(zhuǎn)的效率相差不大[39]。總之， 對 CRISPR/Cas9技術(shù)的研究才剛剛開始，盡管其在不同細胞類型和物種中對DNA片段的編輯效率還有待提高，但該技術(shù)一定會像其他CRISPR技術(shù)一樣很快得到廣泛應(yīng)用。

通常CRISPR技術(shù)用來切割DNA，Ⅱ型Cas9切割 DNA必須要有 PAM位點的存在。O’Connell 等[89]設(shè)計了針對RNA切割位點的含有PAM的互補DNA寡核苷酸，發(fā)現(xiàn)Cas9可以切割RNA。這項研究表明CRISPR技術(shù)也可以用來研究RNA的功能。來自Francisella novicida的Ⅱ型CRISPR/Cas9也有相關(guān)報道，它可以對細菌和病毒RNA進行切割[90, 91]。Ⅰ型和Ⅱ型CRISPR/Cas系統(tǒng)都需要PAM位點的存在[32]，即使基因組上存在很多這樣的PAM位點，但

是有時在實驗設(shè)計時還是會受到 PAM位點選取的限制，而Ⅲ型CRISPR/Cas系統(tǒng)沒有PAM位點的限制，在實驗設(shè)計上更加靈活。對Ⅲ型 CRISPR/Cas系統(tǒng)的應(yīng)用目前已經(jīng)有一些研究報道[92～94]，在III-A 型CRISPR系統(tǒng)中核酸酶在crRNA(CRISPR RNA)的介導(dǎo)下能切割crRNA互補的DNA鏈和靶向DNA序列轉(zhuǎn)錄出來的RNA?？傊?，不斷發(fā)展的CRISPR技術(shù)必將促進靶向DNA片段編輯應(yīng)用，加速人們理解基因表達調(diào)控機制和疾病發(fā)生原因。

[1] Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC,Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H,Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ. Initial sequencing and analysis of the human genome. Nature, 2001, 409(6822): 860–921.

[2] Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng ZM, Di Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan WN, Ge WM, Gong FC, Gu ZP, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke ZX, Ketchum KA, Lai ZW, Lei YD, Li ZY, Li JY, Liang Y, Lin XY, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun JT, Wang ZY, Wang AH, Wang X, Wang J, Wei MH, Wides R, Xiao CL, Yan CH, Yao A, Ye J, Zhan M, Zhang WQ, Zhang HY, Zhao Q, Zheng LS, Zhong F, Zhong WY, Zhu SC, Zhao SY, Gilbert D, Baumhueter S, Spier G, Carter C, Cravchik A, Woodage T, Ali F, An H, Awe A, Baldwin D, Baden H, Barnstead M, Barrow I, Beeson K, Busam D, Carver A, Center A, Cheng ML, Curry L, Danaher S, Davenport L, Desilets R, Dietz S, Dodson K, Doup L, Ferriera S, Garg N, Gluecksmann A, Hart B, Haynes J, Haynes C, Heiner C, Hladun S, Hostin D, Houck J, Howland T, Ibegwam C, Johnson J, Kalush F, Kline L, Koduru S, Love A, Mann F, May D, McCawley S, McIntosh T, McMullen I, Moy M, Moy L, Murphy B, Nelson K, Pfannkoch C, Pratts E, Puri V, Qureshi H, Reardon M, Rodriguez R, Rogers YH, Romblad D, Ruhfel B, Scott R, Sitter C, Smallwood M, Stewart E, Strong R, Suh E, Thomas R, Tint NN, Tse S, Vech C, Wang G, Wetter J, Williams S, Williams M, Windsor S, Winn-Deen E, Wolfe K, Zaveri J, Zaveri K, Abril JF, Guigo R, Campbell MJ, Sjolander KV, Karlak B, Kejariwal A, Mi H, Lazareva B, Hatton T, Narechania A, Diemer K, Muruganujan A, Guo N, Sato S, Bafna V, Istrail S, Lippert R, Schwartz R, Walenz B, Yooseph S, Allen D, Basu A, Baxendale J, Blick L, Caminha M, Carnes-Stine J, Caulk P, Chiang YH, Coyne M, Dahlke C, Mays A, Dombroski M, Donnelly M, Ely D, Esparham S, Fosler C, Gire H, Glanowski S, Glasser K, Glodek A, Gorokhov M, Graham K, Gropman B, Harris M, Heil J, Henderson S, Hoover J, Jennings D, Jordan C, Jordan J, Kasha J, Kagan L, Kraft C, Levitsky A, Lewis M, Liu X, Lopez J, Ma D, Majoros W, McDaniel J, Murphy S, Newman M, Nguyen T, Nguyen N, Nodell M, Pan S, Peck J, Peterson M, Rowe W, Sanders R, Scott J, Simpson M, Smith T, Sprague A, Stockwell T, Turner R, Venter E, Wang M, Wen MY, Wu D, Wu M, Xia A, Zandieh A, Zhu XH. The sequence of the human genome. Science, 2001, 291(5507): 1304–1351.

[3] The ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature, 2012, 489(7414): 57–74.

[4] Stamatoyannopoulos JA. What does our genome encode?. Genome Res, 2012, 22(9): 1602–1611.

[5] Banerji J, Olson L, Schaffner W. A lymphocytespecific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell, 1983, 33(3): 729–740.

[6] Zhang T, Haws P, Wu Q. Multiple variable first exons: a mechanism for cell- and tissue-specific gene regulation. Genome Res, 2004, 14(1): 79–89.

[7] Neph S, Vierstra J, Stergachis AB, Reynolds AP, Haugen E, Vernot B, Thurman RE, John S, Sandstrom R, Johnson AK, Maurano MT, Humbert R, Rynes E, Wang H, Vong S, Lee K, Bates D, Diegel M, Roach V, Dunn D, Neri J, Schafer A, Hansen RS, Kutyavin T, Giste E, Weaver M, Canfield T, Sabo P, Zhang M, Balasundaram G, Byron R, MacCoss MJ, Akey JM, Bender MA, Groudine M, Kaul R, Stamatoyannopoulos JA. An expansive human regulatory lexicon encoded in transcription factor footprints. Nature, 2012, 489(7414): 83–90.

[8] Shen Y, Yue F, McCleary DF, Ye Z, Edsall L, Kuan S, Wagner U, Dixon J, Lee L, Lobanenkov VV, Ren B. Amap of the cis-regulatory sequences in the mouse genome. Nature, 2012, 488(7409): 116–120.

[9] Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, Sheffield NC, Stergachis AB, Wang H, Vernot B, Garg K, John S, Sandstrom R, Bates D, Boatman L, Canfield TK, Diegel M, Dunn D, Ebersol AK, Frum T, Giste E, Johnson AK, Johnson EM, Kutyavin T, Lajoie B, Lee BK, Lee K, London D, Lotakis D, Neph S, Neri F, Nguyen ED, Qu H, Reynolds AP, Roach V, Safi A, Sanchez ME, Sanyal A, Shafer A, Simon JM, Song LY, Vong S, Weaver M, Yan YQ, Zhang ZC, Zhang ZZ, Lenhard B, Tewari M, Dorschner MO, Hansen RS, Navas PA, Stamatoyannopoulos G, Iyer VR, Lieb JD, Sunyaev SR, Akey JM, Sabo PJ, Kaul R, Furey TS, Dekker J, Crawford GE, Stamatoyannopoulos JA. The accessible chromatin landscape of the human genome. Nature, 2012, 489(7414): 75–82.

[10] de Laat W, Duboule D. Topology of mammalian developmental enhancers and their regulatory landscapes. Nature, 2013, 502(7472): 499–506.

[11] Wu Q, Maniatis T. A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell, 1999, 97(6): 779–790.

[12] Wu Q. Comparative genomics and diversifying selection of the clustered vertebrate protocadherin genes. Genetics, 2005, 169(4): 2179–2188.

[13] Li C, Wu Q. Adaptive evolution of multiple-variable exons and structural diversity of drug-metabolizing enzymes. BMC Evol Biol, 2007, 7: 69.

[14] Sharp AJ, Cheng Z, Eichler EE. Structural variation of the human genome. Annu Rev Genomics Hum Genet, 2006, 7: 407–442.

[15] Stankiewicz P, Lupski JR. Structural variation in the human genome and its role in disease. Annu Rev Med, 2010, 61: 437–455.

[16] Feuk L. Inversion variants in the human genome: role in disease and genome architecture. Genome Med, 2010, 2(2): 11.

[17] Lupski JR. Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet, 1998, 14(10): 417–422.

[18] Lupski JR, Stankiewicz P. Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoS Genet, 2005, 1(6): e49.

[19] Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H, Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y, Aburatani H, Niki T, Sohara Y, Sugiyama Y, Mano H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature, 2007, 448(7153): 561–566.

[20] Stephens PJ, Greenman CD, Fu BY, Yang FT, Bignell GR, Mudie LJ, Pleasance ED, Lau KW, Beare D, Stebbings LA, McLaren S, Lin ML, McBride DJ, Varela I, Nik-Zainal S, Leroy C, Jia M, Menzies A, Butler AP, Teague JW, Quail MA, Burton J, Swerdlow H, Carter NP, Morsberger LA, Iacobuzio-Donahue C, Follows GA, Green AR, Flanagan AM, Stratton MR, Futreal PA, Campbell PJ. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell, 2011, 144(1): 27–40.

[21] Baca SC, Prandi D, Lawrence MS, Mosquera JM, Romanel A, Drier Y, Park K, Kitabayashi N, MacDonald TY, Ghandi M, Van Allen E, Kryukov GV, Sboner A, Theurillat JP, Soong TD, Nickerson E, Auclair D, Tewari A, Beltran H, Onofrio RC, Boysen G, Guiducci C, Barbieri CE, Cibulskis K, Sivachenko A, Carter SL, Saksena G, Voet D, Ramos AH, Winckler W, Cipicchio M, Ardlie K, Kantoff PW, Berger MF, Gabriel SB, Golub TR, Meyerson M, Lander ES, Elemento O, Getz G, Demichelis F, Rubin MA, Garraway LA. Punctuated evolution of prostate cancer genomes. Cell, 2013, 153(3): 666–677.

[22] Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature, 1985, 317(6034): 230–234.

[23] Thomas KR, Capecchi MR. Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature, 1986, 324(6092): 34–38.

[24] Capecchi MR. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet, 2005, 6(6): 507–512.

[25] Zheng BH, Sage M, Sheppeard EA, Jurecic V, Bradley A. Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol Cell Biol, 2000, 20(2): 648–655.

[26] Spitz F, Herkenne C, Morris MA, Duboule D. Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes. Nat Genet, 2005, 37(8): 889–893.

[27] Wu S, Ying GX, Wu Q, Capecchi MR. Toward simpler and faster genome-wide mutagenesis in mice. Nat Genet, 2007, 39(7): 922–930.

[28] Gupta A, Hall VL, Kok FO, Shin M, McNulty JC, Lawson ND, Wolfe SA. Targeted chromosomal deletions and inversions in zebrafish. Genome Res, 2013, 23(6): 1008–1017.

[29] Xiao A, Wang ZX, Hu YY, Wu YD, Luo Z, Yang ZP, Zu Y, Li WY, Huang P, Tong XJ, Zhu ZY, Lin S, Zhang B.Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res, 2013, 41(14): e141.

[30] Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem, 2014, 83: 409–439.

[31] Yu YJ, Bradley A. Engineering chromosomal rearrangements in mice. Nat Rev Genet, 2001, 2(10): 780–790.

[32] Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol, 2011, 9(6): 467–477.

[33] Mojica FJ, Díez-Villase?or C, García-Martínez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology, 2009, 155(Pt 3): 733–740.

[34] Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010, 468(7320): 67–71.

[35] Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011, 471(7340): 602–607.

[36] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096): 816–821.

[37] Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, Hsu PD, Wu XB, Jiang WY, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819–823.

[38] Mali P, Yang LH, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science, 2013, 339(6121): 823–826.

[39] Li JH, Shou J, Guo Y, Tang YX, Wu YH, Jia ZL, Zhai YA, Chen ZF, Xu Q, Wu Q. Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9. J Mol Cell Biol, 2015, 7(4): 284–298.

[40] Jinek M, East A, Cheng A, Lin S, Ma EB, Doudna J. RNA-programmed genome editing in human cells. eLife, 2013, 2: e00471.

[41] Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol, 2013, 31(3): 230–232.

[42] Wang HY, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 2013, 153(4): 910–918.

[43] Gilbert LA, Larson MH, Morsut L, Liu ZR, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013, 154(2): 442–451.

[44] Li DL, Qiu ZW, Shao YJ, Chen YT, Guan YT, Liu MZ, Li YM, Gao N, Wang LR, Lu XL, Zhao YX, Liu MY. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013, 31(8): 681–683.

[45] Malina A, Mills JR, Cencic R, Yan YF, Fraser J, Schippers LM, Paquet M, Dostie J, Pelletier J. Repurposing CRISPR/Cas9 for in situ functional assays. Genes Dev, 2013, 27(23): 2602–2614.

[46] Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152(5): 1173–1183.

[47] Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 2013, 154(6): 1380–1389.

[48] Wang T, Wei JJ, Sabatini DM, Lander ES. Genetic screens in human cells using the CRISPR-Cas9 system. Science, 2014, 343(6166): 80–84.

[49] Wei CX, Liu JY, Yu ZS, Zhang B, Gao GJ, Jiao RJ. TALEN or Cas9-rapid, efficient and specific choices for genome modifications. J Genet Genomics, 2013, 40(6): 281–289.

[50] Cai M, Yang Y. Targeted genome editing tools for disease modeling and gene therapy. Curr Gene Ther, 2014, 14(1): 2–9.

[51] Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 2014, 346(6213): 1258096.

[52] González F, Zhu Z, Shi ZD, Lelli K, Verma N, Li QV, Huangfu D. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell, 2014, 15(2): 215–226.

[53] Harrison MM, Jenkins BV, O'Connor-Giles KM, Wildonger J. A CRISPR view of development. Genes Dev, 2014, 28(17): 1859–1872.

[54] Hsu PD, Lander ES, Zhang F. Development andapplications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157(6): 1262–1278.

[55] Kiani S, Beal J, Ebrahimkhani MR, Huh J, Hall RN, Xie Z, Li YQ, Weiss R. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat Methods, 2014, 11(7): 723–726.

[56] Niu YY, Shen B, Cui YQ, Chen YC, Wang JY, Wang L, Kang Y, Zhao XY, Si W, Li W, Xiang AP, Zhou JK, Guo XJ, Bi Y, Si CY, Hu B, Dong GY, Wang H, Zhou ZM, Li TQ, Tan T, Pu XQ, Wang F, Ji SH, Zhou Q, Huang XX, Ji WZ, Sha JH. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 2014, 156(4): 836–843.

[57] Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 2014, 343(6166): 84–87.

[58] Shen B, Zhang WS, Zhang J, Zhou JK, Wang JY, Chen L, Wang L, Hodgkins A, Iyer V, Huang XX, Skarnes WC. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods, 2014, 11(4): 399–402.

[59] Zhou YX, Zhu SY, Cai CZ, Yuan PF, Li CM, Huang YY, Wei WS. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature, 2014, 509(7501): 487–491.

[60] Wan HF, Feng CJ, Teng F, Yang SH, Hu BY, Niu YY, Xiang AP, Fang WZ, Ji WZ, Li W, Zhao XY, Zhou Q. One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system. Cell Res, 2015, 25(2): 258–261.

[61] Wu YX, Zhou H, Fan XY, Zhang Y, Zhang M, Wang YH, Xie ZF, Bai MZ, Yin Q, Liang D, Tang W, Liao JY, Zhou CK, Liu WJ, Zhu P, Guo HS, Pan H, Wu CL, Shi HJ, Wu LG, Tang FC, Li JS. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res, 2015, 25(1): 67–79. [62] Fujii W, Kawasaki K, Sugiura K, Naito K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013, 41(20): e187.

[63] Canver MC, Bauer DE, Dass A, Yien YY, Chung J, Masuda T, Maeda T, Paw BH, Orkin SH. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J Biol Chem, 2014, 289(31): 21312–21324.

[64] Kraft K, Geuer S, Will AJ, Chan WL, Paliou C, Borschiwer M, Harabula I, Wittler L, Franke M, Ibrahim DM, Kragesteen BK, Spielmann M, Mundlos S, Lupiá?ez DG, Andrey G. Deletions, inversions, duplications: engineering of structural variants using CRISPR/Cas in Mice. Cell Rep, 2015, 10(5): 833–839.

[65] Li YX, Park AI, Mou HW, Colpan C, Bizhanova A, Akama-Garren E, Joshi N, Hendrickson EA, Feldser D, Yin H, Anderson DG, Jacks T, Weng ZP, Xue W. A versatile reporter system for CRISPR-mediated chromosomal rearrangements. Genome Biol, 2015, 16: 111.

[66] Wang S, Sengel C, Emerson MM, Cepko CL. A gene regulatory network controls the binary fate decision of rod and bipolar cells in the vertebrate retina. Dev Cell, 2014, 30(5): 513–527.

[67] Incontro S, Asensio CS, Edwards RH, Nicoll RA. Efficient, complete deletion of synaptic proteins using CRISPR. Neuron, 2014, 83(5): 1051–1057.

[68] Straub C, Granger AJ, Saulnier JL, Sabatini BL. CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons. PLoS One, 2014, 9(8): e105584.

[69] Ho TT, Zhou NJ, Huang JG, Koirala P, Xu M, Fung R, Wu FT, Mo YY. Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines. Nucleic Acids Res, 2015, 43(3): e17.

[70] Essletzbichler P, Konopka T, Santoro F, Chen D, Gapp BV, Kralovics R, Brummelkamp TR, Nijman SMB, Bürckstummer T. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res, 2014, 24(12): 2059–2065.

[71] Fu YF, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013, 31(9): 822–826.

[72] Pattanayak V, Lin S, Guilinger JP, Ma EB, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol, 2013, 31(9): 839–843.

[73] Choi PS, Meyerson M. Targeted genomic rearrangements using CRISPR/Cas technology. Nat Commun, 2014, 5: 3728.

[74] Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA, Han YC, Ogrodowski P, Crippa A, Rekhtman N, de Stanchina E, Lowe SW, Ventura A. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature, 2014, 516(7531): 423–427.

[75] Guo Y, Monahan K, Wu HY, Gertz J, Varley KE, Li W, Myers RM, Maniatis T, Wu Q. CTCF/cohesin-mediated DNA looping is required for protocadherin alpha promoter choice. Proc Natl Acad Sci USA, 2012, 109(51):21081–21086.

[76] Guo Y, Xu Q, Canzio D, Shou J, Li JH, Gorkin DU, Jung I, Wu HY, Zhai Y, Tang YX, Lu YC, Wu YH, Jia ZL, Li W, Zhang MQ, Ren B, Krainer AR, Maniatis T, Wu Q. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell, 2015, 162(4): 900–910.

[77] Lupiá?ez DG, Kraft K, Heinrich V, Krawitz P, Brancati F, Klopocki E, Horn D, Kayserili H, Opitz JM, Laxova R, Santos-Simarro F, Gilbert-Dussardier B, Wittler L, Borschiwer M, Haas SA, Osterwalder M, Franke M, Timmermann B, Hecht J, Spielmann M, Visel A, Mundlos S. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell, 2015, 161(5): 1012–1025.

[78] Byrne SM, Ortiz L, Mali P, Aach J, Church GM. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res, 2015, 43(3): e21.

[79] Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol, 2015, 33(5): 538–542.

[80] Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol, 2015, 33(5): 543–548.

[81] Mitelman F, Johansson B, Mertens F. The impact of translocations and gene fusions on cancer causation. Nat Rev Cancer, 2007, 7(4): 233–245.

[82] Mani RS, Chinnaiyan AM. Triggers for genomic rearrangements: insights into genomic, cellular and environmental influences. Nat Rev Genet, 2010, 11(12): 819–829.

[83] Nowell PC, Hungerford DA. Chromosome studies on normal and leukemic human leukocytes. J Natl Cancer Inst, 1960, 25: 85–109.

[84] Weinstock DM, Elliott B, Jasin M. A model of oncogenic rearrangements: differences between chromosomal translocation mechanisms and simple double-strand break repair. Blood, 2006, 107(2): 777–780.

[85] Brunet E, Simsek D, Tomishima M, DeKelver R, Choi VM, Gregory P, Urnov F, Weinstock DM, Jasin M. Chromosomal translocations induced at specified loci in human stem cells. Proc Natl Acad Sci USA, 2009, 106(26): 10620–10625.

[86] Simsek D, Jasin M. Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation. Nat Struct Mol Biol, 2010, 17(4): 410–416.

[87] Ghezraoui H, Piganeau M, Renouf B, Renaud JB, Sallmyr A, Ruis B, Oh S, Tomkinson AE, Hendrickson EA, Giovannangeli C, Jasin M, Brunet E. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell, 2014, 55(6): 829–842.

[88] Lagutina IV, Valentine V, Picchione F, Harwood F, Valentine MB, Villarejo-Balcells B, Carvajal JJ, Grosveld GC. Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease. PLoS Genet, 2015, 11(2): e1004951.

[89] O'Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature, 2014, 516(7530): 263–266.

[90] Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature, 2013, 497(7448): 254–257. [91] Price AA, Sampson TR, Ratner HK, Grakoui A, Weiss DS. Cas9-mediated targeting of viral RNA in eukaryotic cells. Proc Natl Acad Sci USA, 2015, 112(19): 6164–6169.

[92] Tamulaitis G, Kazlauskiene M, Manakova E, Venclovas C, Nwokeoji AO, Dickman MJ, Horvath P, Siksnys V. Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. Mol Cell, 2014, 56(4): 506–517.

[93] Staals RHJ, Zhu YF, Taylor DW, Kornfeld JE, Sharma K, Barendregt A, Koehorst JJ, Vlot M, Neupane N, Varossieau K, Sakamoto K, Suzuki T, Dohmae N, Yokoyama S, Schaap PJ, Urlaub H, Heck AJR, Nogales E, Doudna JA, Shinkai A, van der Oost J. RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Mol Cell, 2014, 56(4): 518–530.

[94] Samai P, Pyenson N, Jiang WY, Goldberg GW, Hatoum-Aslan A, Marraffini LA. Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity. Cell, 2015, 161(5): 1164–1174.

(責(zé)任編委: 張博)

DNA fragment editing of genomes by CRISPR/Cas9

Jinhuan Li, Jia Shou, Qiang Wu
Key Laboratory of Systems Biomedicine (Ministry of Education), Center for Comparative Biomedicine, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system from bacteria and archaea emerged recently as a new powerful technology of genome editing in virtually any organism. Due to its simplicity and cost effectiveness, a revolutionary change of genetics has occurred. Here, we summarize the recent development of DNA fragment editing methods by CRISPR/Cas9 and describe targeted DNA fragment deletions, inversions, duplications, insertions, and translocations. The efficient method of DNA fragment editing provides a powerful tool for studying gene function, regulatory elements, tissue development, and disease progression. Finally, we discuss the prospects of CRISPR/Cas9 system and the potential applications of other types of CRISPR system.

CRISPR/Cas9 system; DNA fragment editing; gene function; regulatory elements; diseases

2015-06-23;

2015-08-27

國家自然科學(xué)基金項目(編號：31171015，31470820)和上海市科學(xué)技術(shù)委員會(編號：13XD1402000，14JC1403600)資助

李金環(huán)，博士研究生，專業(yè)方向：遺傳學(xué)。 E-mail: lijinhuan163@126.com 壽 佳，博士研究生，專業(yè)方向：遺傳學(xué)。 E-mail: shoujia1106@163.com李金環(huán)和壽佳同為第一作者

吳強，博士，教授，博士生導(dǎo)師，研究方向：基因表達調(diào)控及神經(jīng)發(fā)育。E-mail: qwu123@gmail.com

10.16288/j.yczz.15-291

時間:2015-9-1 9:32:39

URL:http://www.cnki.net/kcms/detail/11.1913.R.20150901.0932.002.html