李國玲，楊善欣，吳珍芳,，張獻偉

綜 述

提高CRISPR/Cas9介導(dǎo)的動物基因組精確插入效率研究進展

李國玲1，楊善欣1，吳珍芳1,2，張獻偉2

1. 華南農(nóng)業(yè)大學(xué)動物科學(xué)學(xué)院，國家生豬種業(yè)工程技術(shù)研究中心，廣州 510642 2. 溫氏食品集團股份有限公司，新興 527439

基因編輯技術(shù)是指通過人為方式在基因組插入、缺失或替換特定堿基，對遺傳物質(zhì)進行精確修飾和定向編輯的一種技術(shù)。近年來，鋅指核酸內(nèi)切酶(zinc-finger endonuclease, ZFN)、類轉(zhuǎn)錄激活因子效應(yīng)物核酸酶(transcription activator-like effector nuclease, TALEN)、成簇規(guī)律間隔短回文重復(fù)序列及其相關(guān)系統(tǒng)(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9, CRISPR/Cas9)等基因編輯技術(shù)的出現(xiàn)，使特異性靶向修飾動物基因組序列成為可能。雖然利用CRISPR/Cas9等基因編輯工具可以在細胞基因組高效產(chǎn)生雙鏈斷裂(double-strand breaks, DSB)，但利用同源定向修復(fù)(homology directed repair, HDR)介導(dǎo)的精確插入(knock in, KI)效率卻十分低下。本文結(jié)合當前基因編輯技術(shù)的發(fā)展現(xiàn)狀，對目前提高CRISPR/Cas9介導(dǎo)的動物基因組KI策略進行了綜述，以期為人類疾病模型制備、基因治療和家畜遺傳改良等提供借鑒。

基因編輯；CRISPR/Cas9；精確插入；同源定向修復(fù)；非同源末端連接

基因編輯技術(shù)是指通過人為方式在基因組插入、缺失或替換特定堿基，對遺傳物質(zhì)進行精確修飾和定向編輯的一種技術(shù)。傳統(tǒng)的基因編輯方法利用隨機整合或同源重組(homologous repair, HR)等方式將DNA片段插入基因組，這種方式存在精確插入(knock in, KI)效率低和外源基因表達不穩(wěn)定等問題，嚴重制約了其在農(nóng)業(yè)和醫(yī)學(xué)領(lǐng)域的應(yīng)用。近年來，隨著鋅指核酸內(nèi)切酶(zinc-finger endonuclease, ZFN)、類轉(zhuǎn)錄激活因子效應(yīng)物核酸酶(transcription activator- like effector nuclease, TALEN)和成簇規(guī)律間隔短回文重復(fù)序列及其相關(guān)系統(tǒng)(clustered regularly inter-spaced short palidromic repeats/CRISPR-associated protein 9, CRISPR/Cas9)等基因編輯技術(shù)的出現(xiàn)，以其高效率、特異性靶向等特征在農(nóng)業(yè)和醫(yī)學(xué)領(lǐng)域得到廣泛應(yīng)用，先后在大腸桿菌()、酵母()、果蠅()、斑馬魚()、小鼠()、大鼠()、豬()和恒河猴()等物種中顯示了強大的基因編輯能力，展示出廣闊的應(yīng)用前景[1~7]。動物細胞基因組產(chǎn)生雙鏈斷裂(double-strand breaks, DSB)后，主要激活體內(nèi)非同源末端連接(non-homologous end joining, NHEJ)或同源定向修復(fù)(homology directed repair, HDR)兩種不同的修復(fù)機制，其中HDR介導(dǎo)的KI在人類疾病模型制備、基因治療和農(nóng)業(yè)遺傳改良等方面具有重要作用，但是其效率十分低下[7]，因此在CRISPR/Cas9高效產(chǎn)生DSB的前提下，如何提高動物基因組KI效率仍然充滿挑戰(zhàn)。本文結(jié)合當前基因編輯技術(shù)的發(fā)展，對目前提高CRISPR/Cas9介導(dǎo)的動物基因組KI策略進行了綜述，以期為人類疾病模型制備、基因治療和家畜遺傳改良等提供借鑒。

1 基因編輯技術(shù)

ZFN是由一系列鋅指蛋白(zinc finger protein, ZFP)與I限制性核酸內(nèi)切酶活性區(qū)融合而成，其中ZFP負責(zé)特異性識別3個連續(xù)堿基，I在兩個ZFP識別位點相隔5~8 bp時發(fā)生二聚體化，發(fā)揮核酸酶活性[8,9]。TALEN原理與ZFN相似，激活因子樣效應(yīng)物(transcription activator-like effector, TALE)負責(zé)特異性識別靶位點[10~12]，I隨后切割DNA序列，產(chǎn)生DSB[13]。相對ZFN和TALEN，CRISPR/ Cas9具有切割效率高、構(gòu)建簡單等特征，是目前研究和應(yīng)用最廣泛的基因編輯系統(tǒng)[14,15]。來自化膿性鏈球菌()的II型CRISPR/Cas9系統(tǒng)主要由sgRNA(single guide RNA)和SpCas9蛋白組成，其中sgRNA通過堿基互補配對原則識別DNA序列，SpCas9蛋白在sgRNA引導(dǎo)下，切割含有NGG或NAG的靶點位[16,17]。為了提高CRISPR/ Cas9系統(tǒng)特異性或靶向的DNA覆蓋范圍等，科研人員構(gòu)建不同的SpCas9突變體，如eSpCas9[18]、SpCas9-HF1[19]、EvoCas9[20]、Sniper Cas9[21]、VRER SpCas9[22]及xCas9[23]等。一些結(jié)構(gòu)更簡單、容量更小和切割效率相當或更高的基因編輯工具被逐漸開發(fā)，如SGN[24]、AsCpf1[25]、StCas9[26]、NmCas9[27]、SaCas9[28]、CjCas9[29]和CasX[30]等。此外在CRISPR/ Cas9系統(tǒng)基礎(chǔ)上升級開發(fā)的堿基編輯器，如胞嘧啶堿基編輯器(cytosine base editor, CBE)利用胞嘧啶脫氨酶實現(xiàn)靶位點一定范圍內(nèi)C?G到T?A的替換[31]，腺嘌呤堿基編輯器(adenine base editor, ABE)利用腺嘌呤脫氨酶實現(xiàn)靶位點一定范圍內(nèi)A?T到G?C的替換[32]，有效克服了CRISPR/Cas9介導(dǎo)的單堿基編輯效率低的弊端。目前升級改造的堿基編輯器ABE4max[33]和AncBE4max[34]已經(jīng)可以高效實現(xiàn)哺乳動物細胞特定位置A?T與G?C之間堿基的置換。盡管SpCas9突變體或其他基因編輯技術(shù)一定程度上克服了CRISPR/Cas9系統(tǒng)轉(zhuǎn)染效率低、脫靶效率高、安全性低和靶向位點有限等缺點，但也存在切割效率低、甲基化位點敏感和不適合高通量編輯等弊端[35,36]；此外，雖然ABE和CBE不需要供體模板和引入DSB即可實現(xiàn)單堿基編輯，但不斷增大的系統(tǒng)結(jié)構(gòu)也導(dǎo)致病毒難以包裝和傳送；同時ABE和CBE存在編輯窗口有限、不能實現(xiàn)長片段KI或存在明顯的DNA/RNA脫靶等問題[35,36]，因此如何升級和完善基因編輯技術(shù)仍然充滿挑戰(zhàn)。

2 提高CRISPR/Cas9介導(dǎo)的動物基因組KI策略

自然或人為在基因組中引入DSB是KI發(fā)生的前提，傳統(tǒng)的基因編輯技術(shù)利用供體模板和自然產(chǎn)生的DSB，HDR效率僅為10–7~10–5，對KI技術(shù)的應(yīng)用與研究帶來極大困難[7]。近年來，ZFN、TALEN和CRISPR/Cas9等基因編輯工具的發(fā)現(xiàn)和應(yīng)用，可以在動物基因組高效產(chǎn)生DSB，為實現(xiàn)KI的廣泛應(yīng)用提供了契機。細胞基因組產(chǎn)生DSB后，主要激活體內(nèi)NHEJ或HDR兩種不同的修復(fù)機制，其中HDR利用同源序列作為供體模板可以實現(xiàn)堿基對的準確插入、缺失或突變，而NHEJ介導(dǎo)的DNA修復(fù)容易出錯，常常會引入堿基對的隨機插入或缺失[37]。但是HDR是細胞KI最為依賴的修復(fù)機制，在人類疾病模型制備、基因治療和家畜遺傳改良等方面具有重要的研究價值[37]。在家畜遺傳改良中，KI轉(zhuǎn)基因動物具有目的基因表達穩(wěn)定和有效提高產(chǎn)肉量、改善肉質(zhì)、抗病能力等特點；同時不引入目的基因外的外源片段，可以減少公眾對轉(zhuǎn)基因食品的擔(dān)憂。Gao等[38]將結(jié)核抗性基因精確插入到奶?；蚪M，培育了抗牛結(jié)核病的新型奶牛品種；Zheng等[39]將小鼠基因插入豬基因組，培育了抵抗寒冷應(yīng)激的新型豬品種；Hu等[40]將干擾口蹄疫病毒(foot-and-mouth disease virus, FMDV)的shRNA插入豬基因位點，培育了抗FMDV的KI豬。同時KI技術(shù)可以快速獲得具有特定功能的人類疾病模型，對疾病治療方法的研究起到推動作用。Yan等[41]將人源突變的基因替換豬基因，成功制備亨廷頓病(huntington’s disease, HD)模型，可以用于研究大型哺乳動物神經(jīng)性疾病的發(fā)病機制及其治療方案。此外，利用KI技術(shù)糾正由定點突變導(dǎo)致的人類疾病，是細胞和體內(nèi)基因治療所必需的關(guān)鍵[42,43]。KI技術(shù)將免疫細胞或骨髓造血干細胞等分離出來，進行體外培養(yǎng)和擴增后進行精確修復(fù)，隨后將KI細胞回輸入體內(nèi)達到治療效果。這種方式目前已經(jīng)被用于腫瘤[44]、視網(wǎng)膜色素變性[45]、重度聯(lián)合免疫缺陷病[46]等體外基因治療，而體內(nèi)基因治療利用供體模板直接修復(fù)機體致病突變，目前在I型遺傳性酪氨酸血癥[47]、B型血友病[48]、致命性高血氨癥[49]、鐮刀型紅細胞貧血[50]和白內(nèi)障[51]等均有應(yīng)用。但是細胞利用HDR實現(xiàn)KI效率十分低下，僅有0.5%~20%，而與之競爭的NHEJ效率高達60%[7]。僅僅依靠機體的HDR機制實現(xiàn)KI相對比較困難，因此利用CRISPR/Cas9高效產(chǎn)生DSB的前提下，KI效率仍然需要進一步改進。

2.1 靶位點選擇

基因組高效產(chǎn)生DSB是實現(xiàn)外源DNA片段KI的前提。目前種類繁多的sgRNA設(shè)計軟件為科研人員提供了快速、便捷和高效的sgRNA選擇。Hsu等[52]開發(fā)的CRISPR Design (http://crispr.mit.edu/)可以針對23~500 nt的DNA序列，設(shè)計20 nt的sgRNA，同時對人、小鼠和大鼠等物種基因組進行脫靶評估，最終按靶向效率高低進行排序，并用紅、綠、黃3種顏色標記脫靶的特異性高低。Stemmer等[53]開發(fā)的CCTop (https://crispr.cos.uni-heidelberg.de/index. html)可以針對不大于500 nt的DNA序列設(shè)計PAM可變、核心區(qū)域可選擇的sgRNA序列，同時可對擬南芥、人和斑馬魚等物種的基因組進行脫靶評估。Heigwer等[54]開發(fā)的E-CRISP (http://www.e-crisp. org/E-CRISP/index.html)可設(shè)計考慮PAM類型、內(nèi)含子、CpG島等，并評估人、小鼠和豬等物種基因組脫靶效應(yīng)的sgRNA序列。由于不同軟件所基于的數(shù)據(jù)和算法不同，導(dǎo)致參考靶向效率評分存在差異。同時最佳效率的sgRNA序列還需要結(jié)合體外T7E1酶切法、SSA (single-strand annealing)報告載體檢測或Sanger測序法等進一步篩選。此外，選擇不同靶基因和同一靶基因不同靶位點，KI效率也存在一定差異。Li等[55]利用CRISPR/Cas9將長片段DNA插入基因和基因位點，發(fā)現(xiàn)其效率存在顯著性差異。同時靶位點的組蛋白乙酰化水平會影響染色質(zhì)的致密程度，直接抑制細胞發(fā)生HDR。Bin等[56]發(fā)現(xiàn)，隨著HDAC1、HDAC2蛋白質(zhì)乙酰化水平的增加，染色質(zhì)開放程度逐漸增加，KI效率顯著提高，因此靶位點的染色質(zhì)致密程度可能是阻礙KI發(fā)生的潛在因素。

2.2 SpCas9蛋白改造

SpCas9蛋白是CRISPR/Cas9系統(tǒng)發(fā)揮核酸酶活性的關(guān)鍵，主要包括Ruvc和HNH兩個切割DNA正反鏈的結(jié)構(gòu)域[17]?；赟pCas9蛋白切割結(jié)構(gòu)域特點，Jinek等[57]將SpCas9改造為一個單切口酶D10A Cas9 (Cas9n)，該酶在DNA特定位置制造單鏈切口；只被一個Cas9n切割產(chǎn)生的單鏈缺口只會進行高保真性的HR途徑，這樣基本不會引起NHEJ，更有利于實現(xiàn)外源基因KI。同時有研究表明，Cas9蛋白切割域構(gòu)象也會影響HDR和NHEJ的比例。例如，Kato-Inui等[58]比較了WT-SpCas9、eSpCas9、SpCas9- HF1和HypaCas9共4種SpCas9蛋白，發(fā)現(xiàn)不同細胞HDR/NHEJ比值存在明顯差異，其中HypaCas9顯著提高HEK293T和HeLa的KI效率，分別為6.9倍和7.7倍。因此，可以推斷不同的CRISPR/Cas9系統(tǒng)對KI效率存在差異，高效發(fā)生KI的SpCas9突變體還有待探索。

DSB附近DNA供體模板的可使用性可能是限制KI效率的重要因素，通過改造SpCas9蛋白富集供體模板在靶位點的局部濃度可以提高KI效率。Carlson-Stevermer等[59]將生物素–鏈霉素親和素特異性識別的RNA序列加入sgRNA莖環(huán)構(gòu)建了S1mplex策略，sgRNA引導(dǎo)SpCas9蛋白結(jié)合靶位點的同時，可以將生物素–鏈霉素親和素修飾的DNA模板富集在DSB附近。研究結(jié)果表明，S1mplex策略可以提高HEK293T細胞等位基因KI效率18倍；基于類似的策略，Ma等[60]將SpCas9蛋白與親和素融合表達構(gòu)建Cas9-Avidin/Biotin(CAB)系統(tǒng)，通過Avidin富集生物素標記的供體模板，有效將1 kb片段高效KI小鼠基因組；Gu等[61]在小鼠胚胎2-細胞期注射SpCas9-鏈霉素親和素融合蛋白和生物素修飾的供體模板，發(fā)現(xiàn)KI效率提高了10倍。此外，Savic等[62]將SpCas9蛋白與SNAP蛋白融合表達構(gòu)建的Cas9-SNAP系統(tǒng)，通過SNAP結(jié)合O6-benzyl-guanine (BG)標記的DNA模板，將HEK293T細胞的KI效率提高了24倍；Aird等[63]將圓環(huán)病毒2 (porcine circovirus type 2, PCV2) DNA識別域與SpCas9蛋白融合表達構(gòu)建Cas9-PCV2系統(tǒng)，發(fā)現(xiàn)當DNA模板存在PCV2識別靶序列時，KI效率顯著提高15~30倍。

DNA連接酶IV(DNA-ligase IV, LIG4)是NHEJ途經(jīng)的關(guān)鍵因子，其與Xrcc4類似因子(Xrcc4-like fators, XLF)結(jié)合形成Xrcc4-XLF-LIG4復(fù)合體，強行將2個DNA斷端連接起來，修復(fù)DSB[64]。Chu等[65]將SpCas9蛋白與具有降解LIG4蛋白作用的腺病毒蛋白4E1B55K和E4orf6融合表達，顯著提高人和小鼠細胞系KI效率3.5~5倍；同時通過siRNA和shRNA干擾表達也可以提高KI效率2~3倍；Zhang等[66]也發(fā)現(xiàn)融合表達4E1B55K和E4orf6的CRISPR/Cas9系統(tǒng)可以顯著提高誘導(dǎo)多能干細胞(induced pluripotent stem cells, iPSCs)的KI效率2.5~4倍。此外，Rad51、CtIP、Rad50和Rad52等蛋白是DSB修復(fù)的直接參與者，在HDR途徑中起到關(guān)鍵作用，因此能否通過融合SpCas9蛋白為DSB修復(fù)創(chuàng)造有利環(huán)境也是值得嘗試的。2017年，Shao等[67]利用酵母來源的yRad52與SpCas9融合構(gòu)建yRad52-Cas9，將KI效率顯著提高40%左右；Charpentier等[68]將CtIP與SpCas9融合表達構(gòu)建Cas9-HE系統(tǒng)顯著提高人類細胞系、iPSCs和大鼠受精卵KI效率；Jayavaradhan等[69]將SpCas9蛋白與53BP1的顯性失活突變體DN1S融合構(gòu)建Cas9- DN1S，顯著減少了NHEJ發(fā)生頻率，同時顯著提高了不同人類細胞的KI效率；Tran等[70]發(fā)現(xiàn)SpCas9蛋白與CtIP、Rad52和Mre11蛋白，而非Rad51C蛋白融合表達，可以提高HEK293T細胞KI效率2倍。同時將包含噬菌體MS2被殼蛋白結(jié)合環(huán)的sgRNA與Cas9-CtIP融合蛋白組裝，通過MS2招募CtIP蛋白完成DNA修復(fù)，發(fā)現(xiàn)MS2-CtIP系統(tǒng)進一步提高報告系統(tǒng)KI效率。

NHEJ和HDR分別在不同的細胞周期階段(G1和G2/S)占主導(dǎo)地位，因此SpCas9蛋白時間特異性表達在S期和G2期，可以一定程度提高HDR介導(dǎo)的KI效率。Gutschner等[71]將SpCas9蛋白與人Geminin N(hGem)末端區(qū)域的氨基酸序列融合表達。Cas9-hGem融合蛋白可以作為E3泛素連接酶復(fù)合物APC/Cdh1的底物，促進其在細胞周期G1期降解，而在S/G2期維持高水平表達。由于G1期不存在HDR，Cas9-hGem策略可以提高KI效率1.42倍。但Howden等[72]利用Cas9-hGem系統(tǒng)處理iPSCs，發(fā)現(xiàn)Cas9-hGem并沒有增加HDR頻率，但其在靶位點誘導(dǎo)NHEJ介導(dǎo)的插入缺失的能力顯著降低。盡管SpCas9突變體和融合表達蛋白一定程度上提高了KI效率，但不斷增大的系統(tǒng)結(jié)構(gòu)也為后續(xù)的應(yīng)用提出了新的挑戰(zhàn)。

2.3 dsDNA供體模板優(yōu)化

雙鏈DNA (double strand DNA, dsDNA)模板的拓撲結(jié)構(gòu)、同源臂長度等是影響基因組KI效率的關(guān)鍵因素，其中線性化的供體質(zhì)?？梢赃M一步提高KI效率。1989年，Bollag等[73]首次發(fā)現(xiàn)在供體模板和基因組靶位點附近引入DSB，可提高HR約100倍；隨后Shin等[74]結(jié)合TALEN技術(shù)對斑馬魚和基因進行打靶，發(fā)現(xiàn)體外線性化供體模板可以將KI效率提高10%；Auer等[75]發(fā)現(xiàn)線性化的供體模板KI效率顯著高于超螺旋的質(zhì)粒；Yao等[76]利用CRISPR/Cas9技術(shù)進一步證明了體外線性化的供體模板比環(huán)狀供體具有更高的KI效率，同時體外線性化供體模板在不同細胞系都獲得極高的KI效率；Cristea等[77]發(fā)現(xiàn)當供體模板加入ZFN識別位點時，模板在細胞內(nèi)被線性化可以顯著提高外源基因的KI效率，而Zhang等[66]同樣發(fā)現(xiàn)供體同源臂加入CRISPR/Cas9識別的靶位點，細胞內(nèi)切割供體長同源臂產(chǎn)生短同源臂時更有利于實現(xiàn)KI。上述研究均表明供體質(zhì)粒體內(nèi)或體外線性化對提高KI效率具有一定提高作用。供體同源臂長度對KI效率的提高具有重要的作用。前期研究表明，同源臂長度在200 bp以下時，HDR修復(fù)效率有明顯下降趨勢；當同源臂長度達到約14 kb以上時，這種線性關(guān)系才逐漸不明顯[78]。但在KI細胞篩選過程中，由于同源臂過長會增加細胞鑒定的難度，權(quán)衡KI效率和鑒定難度，傳統(tǒng)的供體模板同源臂長度一般都在6~7 kb。隨著ZFN、TALEN和CRISPR/Cas9等基因編輯技術(shù)的出現(xiàn)，對這一結(jié)論提出了新的挑戰(zhàn)。Orlando等[79]結(jié)合ZFN技術(shù)設(shè)計同源臂長度在500~1500 bp的供體模板，發(fā)現(xiàn)500 bp的同源臂模板也能保持較高的KI效率；Byrne等[80]利用CRISPR/Cas9系統(tǒng)設(shè)計同源臂長度100 bp~5 kb的供體模板，發(fā)現(xiàn)在iPSCs細胞中同源臂長度在2 kb時，基因置換為小鼠基因的效率最高，并隨著同源臂長度變短，效率逐漸降低，同時長于2 kb的同源臂并沒有明顯提高KI效率；隨后Shin等[74]和Chu等[65]也證實KI長片段的供體模板同源臂需要在1~2 kb。當同源臂長度小于350 bp時，HDR效率降低2.5倍；但Shy等[81]在小鼠胚胎干細胞(mouse embryonic stem cell, mESCs)中發(fā)現(xiàn)短同源臂(<200 bp)和長同源臂(>1 kb)搭配，KI效率更高。2014年，Nakade等[82]提出一種全新的KI策略——微同源介導(dǎo)的末端連接(micro-homology mediated end joining, MMEJ)，即利用5~40 bp短同源臂高效介導(dǎo)外源基因(<1000 bp)精確插入；隨后該團隊繼續(xù)對這種策略進行優(yōu)化，發(fā)現(xiàn)利用5~25 bp可以將長片段(5.7~9.6 kb)外源DNA片段高效插入中國倉鼠卵巢細胞(Chinese hamster ovary cells, CHO)基因組，其效率為10%~17%[83,84]；Paix等[85]也發(fā)現(xiàn)36 bp短同源臂可以將739 bpDNA片段高效插入小鼠受精卵基因組，而在HEK293T細胞中，同源臂長度是33 bp或518 bp 時KI效率是相同的。因此，同源臂的長度可能不是一成不變的，它可能需要根據(jù)所使用的基因編輯工具類型、細胞來源、插入片段大小和插入方式等來確定其最優(yōu)長度。

2.4 ssODN供體模板設(shè)計與優(yōu)化

相比dsDNA模板，單鏈寡核苷酸(single-strand oligodeoxynucleotide, ssODN)作為供體模板具有更高的KI效率。Liang等[86]對ssODN模板側(cè)翼序列進行優(yōu)化，發(fā)現(xiàn)側(cè)翼為30~40 nt的ssODN模板在HEK293T細胞可以獲得最佳的KI效率；Rivera- Torres等[87]發(fā)現(xiàn)側(cè)翼為35~50 nt的ssODN模板在HCT116細胞KI效率最佳，而Wang等[1]研究表明側(cè)翼分別是45 nt和71 nt的ssODN在豬胎兒成纖維細胞(porcine fetal fbroblasts, PFFs)具有最高的KI效率(>10%)，而過長的ssODN模板對KI效率并沒有顯著的促進作用；Richardson等[88]通過優(yōu)化ssODN供體模板的長度和方向，發(fā)現(xiàn)利用不對稱的ssODN模板(側(cè)翼分別為91 nt和36 nt)在HEK293T獲得高達60% KI效率，而Yumlu等[89]和Yang等[90]進一步研究表明采用不對稱的ssODN模板在iPSCs可以獲得最佳KI效率，但是Moreno-Mateos等[91]發(fā)現(xiàn)對稱和不對稱ssODN模板在斑馬魚的效率是一致的，因此不同來源的細胞系對ssODN側(cè)翼長度和方向要求可能是不一致的，在特定細胞系采用何種方式還有待優(yōu)化。此外化學(xué)修飾的ssODN模板可以減慢其在細胞與胚胎內(nèi)的降解速度，阻礙NHEJ發(fā)生，進一步提高KI效率。Prykhozhij等[92]發(fā)現(xiàn)硫代磷酸化標記的ssODN模板可顯著提高斑馬魚KI效率；同時有研究表明通過化學(xué)修飾的sgRNA[93]或采用其他修飾方式的ssODN模板[94]也可以顯著提高KI效率。為了解決ssODN插入片段較短等問題，Miura等[95]和Quadros等[96]開發(fā)了Easi-CRISPR (efficient addi-tions with ssDNA inserts-CRISPR)策略：通過先體外轉(zhuǎn)錄再逆轉(zhuǎn)錄的方法制備長約4~5 kb的ssODN模板；Yoshimi等[97]開發(fā)了2H2OP (Two-hit by sgRNA and two oligos with a targeting plasmid)策略：sgRNA同時切割基因組和不具有同源序列的供體質(zhì)粒，隨后兩個提供同源堿基的ssODN以“創(chuàng)可貼”形式將供體質(zhì)粒整合至基因組，為插入更長的DNA片段提供了可能。但是相對dsDNA，長片段的ssODN難以制備，且成本相對較高，因此ssODN常應(yīng)用于插入片段小于100 bp的基因精確修復(fù)。

2.5 小分子化合物調(diào)控DNA修復(fù)

小分子化合物是一個相對于高分子化合物的概念，通常指相對分子質(zhì)量小于10,000的一類化合物，如營養(yǎng)素、代謝產(chǎn)物、天然產(chǎn)物和合成的藥理學(xué)因子等。由于小分子化合物具有結(jié)構(gòu)和功能多樣性的特點，給予其無限潛力去控制分子間、蛋白質(zhì)間的識別及相互作用。近年來，研究人員利用小分子化合物調(diào)控DNA修復(fù)通路關(guān)鍵蛋白，在提高基因組KI效率方面取得了一定進展[2,7,98~101]。Maruyama等[7]使用Scr7(LIG4抑制劑)處理MelJuSo細胞和A549細胞，將ssODN介導(dǎo)的KI效率提高3~19倍，同時直接注射小鼠受精卵將KI效率提高了2倍；Li等[98]利用小分子化合物Scr7提高豬成纖維細胞(porcine fetal fbroblasts, PFFs)基因組KI效率2~3倍；Ma等[100]使用VE-822(Rad3相關(guān)激酶抑制劑)和AZD-7762(檢查點激酶CHEK1特異性抑制劑)將人ESCs CRISPR- Cpf1介導(dǎo)的KI效率提高3~6倍；Yu等[101]通過高通量篩選，發(fā)現(xiàn)Brefeldin A (蛋白轉(zhuǎn)動抑制劑)或L755507 (β3腎上腺素受體激動劑)顯著提高小鼠ESCs、人源HeLa、K562和iPSCs KI效率1.3~2倍；Song等[2]結(jié)合CRISPR/Cas9和TALEN技術(shù)，利用Rad51蛋白的激活劑RS-1將基因插入大鼠基因效率提高2~5倍，隨后Pinder等[102]在HEK293A細胞也進一步證實了該結(jié)果；Lin等[103]通過PD0325901和CHIR99021分別抑制mESCs MEK和GSK3b信號通路，提高KI效率1~5倍；Robert等[104]使用DNA-PK抑制劑NU7441和KU-57788顯著降低NHEJ效率，同時顯著提高CRISPR/Cas9介導(dǎo)的KI效率。DSB修復(fù)表面上是通過NHEJ/HDR途徑實現(xiàn)，但細胞所在的細胞周期會顯著影響DNA修復(fù)途徑的選擇。在DNA修復(fù)過程中，NHEJ可以發(fā)生在細胞分裂的任一時期，而HDR主要發(fā)生在G2和S期[15]。利用這個規(guī)律，通過化合物阻滯細胞周期可以為研究者提供一條提高基因組KI效率的思路。Urnov等[105]通過添加長春花堿(vinblastine)將細胞同步化至G2期顯著提高HR發(fā)生頻率約7倍；隨后Rahman等[106]利用indirubin-3?-monoxime抑制劑將HeLa、HT-1080和U-2 OS細胞周期停滯在G2期，有效提高了I-SceI和ZFN的KI效率2~5倍，但目前并沒有CRISPR/Cas9相關(guān)研究報道。Lin等[99]在多種人源細胞中添加能使細胞停滯在G2/S期的Aphidicolin和Nocodazole，顯著提高了ssODN介導(dǎo)的KI效率；Yang等[107]利用化合物ABT-751誘導(dǎo)人iPSCs細胞周期停滯在G2期，隨后將2~5 kb的序列分別整合至人類基因組5個區(qū)域，KI效率提高了3~6倍。盡管一些化合物可將細胞周期顯著停滯在G2期，如LiCl[108]，但這些化合物并不能提高CRISPR/Cas9介導(dǎo)的KI效率。一些被證實可以將細胞周期顯著阻滯在S期的化合物，如硫酸羥脲(hydroxyurea)[109]和2?,3?- 雙脫氧胞苷(dideoxycytidine, ddC)[110]等，是否能有效提高哺乳動物細胞KI效率還有待進一步探索。

雖然已經(jīng)有較多小分子化合物被應(yīng)用于提高KI效率，但一些小分子化合物在不同細胞系，甚至是同一細胞系的結(jié)果都是不一致的。其原因可能是：(1)不同物種來源細胞NHEJ/HDR活性存在差異，導(dǎo)致實驗結(jié)果有偏差[111]，如SCR7可以顯著提高大部分人源細胞如HEK293T、A549和MelJuSo等的KI效率，但對兔子胚胎和CHO的KI效率提高并不理想[2,7,108]。(2)采用CRISRR/Cas9系統(tǒng)方式不一致，導(dǎo)致SpCas9蛋白在體內(nèi)表達時間存在明顯差異影響結(jié)果[112]。如Lin等[99]通過轉(zhuǎn)染Cas9 RNP復(fù)合物顯著提高HEK293T ssODN介導(dǎo)的KI效率，而Yan等[113]通過轉(zhuǎn)染SpCas9質(zhì)粒卻不能提高。(3)供體模板形式不同導(dǎo)致細胞KI效率和修復(fù)方式有差異影響結(jié)果。如前文所述，ssODN介導(dǎo)的KI效率明顯高于dsDNA[88]；同時也有研究表明，ssODN修復(fù)基因組DSB并不是通過HDR途徑實現(xiàn)的，而是通過Fanconi anemia(FA)途徑[114]，這些可能是導(dǎo)致HDR激活劑RS-1不能顯著提高斑馬魚胚胎ssODN介導(dǎo)的KI效率的原因。(4)化合物工作濃度不同導(dǎo)致差異。Li等[98]利用100 μmol/L Scr7顯著提高PFFs細胞HDR效率1.9倍，而10 μmol/L時效果不明顯(從26.2%提高至28.1%)；而Xie等[115]使用5 μmol/L Scr7處理PFFs細胞，提高KI效果不理想。此外提高KI效率固然很重要，但是小分子化合物對細胞DNA損傷仍然需要考慮。有研究表明Scr7可顯著提高KI效率，但其存在許多未知的風(fēng)險，如劑量過大具有明顯了細胞毒性，而直接注射胚胎會導(dǎo)致胚胎停滯在桑椹胚時期等[116]。Nocodazole、vinblastine和ABT-751等通過與細胞微管蛋白結(jié)合競爭性抑制微管蛋白的聚合，導(dǎo)致分裂的細胞不能形成紡錘體微管而使細胞分裂停止，隨后引起細胞內(nèi)谷胱甘肽/活性氧失衡，導(dǎo)致細胞凋亡和DNA損傷[117~119]，因此細胞毒性更小、效率更高的小分子化合物還有待發(fā)現(xiàn)(見表1)。

2.6 利用NHEJ途徑實現(xiàn)KI

相對低頻率的HDR途徑，NHEJ在不同類型的細胞都高度活躍。He等[127]將供體模板側(cè)翼同源臂替換為sgRNA靶位點開發(fā)HITI (homology-indepen-dent targeted integration)策略，利用NHEJ途徑將4.6 kb的ires-片段插入LO2細胞和人胚胎干細胞(human embryonic stem cells, hESCs)的位點；同樣，Auer等[128]利用HITI方式高效地將>5.7 kb的DNA片段插入斑馬魚基因組；Lackner等[129]利用該方法成功對內(nèi)源基因?qū)崿F(xiàn)了NanoLuc luciferase和Turbo GFP標記?；谙嗤牟呗?，Suzuki等[130]成功將DNA片段精確插入到非分裂細胞基因組位點上；隨后該團隊對HITI策略進行優(yōu)化：保留一側(cè)sgRNA序列，另一側(cè)sgRNA序列替換或增加同源臂，開發(fā)了SATI (single homology arm donor mediated intron-targeting integration)策略[131]。SATI策略顯著提高細胞發(fā)生KI效率，同時一定程度克服了HITI策略靶位點插入片段前后顛換等問題。SpCas9蛋白產(chǎn)生的平末端切口不適合被DNA修復(fù)蛋白捕獲，而ZFN和TALEN產(chǎn)生的粘性末端更適合切口捕獲。Maresca等[132]開發(fā)了ObLiGaRe (obligate ligation- gated recombination)策略，通過兩對ZFP蛋白分別識別供體模板和基因組靶位點，只有I核酸酶發(fā)生異源二聚化時才能切割DNA。線性化的供體模板插入基因組靶位點時，I核酸酶同源二聚化無法切割KI的DNA片段，因此有利于提高KI效率；隨后Tsai等[133]和Guilinger等[134]采用類似的策略，將dCas9與I核酸酶融合表達，發(fā)現(xiàn)dCas9-I顯著提高了KI效率。盡管HITI策略、SATI策略和ObLiGaRe策略成功克服了HDR介導(dǎo)KI低的障礙，但由NHEJ介導(dǎo)的KI方式，可能會導(dǎo)致外源基因隨機插入到基因組的其他位置，同時靶位點容易插入或缺失少量堿基，對研究應(yīng)用依然充滿技術(shù)挑戰(zhàn)。

3 結(jié)語與展望

ZFN、TALEN、CRISPR/Cas9和基于CRISPR/ Cas9改造的CBE和ABE系統(tǒng)極大地豐富了基因組編輯的應(yīng)用范圍，但不同的基因編輯系統(tǒng)有著其獨特的優(yōu)點，因此針對不同的物種以及不同的細胞系、轉(zhuǎn)染效率和基因位點序列信息選擇合適的基因編輯系統(tǒng)尤為重要。總體而言，ZFN編碼的序列更小，更容易實現(xiàn)病毒包裝與傳送。相對ZFN，TALEN特異性更高，但其設(shè)計同樣繁瑣和不適合高通量篩選；同時TALEN蛋白結(jié)構(gòu)更大，只能通過腺病毒或電轉(zhuǎn)染等方式傳送至細胞。CRISPR/Cas9系統(tǒng)擺脫了合成和組裝具有特異性DNA識別能力蛋白模塊的繁瑣操作，以其高效率、易設(shè)計構(gòu)建等特征在生物、農(nóng)業(yè)和醫(yī)學(xué)領(lǐng)域得到廣泛應(yīng)用。ABE和CBE是CRISPR/Cas9系統(tǒng)的進一步提升，其不需要供體模板和引入DSB即可實現(xiàn)單堿基編輯，但不斷增大的系統(tǒng)結(jié)構(gòu)也導(dǎo)致病毒難以包裝和傳送；同時ABE和CBE仍然存在編輯窗口有限、不能實現(xiàn)長片段KI或存在明顯的DNA/RNA脫靶等問題[35~37]。2019年，美國哈佛大學(xué)David Liu實驗室開發(fā)的全新堿基基因編輯器PE (prime editors)，其無需額外的DNA模板即可實現(xiàn)所有12種單堿基的自由轉(zhuǎn)換，且能有效實現(xiàn)多堿基的KI與基因敲除(knock out, KO)[135]。PE基因編輯器的出現(xiàn)極大地豐富了單堿基與小片段增刪的基因編輯系統(tǒng)，但目前沒有來自其他實驗室重復(fù)數(shù)據(jù)的報道，其基因編輯效率還需謹慎對待。

表1 小分子化合物對CRISRR/Cas9介導(dǎo)的KI效率的影響

dsDNA：雙鏈DNA；ssODN：單鏈寡核苷酸；NA表示小分子化合濃度不詳，None表示KI效率沒有提高。

細胞基因組產(chǎn)生DSB后，主要激活體內(nèi)NHEJ或HDR兩種不同的修復(fù)機制，其中HDR介導(dǎo)的KI效率十分低下，而與之競爭的NHEJ效率卻非常高。HDR是細胞KI最為依賴的修復(fù)機制，其在人類疾病模型制備，基因治療和家畜遺傳改良等具有重要的研究價值，如KI功能基因達到提高經(jīng)濟動物產(chǎn)肉量、改善肉質(zhì)、抗病能力等；同時利用KI技術(shù)可以制備特定功能的人類疾病模型，和糾正定點突變導(dǎo)致的人類疾病，為研究疾病的發(fā)病機制和基因治療提供方案。但是KI效率的低下極大地限制了其廣泛應(yīng)用，目前已經(jīng)有多種策略用于提高KI效率，如靶位點選擇、SpCas9蛋白改造、dsDNA供體模板優(yōu)化、ssODN供體模板設(shè)計與優(yōu)化、小分子化合物調(diào)控DNA修復(fù)和NHEJ途徑實現(xiàn)KI等。其中研究人員將SpCas9與其他功能性蛋白融合，通過融合蛋白招募DNA修復(fù)因子、調(diào)控Cas9蛋白周期特異性降解或富集供體模板等形式一定程度上提高基因組的KI效率，但不斷增大的系統(tǒng)容量也為后續(xù)病毒包裝和傳送增加了困難。同時研究也發(fā)現(xiàn)不同細胞采用不同類型的修復(fù)方式對dsDNA/ssODN供體模板的拓撲結(jié)構(gòu)、同源臂的長度等也有要求，其中HDR介導(dǎo)的dsDNA供體模板同源臂需要在500 bp~1 kb；MMEJ介導(dǎo)的同源臂只需要5~40 bp；而NHEJ介導(dǎo)的KI需要根據(jù)HITI、SATI或ObLiGaRe等策略制定其獨特的供體模板；盡管NHEJ介導(dǎo)的KI效率非常高，但其DNA片段容易隨機插入到基因組的其他位置，增加潛在的安全風(fēng)險；相比dsDNA供體模板，ssODN插入或替換少量堿基具有更高的編輯效率，而通過化學(xué)修飾的ssODN將進一步提高KI效率，但是ssODN模板難以合成，只適用于少量堿基的編輯；此外，使用小分子化合物激活HDR途徑關(guān)鍵蛋白、抑制NHEJ途徑關(guān)鍵蛋白或阻滯細胞周期至S/G2期也可以提高HDR介導(dǎo)的KI效率，但一些小分子化合物在不同細胞系，甚至同一細胞系的研究結(jié)果都有所差異。同時小分子化合物對細胞DNA損傷仍然需要考慮，細胞毒性更小、效率更高的小分子化合物還有待發(fā)現(xiàn)。總之，不管采用哪種策略都有其優(yōu)勢和劣勢，因此繼續(xù)完善和提高KI效率仍然需要科研人員努力，這將對家畜遺傳改良、人類疾病模型制備和基因治療等具有重要的意義和價值。

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Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals

Guoling Li1, Shanxin Yang1, Zhenfang Wu1,2, Xianwei Zhang2

Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), trans-cription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.

gene editing; CRISPR/Cas9; knock in; homology directed repair; non-homologous end joining

2020-03-04;

2020-04-24

國家轉(zhuǎn)基因重大專項(編號：2016ZX08006002)資助[Supported by the National Transgenic Major Projects (No. 2016ZX08006002)]

李國玲，在讀博士研究生，專業(yè)方向：基因編輯。E-mail: 792268184@qq.com

張獻偉，博士，碩士生導(dǎo)師，研究方向：遺傳育種。E-mail: zxianw@163.com

10.16288/j.yczz.20-056

2020/6/2 11:50:24

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20200601.1621.003.html

(責(zé)任編委: 谷峰)