哺乳动物基因组靶向修饰阳性细胞富集的报告载体系统研究进展

基因组靶向修饰技术对基因功能研究、基因治疗以及转基因育种研究都具有重要的意义和价值。近年来发展起来的人工核酸酶如ZFNs、TALENs和CRISPR/Cas9等的应用大大提高了基因组靶向修饰的效率。但是由于核酸酶表达载体转染效率、核酸酶表达效率及活性以及基因组被打靶后的修复效率等因素在一定程度上制约着基因组靶向修饰阳性细胞的获得。因此富集和筛选基因组靶向修饰阳性细胞是一个亟待解决的问题。报告载体系统可以间接地反映核酸酶的工作效率并有效富集核酸酶修饰的阳性细胞,进而提高基因组靶向修饰阳性细胞的富集和筛选效率。本文主要针对由非同源末端连接(Non-homologous end joining,NHEJ)和单链退火(Single-strand annealing,SSA)两种修复机制分别介导的报告载体系统的原理和应用进行了详细的介绍,以期为以后的相关研究提供借鉴和参考。     

体外验证SSA报告载体系统对CRISPR/Cas9的切割效率

目的根据单链退火(single strand annealing, SSA)修复途径原理,构建萤火虫荧光素酶荧光报告基因系统,体外检测SSA报告载体对CRISPR/Cas9的切割效率及gRNA的特异性和剪切活性。方法基于SSA修复DNA双链断裂损伤需要断裂末端包含一段同源重复序列的原理,构建4个萤火虫萤光素酶luciferase基因报告质粒,其中luciferase基因被分成前后两个片段;PCR先扩增luciferase基因319～1 653 bp片段并连接至巨细胞病毒(cytomegalovirus, CMV)启动子表达质粒,随后再分别连接luciferase基因不同长度前段序列,分别为1～579 bp、1～858 bp、1～1 188 bp和1～1 317 bp,每个片段中均含有一段重复序列,并且被终止密码子提前终止为无活性的luciferase基因;设计了261、540、870和900 bp等4个同源臂,将特异性的gRNA连入两段序列中,并加入有活性的Cas9/gRNA,通过检测荧光活性来检测gRNA的特异性和剪切活性。结果成功构建了4个荧光报告质粒pLuc-N1-1～4;用海参荧光素酶作为内参,在293T细胞转染4个荧光报告质粒,在24 h时检测4个质粒的相对荧光强度,均无荧光活性物质产生;在荧光报告质粒中加入特异性的靶点序列,并表达Cas9/gRNA,这些报告基因具有显著荧光强度升高(P0.000 1),且不受同源臂长度影响。结论成功构建的基于luciferase基因的SSA报告系统,可提供一种简单而快速方法来评估和比较gRNA在CRISPR/Cas9系统引入的indel突变诱导效率,为选择最有效gRNA减少不确定性,大大扩展CRISPR/Cas9介导的模式动物基因工程的实际应用提供可能性。     

单链退火修复及其在疾病治疗和基因编辑中的作用

DNA双链断裂(double strand breaks,DSBs)对细胞生存是致命的。细胞内经典非同源末端连接(classical non-homologous end joining,C-NHEJ)和选择性非同源末端连接(alternative non-homologous end joining,A-NHEJ)、重组修复(homology-directed repair,HDR)、单链退火修复(single-strand annealing,SSA)等通路可竞争性修复DNA双链断裂损伤。其中,SSA途径不使用同源染色体或姐妹染色单体,仅依赖于重复序列彼此退火配对,并涉及遗传信息的丢失,是容易出错的修复过程,具有诱变性。相比其他修复途径,在细胞周期S和G_2期中,末端切除暴露出更长的同源重复序列(20 bp),有利于细胞选择SSA途径进行修复。在一些SSA活性升高的同源重组(homologous recombination,HR)缺陷癌症中,癌症细胞可利用SSA途径获得耐药性,也预示着疾病风险的提高。因此,靶向SSA途径的抑制剂具有抑制癌症进展,以及逆转肿瘤细胞对聚腺苷二磷酸核糖聚合酶(poly ADP-ribose polymerase,PARP)抑制剂耐药的作用,是一种新型的治疗策略,可能成为特定同源重组缺陷癌症风险评估的有力工具。检测SSA活性将有助于更好区分癌症的发生和进展。同时随着基因编辑技术的发展,基于SSA途径的荧光报告基因方法,用于检测规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR-Cas9)系统中引导RNA(gRNA)的特异性和裂解活性被证明是有效、可靠的策略,同时结合CRISPR-Cas9靶向和SSA诱导的DNA编辑,可以特定模式表达多个gRNA并实现多种细胞类型特异性操作和组合遗传靶向。尽管对SSA途径的研究与基于SSA途径的技术应用已取得不错的进展,但仍有许多问题有待阐明。     

快速构建CRISPR/Cas9基因组靶向编辑系统

CRISPR/Cas9核酸酶作为一种新的基因组靶向编辑技术,已成功应用于多种动植物基因组修饰研究. CRISPR/Cas9作用后的阳性细胞筛选和富集是该技术的关键之一. 本研究以鸡EAV-HP（endogenous avian retrovirus-HP）基因和MSTN（myostatin）基因为例,从靶位点的选择、表达载体构建、双基因报告载体构建和核酸酶活性验证4个方面,系统研究了CRISPR/Cas9核酸酶技术平台. 结果表明,利用寡聚核苷酸直接退火方法,构建表达载体和报告载体的阳性率分别高达100%和89.5%. 报告载体的PuroR（puromycin resistant gene）和eGFP（enhanced green fluorescent protein）基因的成功表达表明,构建的CRISPR/Cas9系统能有效切割靶序列,并用于后续阳性克隆的筛选和富集. 本方法摒弃了传统分子克隆的PCR扩增和酶切处理目标基因的方法,而是利用寡聚核苷酸直接退火获得含有黏性末端的目标DNA,简化了载体构建过程,低成本且快速获得CRISPR/Cas9基因组靶向编辑系统.     

组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用

DNA双链断裂(double strand breaks, DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶（histone deacetylase）作为抗肿瘤治疗的新靶标,其抑制剂(histonedeacetylase inhibitors, HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.     

HPD基因启动子报告基因载体构建与转录活性分析

目的:HPD是一种酪氨酸分解代谢途径中的关键酶,特异表达于肝脏。目前对HPD的研究主要集中在HPD与酪氨酸血症等疾病的关系上,而对其转录调控的报道较少,并且对HPD在肝脏中特异性表达的转录调控机制尚不清楚,也未见报道。通过构建HPD基因启动子荧光素酶报告基因载体,检测其在肝源细胞中的特异转录活性,进而揭示其肝脏特异性表达的调控机制。方法:运用UCSC在线数据库分析人HPD基因组结构,并获得其5'端上游启动子区域-2000~+39bp的DNA序列。以人的肝癌细胞Hep G2基因组DNA为模板,利用PCR扩增该序列,并克隆到p GL3-Basic荧光素酶报告基因载体上。通过设计不同引物,进一步构建系列缺失体,共获得7个不同长度的荧光素酶报告基因载体。将构建的载体分别转染Hep G2、L02及HEK293细胞,通过双荧光素酶活性分析实验检测不同缺失体的转录活性。结果:报告基因实验结果显示-600~-400区域在肝源细胞系Hep G2和L02细胞中具有较强转录活性,而在HEK293细胞中活性较低,-400~+39区段则在两种细胞中均有转录活性。生物信息学分析结果显示-600~-400区域内存在较多肝脏特异性转录因子结合位点。结论:成功构建了HPD基因启动子荧光素酶报告基因载体,发现-600~-400存在调控HPD肝脏特异表达的关键元件,为进一步深入揭示HPD肝脏特异性表达的转录调控机制奠定了理论基础。     

DNA双链断裂损伤反应及它的医学意义

DNA损伤应激反应是维持基因组稳定性的基石.细胞在长期进化中形成了由损伤监视、周期调控、损伤修复、凋亡诱导等在内的自稳平衡机制.一方面,借助感应、识别并启动精细而复杂的修复机制修复损伤;另一方面,通过DNA损伤应激活化的细胞周期检查点机制,延迟或阻断细胞周期进程,为损伤修复提供时间,使细胞能安全进入新一轮细胞周期;损伤无法修复时则诱导细胞凋亡.DNA双链断裂(double strand breaks,DSBs)是真核基因组后果最严重的损伤类型之一,其修复不利,同肿瘤等人类疾病的发生发展密切相关.新进展揭示:DSBs损伤反应信号分子ATM-Chk2-p53、H2AX等的组成性活化,是肿瘤形成早期所激活的细胞内可诱导的抗癌屏障,其信号网络的精确、精细调控在基因组稳定性维持中发挥重要作用.此外,HIV病毒整合进入宿主细胞基因组的过程也依赖于宿主细胞中ATM介导的DSBs损伤反应信号转导;ATM特异性的小分子抑制剂在抗HIV感染中显示重要的功能意义.文中重点讨论调控DSBs损伤应激反应信号网络的主要研究进展,及其在肿瘤发生、发展及抗HIV感染中的新医学意义.     

解偶联蛋白UCP1启动子荧光素酶报告基因载体的构建

目的:构建解偶联蛋白UCP1启动子荧光素酶报告基因载体,为寻找调控UCP1表达的小分子化合物提供有效工具。方法:从小鼠基因组DNA中PCR扩增小鼠UCP1启动子上游2000 bp序列,并将该序列连接到荧光素酶报告基因载体p GL3-basic中,构建p GL3-UCP1启动子。测序正确后,提取质粒,然后将上述载体与p RL-TK载体共转染至HEK293细胞、小鼠白色脂肪前体细胞和小鼠棕色脂肪前体细胞,48 h后裂解细胞检测荧光素酶的活性。结果:通过PCR成功扩增获得了目的片段,并将其克隆至p GL3-basic中。与细胞内源UCP1表达水平相似,荧光素酶报告系统表明构建的p GL3-UCP1在棕色脂肪细胞中启动子活性最高,在白色脂肪细胞中活性较低,在HEK293细胞中基本没有活性。同时β3肾上腺素受体激动剂CL 316,243同样能够上调p GL3-UCP1的启动子活性。结论:成功构建了小鼠UCP1启动子荧光素酶报告基因载体,并证明在棕色脂肪细胞中,该启动子具有很强的启动子活性,而在白色脂肪和HEK293细胞中,启动子活性很低。该启动子报告系统有望为寻找激活UCP1的小分子化合物提供重要平台。     

含VEGF启动子的报告基因的构建及雌激素受体对其活性的影响

目的：构建含血管内皮生长因子（VEGF）基因启动子的荧光素酶报告基因载体,并检测其在雌激素受体作用下的转录活性。方法：以乳腺癌细胞系MCF-7基因组为模板,扩增VEGF启动子片段,克隆到荧光素酶报告基因载体pGL3-basic中。用脂质体介导的基因瞬时转染法,将重组正确的报告基因载体转染293T细胞,检测重组质粒中荧光素酶报告基因的表达。结果：酶切鉴定和DNA序列分析表明构建了正确的pGL3-basic—VEGF报告基因载体;转录活性实验表明构建的报告基因载体具有启动子活性,雌激素受体α（ERα）能以剂量依赖的方式升高VEGF启动子调控下的报告基因的转录。结论：克隆了VEGF启动子,为ERα共调节子的功能研究奠定了基础。     

High-frequency homologous recombination in plants mediated by zinc-finger nucleases

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.     

The HSV-1 Exonuclease, UL12, Stimulates Recombination by a Single Strand Annealing Mechanism

Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5′-to-3′ exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.     

人工核酸酶介导的基因组编辑技术

传统的基因组编辑技术是基于胚胎干细胞和同源重组实现生物基因组定向改造,但是该技术打靶效率低,严重制约了生命科学以及医学的研究.因此,研究新的基因组编辑技术十分重要.人工核酸酶介导的基因组编辑技术是通过特异性识别靶位点造成DNA双链断裂,引起细胞内源性的修复机制实现靶基因的修饰.与传统的基因组编辑技术相比,人工核酸酶技术打靶效率高,这对于基因功能的研究、构建人类疾病动物模型以及探索新型疾病治疗方案有着重要的意义.人工核酸酶技术有3种类型：锌指核酸酶(ZFN)、类转录激活因子核酸酶(TALEN)及规律成簇的间隔短回文重复序列(CRISPR).本文将对以上3种人工核酸酶技术的原理以及在生命科学和医学研究的应用进行综述.     

Exo1 independent DNA mismatch repair involves multiple compensatory nucleases

Functional DNA mismatch repair (MMR) is essential for maintaining the fidelity of DNA replication and genetic stability. In hematopoiesis, loss of MMR results in methylating agent resistance and a hematopoietic stem cell (HSC) repopulation defect. Additionally MMR failure is associated with a variety of human malignancies, notably Lynch syndrome. We focus on the 5′ → 3′ exonuclease Exo1, the primary enzyme excising the nicked strand during MMR, preceding polymerase synthesis. We found that nuclease dead Exo1 mutant cells are sensitive to the O6-methylguanine alkylating agent temozolomide when given with the MGMT inactivator, O6benzylguanine (BG). Additionally we used an MMR reporter plasmid to verify that Exo1^mut MEFs were able to repair G:T base mismatches in vitro. We showed that unlike other MMR deficient mouse models, Exo1^mut mouse HSC did not gain a competitive survival advantage post temozolomide/BG treatment in vivo. To determine potential nucleases implicated in MMR in the absence of Exo1 nuclease activity, but in the presence of the inactive protein, we performed gene expression analyses of several mammalian nucleases in WT and Exo1^mut MEFs before and after temozolomide treatment and identified upregulation of Artemis, Fan1, and Mre11. Partial shRNA mediated silencing of each of these in Exo1^mut cells resulted in decreased MMR capacity and increased resistance to temozolomide/BG. We propose that nuclease function is required for fully functional MMR, but a portfolio of nucleases is able to compensate for loss of Exo1 nuclease activity to maintain proficiency.     

Targeted genome editing in pluripotent stem cells using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous end-joining (NHEJ). Thus, ZFNs are powerful tools for targeted genome engineering in a variety of mammalian cell types, including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). As a paradigm for genome editing in pluripotent stem cells, we describe the use of ZFNs in murine ESCs for generating knockout alleles by NHEJ without selection or by HR employing different selection schemes.     

In vivo evidence for two active nuclease motifs in the double-strand break repair enzyme RexAB of Lactococcus lactis

In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.