靶向miRNA前体不同类型sgRNA的丰度及特异性评估

CRISPR/Cas技术能高效进行基因组定点编辑,但不同细菌来源或人工改造的Cas9以及Cpf1等核酸酶识别的PAM (protospacer adjacent motif)有差异,因此不同的基因编辑核酸酶可能采用不同类型的sgRNAs(small guide RNAs)。MicroRNAs (miRNAs)是一类调控性的小分子非编码RNAs,为了研究miRNA前体中是否可能存在特异性高的sgRNAs靶点,本文利用本课题组前期开发的生物信息学软件CRISPR-offinder,对靶向28 645条miRNA前体的11种不同类型sgRNA的丰度及特异性进行了分析,并利用CRISPR/Cas9慢病毒技术构建了猪miR-302/367基因簇敲除细胞系,对构建的猪miRNA敲除细胞系的效率进行了检测。结果表明,每个miRNA前体中平均存在约8种不同类型sgRNA的靶点;通过评估靶向猪miRNA前体sgRNA的脱靶效应,发现其中特异性高的sgRNA仅占18.2%;通过CRISPR/Cas9慢病毒技术成功构建了猪miR-302/367基因簇敲除细胞系,发现通过该技术构建miRNA敲除细胞系的效率为40%。本研究为利用CRISPR/Cas技术靶向敲除miRNA提供了重要资源。     

Optimized paired‐sgRNA/Cas9 cloning and expression cassette triggers high‐efficiency multiplex genome editing in kiwifruit

Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing. In this study, we developed a new cloning strategy for generating paired‐sgRNA/Cas9 vectors containing four sgRNAs targeting the kiwifruit phytoene desaturase gene (AcPDS). Comparing to the previous method of paired‐sgRNA cloning, our strategy only requires the synthesis of two gRNA‐containing primers which largely reduces the cost. We further compared efficiencies of paired‐sgRNA/Cas9 vectors containing different sgRNA expression devices, including both the polycistronic tRNA‐sgRNA cassette (PTG) and the traditional CRISPR expression cassette. We found the mutagenesis frequency of the PTG/Cas9 system was 10‐fold higher than that of the CRISPR/Cas9 system, coinciding with the relative expressions of sgRNAs in two different expression cassettes. In particular, we identified large chromosomal fragment deletions induced by the paired‐sgRNAs of the PTG/Cas9 system. Finally, as expected, we found both systems can successfully induce the albino phenotype of kiwifruit plantlets regenerated from the G418‐resistance callus lines. We conclude that the PTG/Cas9 system is a more powerful system than the traditional CRISPR/Cas9 system for kiwifruit genome editing, which provides valuable clues for optimizing CRISPR/Cas9 editing system in other plants.     

Expansion of CRISPR targeting sites in Bombyx mori

The CRISPR/Cas9 system has been proven as a revolutionary genome engineering tool. In most cases, single guide RNA (sgRNA) targeting sites have been designed as GN19NGG or GGN18NGG, because of restriction of the initiation nucleotide for RNA Pol III promoters. Here, we demonstrate that the U6 promoter from a lepidopteran model insect, Bombyx mori, effectively expressed the sgRNA initiated with any nucleotide bases (adenine, thymine, guanine or cytosine), which further expands the CRISPR targeting space. A detailed expansion index in the genome was analysed when N20NGG was set as the CRISPR targeting site instead of GN19NGG, and revealed a significant increase of suitable targets, with the highest increase occurring on the Z sex chromosome. Transfection of different types of N20NGG sgRNAs targeting the enhanced green fluorescent protein (EGFP) combined with Cas9, significantly reduced EGFP expression in the BmN cells. An endogenous gene, BmBLOS2, was also disrupted by using various types of N20NGG sgRNAs, and the cleavage efficiency of N20NGG sgRNAs with different initial nucleotides and GC contents was evaluated in vitro. Furthermore, transgenic silkworms expressing Cas9 and sgRNAs targeting the BmBLOS2 gene were generated with many types of mutagenesis. The typical transparent skin phenotype in knock-out silkworms was stable and inheritable, suggesting that N20NGG sgRNAs function sufficiently in vivo. Our findings represent a renewal of CRISPR/Cas9 target design and will greatly facilitate insect functional genetics research.     

NPAS2基因敲除的HepG2细胞系构建及其对肝癌细胞凋亡的影响

目的:利用CRISPR/Cas9基因编辑技术构建生物节律基因NPAS2敲除的HepG2肝癌细胞系,并初步探讨NPAS2基因敲除对肝癌细胞凋亡的影响。方法:利用sgRNA在线设计工具,针对NPAS2设计两条sgRNA;利用PX459质粒构建分别含有两条sgRNA的敲除载体PX459-sgRNA1;PX459-sgRNA2;利用T7核酸内切酶I检测两条sgRNA活性;将活性较高的打靶载体瞬时转染HepG2细胞,经过药物筛选,克隆化培养及基因测序后得到NPAS2敲除的HepG2肝癌细胞系;利用Western blot检测NPAS2蛋白的表达和凋亡相关蛋白Caspase3的活化;利用流式细胞仪检测敲除细胞系的凋亡水平。结果:成功构建了针对NPAS2的打靶载体;并筛选得到了活性较高的打靶载体;经过药物筛选和克隆化培养得到的NPAS2敲除肝癌细胞系未检测到NPAS2蛋白的表达;进一步发现NPAS2敲除的肝癌细胞Caspase3明显活化,凋亡水平显著升高。结论:利用CRISPR/Cas9基因编辑技术成功构建了NPAS2基因敲除的HepG2肝癌细胞系,并发现NPAS2敲除可以促进肝癌细胞凋亡,为进一步研究生物节律基因NPAS2及其它相关基因在肝癌发生发展中的作用机制提供了有力的工具。     

快速构建多重sgRNA载体利用CRISPR/Cas9技术敲除拟南芥IAA2基因

IAA2(Indole Acetic Acid 2)是拟南芥Aux/IAA生长素响应基因大家族中的一员,目前还没有它的突变体的报道,阻碍了对其功能和作用机制的深入研究。在CRISPR/Cas9基因组编辑技术中,1个sgRNA只能靶向基因的1个位点,有时基因敲除的效率并不高。为了提高敲除效率,本文在Golden-Gate克隆技术的基础上,通过两轮PCR扩增,将每3个sgRNA串联到同1个入门载体中,再将入门载体与含Cas9表达框的目标载体LR反应,获得最终的表达载体。结果表明,设计的6个sgRNA有4个发挥了作用,产生了碱基插入突变和大片段缺失突变等多种可遗传的突变。与单个sgRNA相比,多重sgRNA的基因敲除效率高、种系突变多;与其他构建多重sgRNA载体的方法相比,本方法具有快速、高效等优点。本文所得到的5个突变体为后续的IAA2功能研究提供了良好的材料。     

基于新的CRISPR/Cas9技术实现斑马鱼LHX9基因的快速编辑及对F0突变体性腺发育的初筛

目的:CRISPR/Cas9系统在斑马鱼的反向遗传学中的到了广泛应用,但突变基因的表型观察往往需要在突变鱼系的F1中进行,费时较长。LHX9作为LIM家族的一种转录因子,在胚胎早期的泌尿生殖嵴中有广泛分布;且LHX9基因敲除的小鼠存在性腺发育不良。本研究拟通过一种新的CRISPR/Cas9基因编辑技术,采用四条sgRNA对LHX9基因进行VASA转基因斑马鱼的基因敲除,以观察该基因缺陷对斑马鱼性腺发育的影响。方法:利用新的CRISPR/Cas9技术,设计四条针对斑马鱼LHX9基因3号外显子的20bp的sgRNA,通过非克隆体外转录得到靶位点的四条sgRNA。将以上靶位点的四条sgRNA与Cas9核酸酶蛋白同时注射入单细胞期的斑马鱼胚胎内,利用PCR鉴定突变型类型和突变比例。通过对LHX9基因突变体的VASA转基因斑马鱼进行荧光观察,发现LHX9基因缺陷的斑马鱼性腺发育的情况。结果:靶向Exon 3的四条sgRNA可成功编辑斑马鱼LHX9基因,敲除效率高达82%,Sanger测序发现产生10种不同的移码突变类型。通过该方法对VASA转基因斑马鱼的LHX9基因进行编辑,发现LHX9基因突变导致dph6的的斑马鱼原始生殖细胞增殖和迁移受到影响。结论:基于4条sgRNA注射的CRISPR/Cas9技术,可以快速地产生具有突变表型的G0斑马鱼,具有应用潜力。LHX9基因敲除导致原始生殖细胞的发育和迁移受到影响,提示该基因参与了斑马鱼早期性腺的发育。     

Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigera

Helicoverpa armigera, cotton bollworm, is one of the most disastrous pests worldwide, threatening various food and economic crops. Functional genomic tools may provide efficient approaches for its management. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, dependent on a single guide RNA (sgRNA), has been used to induce indels for targeted mutagenesis in cotton bollworm. However, genomic deletions may be more desirable to disrupt the function of noncoding genes or regulatory sequences. By injecting two sgRNAs with Cas9 protein targeting different exons, we obtained predictable genomic deletions of several hundred bases. We achieved this type of modification with different combinations of sgRNA pairs, including HaCad and HaABCC2. Our finding indicated that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H. armigera.     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

利用CRISPR/Cas9技术建立敲除JAK2基因K562细胞系

目的:利用CRISPR/Cas9技术对K562细胞系JAK2基因进行编辑,构建JAK2基因敲除的K562细胞系。方法:使用CRISPR在线设计工具,针对JAK2基因设计sgRNA,构建Cas9-sgRNA共表达质粒。使用第二代慢病毒包装系统包装慢病毒并感染K562细胞,提取细胞基因组DNA,Sanger测序和TA克隆检测基因编辑活性。无限稀释法将编辑阳性的细胞接种于96孔板并扩培得到单克隆细胞株,提取基因组DNA,Sanger测序和TA克隆分析敲除JAK2单克隆细胞的基因型。结果:成功构建靶向敲除JAK2基因的lentiCRISPRv2-sgRNA3-1质粒。优化方案得到低细胞毒性高转染效率的感染K562细胞慢病毒量。CRISPR/Cas9系统成功在JAK2基因sgRNA3-1识别位点发挥基因组编辑活性,获得纯合敲除JAK2基因细胞株K562-JAK2~(-/-)(两个等位分别发生移码突变,预期编码没有功能的JAK2蛋白)。结论:CRIAPR/Cas9系统通过慢病毒感染方式获得JAK2基因纯合敲除的K562细胞株,该细胞模型可用于研究在慢性髓系白血病中JAK2基因的作用,为构建K562敲除其他基因细胞系提供实验依据,为探究造血分化机制的研究奠定实验基础。     

CRISPcut: A novel tool for designing optimal sgRNAs for CRISPR/Cas9 based experiments in human cells

The ability to direct the CRISPR/Cas9 nuclease to a unique target site within a genome would have broad use in targeted genome engineering. However, CRISPR RNA is reported to bind to other genomic locations that differ from the intended target site by a few nucleotides, demonstrating significant off-target activity. We have developed the CRISPcut tool that screens the off-targets using various parameters and predicts the ideal genomic target for –guide RNAs in human cell lines. sgRNAs for four different types of Cas9 nucleases can be designed with an option for the user to work with different PAM sequences. Direct experimental measurement of genome-wide DNA accessibility is incorporated that effectively restricts the prediction of CRISPR targets to open chromatin. An option to predict target sites for paired CRISPR nickases is also provided. The tool has been validated using a dataset of experimentally used sgRNA and their identified off-targets.URL: http://web.iitd.ac.in/crispcut     

sgRNAcas9软件图形用户界面开发及应用

CRISPR/Cas9系统介导的基因组编辑技术是新一代功能强大的基因修饰技术。然而,脱靶效应(Off-target effects)是目前CRISPR/Cas9技术面临的最大问题。因此,设计脱靶风险低的sgRNA(single guide RNA)就成为关键。sgRNAcas9是一款专门用于sgRNA设计和评估脱靶效应的软件包。针对其核心运行程序,我们利用Java程序语言开发了其图形用户界面。此外,依据脱靶位点碱基数和sgRNA种子序列(seed sequences)的特异性,通过设置不同的风险等级对sgRNA的脱靶效应进行评估。随后,利用此软件设计了34 124条靶向人、小鼠、大鼠、猪和鸡中共4691个microRNA(miRNA)前体的sgRNAs。此外,随机挑选了一个靶向人miR-206前体的sgRNA进行脱靶效应评估和验证。结果发现,sgRNAcas9软件人机交互界面友好,大多数miRNA前体可通过该软件寻找到sgRNA,且他们的GC%含量范围集中于40%~60%。利用sgRNAcas9软件设计的靶向miR-206的sgRNA,其基因组编辑活性和脱靶位点可被实验验证。本研究表明sgRNAcas9图形用户界面软件能针对任意物种设计特异性的sgRNA,其可在BiooTools(http://www.biootools.com/)网站下载。     

Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy,a web‐based target finding tool

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site‐specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome‐editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user‐friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO‐K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off‐target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

CRISPR/Cas9的应用及脱靶效应研究进展

CRISPR/Cas9基因编辑技术在生命科学领域掀起了一场全新的技术革命,该技术可以对基因组特定位点进行靶向编辑,包括缺失、插入、修复等。CRISPR/Cas9比锌指核酸酶 (ZFNs)和转录激活因子样效应物核酸酶(TALENs)技术更易于操作,而且更高效。CRISPR/Cas9系统中的向导RNA(Single guide RNA, sgRNA)是一段与目标DNA片段匹配的RNA序列,指导Cas9蛋白对基因组进行识别。研究发现,设计的sgRNA会与非靶点DNA序列错配,引入非预期的基因突变,即脱靶效应(Off-target effects)。脱靶效应严重制约了CRISPR/Cas9基因编辑技术的广泛应用。为了避免脱靶效应,研究者对影响脱靶效应的因素进行了系统研究并提出了许多降低脱靶效应的方法。文章总结了CRISPR/Cas9系统的应用及脱靶效应研究进展,以期为相关领域的工作提供参考。     

利用CRISPR/Cas9系统构建稳定敲除TrxR1基因的HCT-116细胞株北大核心CSCD

为更好地研究靶向硫氧还蛋白还原酶1的小分子化合物的细胞内靶点选择性,利用CRISPR/Cas9系统构建稳定敲除TrxR1基因(编码硫氧还蛋白还原酶1)的HCT-116细胞株。首先根据TrxR1基因序列和CRISPR/Cas9靶点设计原则,设计并选择合适的敲除位点,再根据敲除位点序列设计敲除TrxR1基因的sgRNA干扰序列,以pCasCMV-Puro-U6空质粒载体为骨架构建能表达该sgRNA干扰序列的重组质粒。质粒共转染至HCT-116细胞后,利用嘌呤霉素筛选TrxR1敲除的HCT-116细胞,通过DNA测序、免疫蛋白印迹、TRFS-green荧光探针和细胞内TrxR1酶活力检测等方法鉴定和验证HCT-116细胞的TrxR1基因敲除效果。进一步通过CCK-8实验初步研究靶向TrxR1小分子化合物对细胞内TrxR1酶活力和细胞增殖力抑制的相关性。结果显示,表达sgRNA干扰序列的重组质粒可以敲除HCT-116细胞中TrxR1基因,筛选获得的稳定敲除细胞HCT116-TrxR1-KO中无TrxR1蛋白表达,而靶向TrxR1小分子抑制剂对该细胞无TrxR1酶活力和细胞增殖力抑制效果。本研究利用CRISPR/Cas9系统成功构建了HCT-116的TrxR1基因敲除的稳定细胞株,为进一步研究TrxR1在相关疾病的发生机制和治疗中的作用奠定了基础。     

Generation of a multiplex mutagenesis population via pooled CRISPR‐Cas9 in soya bean

The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR‐Cas9 as a mutant screening tool. Here, we report a pooled CRISPR‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1‐1/1‐2/1‐3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.