介绍

从2013年首次宣布CRISPR-CAS9系统可以应用于哺乳动物开始，CRISPR技术因其高效率、易操作和低成本，正在基因编辑领域掀起一场新的革命。2015年，张峰首次在NatureMedicine上撰文并总结了CRISPR临床转化所面临的的三个挑战，即基因编辑效率、特异性和递送手段[1]。经过五年发展，2020年JenniferA.Doudna在Nature中的文章针对这三个挑战提出相应的解决方案[2]，尽管CRISPR系统目前在临床转化中仍面临多样的技术瓶颈技术，但其在临床一期实验中对遗传性淀粉样蛋白疾病（hATTR）的成功治疗表明CRISPR系统依然具有光明的应用前景[3]。本文将从基因编辑效率、特异性和递送手段三个方面阐述近几年CRISPR系统的进展。

基因编辑的有效性

CRISPR/Cas9发现之初只能用于双链DNA编辑。2017年,张峰实验室发现Cas13a和Cas13b可在引导RNA(sgRNA)的作用下对RNA进行切割[4,5],此后更多的Cas13家族成员如Cas13d、Cas13X和Cas13Y被发现，拓展了RNA编辑中编辑工具的选择范围[6,7]。除了靶向双链DNA和RNA外，JenniferA.Doudna还发现了一种新的Cas14蛋白，它可以不需要PAM序列而对单链DNA进行非特异性切割，基于此特性该蛋白已成功应用于病毒核酸检测[8]。

基于CRISPR/Cas切割活性的第一代CRISPR编辑系统主要依赖于细胞中非同源末端连接(NHEJ)机制介导基因敲除或依赖于同源重组修复（HDR）机制介导基因敲入。尽管CRISPR系统可通过HDR介导基因敲入，但由于HDR仅在G2和S期细胞中活性较高，因此，对于细胞分裂缓慢或处于有丝分裂后期的细胞，如神经元，基于HDR策略的基因敲入并不适用；另外，HDR介导的基因组编辑方法是依赖Cas9在目标位点引入双链DNA断裂和纠正模板来恢复基因功能，然而双链DNA断裂一旦形成，DNA损伤很容易通过NHEJ途径而不是HDR进行修复[9,10]。因此，上述两种因素导致HDR介导的基因修复效率较低，在体内仅达到0.1%-6.5%的编辑效率[11]。由于第一代CRISPR编辑系统在基因改造如基因点突变、基因片段插入的效率较低，因此其主要应用与基因敲除实验，并以完成一个临床一期试验及多个研究者发起的临床研究（InvestigatorInitialed Trial，IIT）[3,12-14]。

尽管目前CRISPR-Cas9系统能够通过引入框外突变而有效地敲除靶基因，但在进行基因缺失研究时必须注意基因补偿反应(genetic compensation response, GCR)。事实上，通过CRISPR敲除一个基因有时并没有表现出应有的表型差异，而利用RNA干扰手段敲低同一基因却有可能产生明显表型。这种差异是由于利用CRISPR/Cas系统敲除某一基因，但同时导致同一家族中其他成员基因表达上调所致。2019年，两个研究小组证实，CRISPR/Cas系统敲除某一基因产生的短核苷酸序列突变可导致该基因其他同家族成员启动子区域表观遗传学修饰改变，并促进该基因其他家族成员的转录，进而补偿原有基因敲除所诱发的表型改变[15,16]。该机制的发现有助于解释以往基于CRISPR/Cas系统建立的基因敲除模型中一些意想不到的表型结果。

2016年和2017年,刘如谦实验室分别将胞苷脱氨酶或腺苷脱氨酶与Cas9结合从而开创性地建立了第二代基因编辑系统-碱基编辑器(BE)。该系统包含胞嘧啶碱基编辑器（CBE）[17]、腺嘌呤碱基编辑器（ABE）[18]和鸟嘌呤编辑器（GBE）[19-21]可以在不需要模板DNA和双链DNA断裂的情况下分别实现C·T、A·G和C·G核苷酸之间的转化。除了在DNA中进行基因编辑，张峰实验室还开发了一个基于Cas13的REPAIR系统，在RNA水平上实现腺苷A→肌苷I（鸟苷G）置换[22]；魏文胜课题组则通过建立LEAPER系统，利用特殊设计的RNA（ADAR-recruitingRNA,arRNA）招募细胞中内源脱氨酶ADAR，实现靶向目标RNA中腺苷A→肌苷I（鸟苷G）转换[23]；由于上述两种RNA碱基编辑不依赖内源性修复途径。因此，即使在有丝分裂后期的细胞，如神经元，REPAIR和LEAPER系统也同样能够介导有效的RNA编辑。与DNA编辑相反，RNA编辑是短暂的，更容易逆转，允许对编辑结果进行暂时控制。由于碱基编辑器的DNA和RNA靶向是由单一sgRNA主导的，因此，通过转染多个sgRNA就有可能并行地进行多个基因突变。然而，与之对比，利用HDR同时引入多个基因突变是非常困难的，即使在分裂细胞中效率也极低，实用性较差。最新的C·T、A·G和C·G碱基编辑器的基因点突变置换效率可分别达到~50%、~57%和~15%[24-26]。基于目前的碱基编辑技术原则上可以治疗超过70%的人单基因遗传病[27]。

胞苷碱基编辑器可将C转换为T，腺嘌呤碱基编辑器可将A转换为G，鸟嘌呤编辑器可将C转换G。但是还有9种其它类型的核苷酸变化：如T转换为A和A转换为T，是这三种碱基编辑器无法完成的操作；另外，碱基编辑器也无法实现碱基片段的插入和删除。2019年刘如谦课题组开发了先导编辑器，通过将nickaseCas9蛋白与逆转录酶形成复合物，在向导RNA的引导下，该复合物可以在特定DNA区域添加、改变、或者移除目标核苷酸，也可实现所有12种单核苷酸转换，插入最长为44个核苷酸的新DNA序列，或删除长达80个碱基对的序列[28]。之后该课题组在2021年进一步发现DNA错配修复(MMR）途径可强烈抑制先导编辑效率，并通过瞬时表达显性负相关MMR蛋白(MLH1dn)，使其达到了30%~50%的编辑效率[29,30]。先导基因编辑技术原则上可以治疗超过89%的人类遗传病[28]。

除细胞核内基因组突变外，线粒体基因组由母系遗传，其突变可导致包括莱伯遗传性视神经病变和线粒体脑肌病伴高乳酸血症和卒中样发作等多种人类遗传病，因此开发针对线粒体的基因编辑具有重要意义。然而，由于向导RNA无法穿过线粒体膜，因此CRISPR-Cas平台不适用于编辑线粒体中的这类突变。2020年和2022年刘如谦和Jin-SooKim课题组通过分别将胞苷脱氨基酶DddA和腺嘌呤脱氢酶TadA8e和特异的DNA序列结合蛋白TALE构建形成融合蛋白，开发了线粒体胞嘧啶碱基编辑器（DdCBE）和线粒体腺苷酸碱基编辑器（TALED）[31-33]。由于DdCBE和TALED可分别实现C到T，和A到G的转化，原则上可以校正>53%的已知线粒体突变位点。

基因编辑的特异性

虽然CRISPR/Cas9技术在基因编辑方面比ZFN和TALEN表现出更高的保真度，但在生物学或临床应用中其脱靶效应仍是一个主要问题。CRISPR/Cas9编辑技术保真性的提高可通过优化重组Cas9蛋白和改造sgRNA序列实现。例如，新的Cas9变异体如EvoCas9、HypaCas9、Cas9-HF、eSpCas9和SniperCas9[34-38]，表现为较低的脱靶性，尽管与野生型Cas9相比，它们的在靶活性会降低。传统的sgRNA序列包含与目标DNA序列互补的20个核苷酸，然而研究人员发现，使用截短的sgRNA，如17或18核苷酸长度，可以将一些脱靶位点的发生率降低5000倍之多，而不牺牲目标基因组编辑效率[39]。除了缩短sgRNA的长度，D.DewranKocak发现在sgRNA的5'端添加一个发夹结构可以提高Cas9和其他Cas效应蛋白的特异性，平均提高55倍[40]。除了提高CRISPR-Cas9系统的精度，其他Cas效应蛋白也可被用于真核细胞的基因编辑，并展现出比Cas9更高的特异性。例如，Cas12a和Cas12b作为与Cas9在细胞切割活性相似的新型编辑蛋白，其对sgRNA和目标DNA之间的单核苷酸错配具有很高的敏感性，因此，与CRISPR-Cas9工具相比，它们减少了脱靶效应[41,42]。

碱基编辑器自2016年首次报道以来受到了广泛的关注。然而，在2019年，碱基编辑工具的安全性受到了广泛质疑，先是杨辉等研究组报道了胞嘧啶单碱基编辑器存在严重的DNA脱靶[43,44]，接着KeithJoung团队、刘如谦团队和杨辉团队又分别报道了胞嘧啶碱基编辑器和腺嘌呤碱基编辑器存在大量的RNA脱靶效应，其RNA脱靶效应主要由碱基编辑器中和Cas9蛋白偶联的胞苷脱氨酶或腺苷脱氨酶与RNA的偏好性结合导致[45-47]。其中DNA水平的脱靶效应可分为Cas依赖型和Cas非依赖型脱靶。Cas依赖型脱靶在ABEs和CBEs均有发生，可通过使用高特异性的Cas蛋白及突变体、截短型sgRNA而优化。Cas非依赖型脱靶源自脱氨基酶与细胞内基因组中瞬时存在的单链DNA的相互作用，主要发生在CBEs系统中。目前研究者主要通过构建脱氨基酶突变体和不同类型的脱氨基酶筛选对此加以优化。由于CBEs和ABEs系统中的脱氨基酶多可作用于RNA的碱基发挥脱氨基作用，早期开发的单碱基编辑器多在RNA水平存在明显的脱靶效应。目前研究者多通过蛋白质工程构建脱氨基酶的突变体以降低其在RNA水平的脱靶风险。

先导编辑目前研究发现其基因编辑精准度较高，只会产生少量的pegRNA依赖性的脱靶突变，进一步通过全基因组和全转录组水平研究发现先导编辑器不会导致pegRNA非依赖性脱靶突变，证明了先导编辑的安全性[28,48]。

尽管TALED编辑技术没有发现细胞毒性，也不会引起mtDNA的不稳定和细胞核DNA中的脱靶效应[33]；但DdCBE编辑技术中，DdCBE可进入细胞核，导致其在核基因组上产生了严重的脱靶效应[49,50]。

CRISPR系统的递送方法

CRISPR系统体内递送是实现其临床转化的关键，目前有三种主要的传递策略，包括基于质粒的递送、CasmRNA和sgRNA的递送以及Cas蛋白和sgRNA的直接传递(表1)。

基于质粒的CRISPR递送系统,目前最广泛的使用工具是腺相关病毒（AAV），AAV具有低免疫原性、感染效率高和无人致病性等特点,其最大包装长度为4.7kb的基因片段。编码SpCas9蛋白的mRNA大小约为4.3 kb，加上启动子和多聚腺苷化信号等其他必需序列，所需元件长度已达4.7 kb，因此无法实现将Cas9和sgRNA构建到同一AAV载体中[51]。一种解决方案是使用双AAV系统，将编码Cas9蛋白的mRNA和sgRNA分别装入两个AAV载体中。另一种方法使用较小尺寸的Cas蛋白。对于DNA编辑，CasX和CasMINI来源于非致病微生物，其编码序列长度分别约为2.9kb和1.5kb，具有特异的DNA切割特性，可成为潜在的Cas9替代蛋白[52,53]。在RNA编辑方面，Cas13d编码序列大小为2.8kb，比Cas13a、Cas13b等其他Cas13家族成员小，同样也易于AAV包装[6]。

近年来，细胞外泌体已成为新的CRISPR系统递送方法。红细胞无基因组DNA，且外泌体分泌数量较高，另外，来自健康献血者的O型血缺乏表面抗原，因此O型血来源的外泌体具有较低的免疫源性；Cas9mRNA和sgRNA可通过电穿孔方法导入纯化的外泌体中[54]。Cas9核酸酶与sgRNA形成的核糖核蛋白(RNP)复合体可以穿过细胞核进行基因编辑，因此与转染CRISPR质粒或mRNA相比，Cas蛋白直接递送具有作用速度快、免疫源低等独特优点[55]。另外近年来病毒样颗粒作为潜在的基因药物递送载体也备受关注，张峰、刘如谦和蔡宇伽课题组相继开发了用于CRISPR系统递送的SEND、VLP和mLP平台[56-58]；由于病毒样颗粒缺乏病毒的遗传物质，它们可能比其他使用病毒的递送方法更安全。

尽管CRISPR系统能够通过不同的策略被递送到细胞中，但其持续表达可能带来由于脱靶导致的毒副作用。例如，Cas9可在非靶点位置引起大片段删除和染色质破碎[59,60]；Cas9切割双链DNA可引发p53介导的DNA损伤反应，抑制细胞生长[61,62]。因此，在基因编辑完成后，应抑制CRISPR系统活性，以避免脱靶和毒性作用。幸运的是，一些抗Cas蛋白如AcrIIA4、AcrIIC1和AcrIIC3以及硫代修饰的DNA寡核苷酸已经被发现，它们能够通过不同的机制抑制Cas9或Cas12a的在靶活性[63-65]，因此，在临床应用中这两种抑制剂都可作为潜在的CRISPR系统解毒剂。

结论

CRISPR技术的快速发展大大提高了我们对真核细胞基因组进行精确编辑的能力。虽然基因编辑技术目前仍然面临脱靶性和递送效率偏低等多种问题，但相信上述问题将在未来的跨学科整合和科学家的合作中逐步得到解决。基因编辑技术的临床转化研究将推动个性化医疗的快速发展。

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