CRISPR/Cas9技术在生物医学领域的非病毒及病毒载体

规律成簇的间隔短回文重复序列(CRISPR)及CRISPR相关蛋白9(CRISPR/Cas9)系统是一种新型基因组编辑技术,能够靶向干扰或修复基因组的特定基因.来自细菌或人工改造的CRISPR/Cas9系统已经由生物学家发现或构建,Cas9核酸酶及单链导向RNA(sgRNA)是CRISPR/Cas9系统的主要组成成分.该系统被广泛应用于疾病治疗新靶点的发掘,基因功能的鉴定,动物模型的建立以及基因治疗药物的开发.CRISPR/Cas9系统已经通过突变或修正疾病相关基因来部分缓解或彻底治愈某些病症.然而,如何有效递送CRISPR/Cas9至目标细胞及靶器官仍然是运用该技术所面临的挑战之一,这影响着该系统稳定和精准的基因编辑能力.本文主要综述Cas9mRNA,Cas9蛋白或编码Cas9基因及相应sgRNA载体的递送系统.递送Cas9蛋白的非病毒载体能够维持Cas9的靶向作用,减少脱靶效应;递送sgRNA和供体模板的病毒载体能够改进基因编辑及同源修复效率.安全,有效及可规模化生产的递送载体将会推进CRISPR/Cas9技术在人类基因治疗领域中的应用.     

CRISPR/Cas系统在植物基因组编辑中的应用

在基因组水平上进行植物基因人工改造对农作物的改良具有重要意义。一直以来,科学家都未发现有效的植物基因组编辑的方法。最近发现的CRISPR/Cas系统因其简易和有效性被广泛应用于包括植物在内的多种生物的基因组编辑中。CRISPR/Cas系统仅需要短RNA和核酸酶就可以对特定的靶标基因进行突变。目前CRISPR/Cas系统已成功在拟南芥(Arabidopsis thaliana)、烟草(Nicotiana benthamiana)、甜橙(Citrus sinensis)、水稻(Oryza sativa)、小麦(Triticum aestivum)、高粱(Sorghum bicolor)、玉米(Zea mays)以及苔藓植物地钱(Marchantia polymorpha)等植物中实现了定点基因组编辑。综述将对CRISPR/Cas系统在植物中的最新研究进行全面的总结和讨论。     

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。     

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。     

基因组编辑技术在水稻功能基因组和遗传改良中的应用

基因组编辑技术对植物基因功能研究和作物遗传改良具有巨大的潜在价值。CRISPR/Cas9系统是继锌指核酸酶(ZFNs)和类转录激活效应因子核酸酶(TALENs)系统之后的新一代基因组编辑技术系统,具有操作简单和效率高等优点。概述了CRISPR/Cas9系统的技术特点及其在水稻基因功能研究及遗传改良中的应用,并指出了该系统在植物基因精准编辑中需要突破的关键问题。     

机器学习方法在CRISPR/Cas9系统中的应用

基于CRISPR/Cas9系统介导的第三代基因组定点编辑技术,已被广泛应用于基因编辑和基因表达调控等研究领域。如何提高该技术对基因组编辑的效率与特异性、最大限度降低脱靶风险一直是该领域的难点。近年来,机器学习为解决CRISPR/Cas9系统所面临的问题提供了新思路,基于机器学习的CRISPR/Cas9系统已逐渐成为研究热点。本文阐述了CRISPR/Cas9的作用机理,总结了现阶段该技术面临的基因组编辑效率低、存在潜在的脱靶效应、前间区序列邻近基序(PAM)限制识别序列等问题,最后对机器学习应用于优化设计高效向导RNA (sgRNA)序列、预测sgRNA的活性、脱靶效应评估、基因敲除、高通量功能基因筛选等领域的研究现状与发展前景进行了展望,以期为基因组编辑领域的研究提供参考。     

利用CRISPR/Cas9技术对人多能干细胞进行高效基因组编辑

CRISPR/Cas9技术提供了一个全新的基因组编辑体系。本文利用CRISPR/Cas9平台,在人胚胎干细胞株中对选取的一段特定基因组区域进行了多种基因组编辑：通过在基因编码框中引入移码突变进行基因敲除;通过单链DNA提供外源模板经由同源重组定点敲入FLAG序列;通过同时靶向多个位点诱导基因组大片段删除。研究结果表明CRISPR/Cas9可以对多能干细胞进行高效基因编辑,获得的突变干细胞株有助于对基因和基因组区域的功能进行分析和干细胞疾病模型的建立。     

CRISPR/Cas9-mediated correction of human genetic disease

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas) protein 9 system(CRISPR/Cas9) provides a powerful tool for targeted genetic editing. Directed by programmable sequence-specific RNAs,this system introduces cleavage and double-stranded breaks at target sites precisely. Compared to previously developed targeted nucleases, the CRISPR/Cas9 system demonstrates several promising advantages, including simplicity, high specificity,and efficiency. Several broad genome-editing studies with the CRISPR/Cas9 system in different species in vivo and ex vivo have indicated its strong potential, raising hopes for therapeutic genome editing in clinical settings. Taking advantage of non-homologous end-joining(NHEJ) and homology directed repair(HDR)-mediated DNA repair, several studies have recently reported the use of CRISPR/Cas9 to successfully correct disease-causing alleles ranging from single base mutations to large insertions. In this review, we summarize and discuss recent preclinical studies involving the CRISPR/Cas9-mediated correction of human genetic diseases.     

An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida)

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-related nuclease 9(Cas9) system enables precise, simple editing of genes in many animals and plants.However, this system has not been applied to rose(Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single-guide RNAs(sgRNAs) with high editing efficiency for CRISPR/Cas9-mediated gene editing in rose using suspensio...     

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.     

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.     

The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny

The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double‐strand breaks (DSBs) at pre‐selected genomic sites that are repaired by non‐homologous end joining (NHEJ) in an error‐prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas‐mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR‐based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off‐target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.