CRISPR/Cas9系统研究进展

CRISPR/Cas9系统的发展彻底改变了人们编辑DNA序列和调控目标基因表达水平的能力,从而为生物体的精确基因组编辑提供了有力的工具。简化后的CRISPR/Cas9系统由两部分组成:Cas9蛋白和sgRNA。其作用原理为sgRNA通过自身的Cas9把手与Cas9蛋白形成Cas9-sgRNA复合体,Cas9-sgRNA复合体中sgRNA的碱基互补配对区序列与目标基因的靶序列通过碱基互补配对原则进行配对结合,Cas9利用自身的核酸内切酶活性对目标DNA序列进行切割。与传统的基因组编辑技术相比,CRISPR/Cas9系统具有几大明显的优势:易用性、简便性、低成本、可编程性以及可同时编辑多个基因。CRISPR/Cas9基因组编辑技术以及衍生出来的CRISPRi和CRISPRa基因表达调控技术已经广泛应用于多种真核和原核生物中。综述了CRISPR/Cas9系统的起源、作用机理、在生物体中的应用和其衍生出的技术,并概述了其脱靶效应和未来前景。     

利用CRISPR/Cas9核酸酶靶向细菌必需基因的特异性抗菌剂研究（英文）

CRISPR/Cas9核酸酶是一种高效和精确的基因组DNA编辑工具。目前,CRISPR/Cas9被开发成一种新型抗菌剂,通过靶向破坏目标基因,例如抗生素耐药基因来诱导细菌死亡。本研究利用CRISPR携带多靶点优势,同时靶向大肠杆菌必需基因gyrA、gyrB和folA,以达到杀菌作用。本设计针对3个内源基因的CRISPR系列载体,分别含有1个、2个或3个靶点。当IPTG诱导Cas9表达后,CRISPR RNA (crRNA)和反式作用CRISPR RNA (trans-activating CRISPR RNA, tracrRNA)复合物募集Cas9切割靶基因,导致细菌死亡。随着crRNA中靶点数量的增加,杀菌效率显著提高。当含有3个靶点的crRNA作用于细菌基因组时,杀菌效率达到99.35%。此外,在存活的大肠杆菌中,crRNA表达质粒的靶序列和直接重复序列发现碱基缺失,而内源靶基因和Cas9基因均未发生突变。最后,该CRISPR系统还应用于其他大肠杆菌实验室菌株和产肠毒素K88菌株,并表现出高效的杀菌作用。我们设计了一种基于CRISPR/Cas9核酸酶的新型抗菌剂,为抗菌剂的研发提供新策略。     

BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发及应用

碱基编辑技术结合了CRISPR/Cas系统的靶向特异性与碱基脱氨酶的催化活性,因其不产生双链DNA断裂、不需要外源DNA模板、不依赖同源重组修复,自开发以来,便受到研究者的追捧,在哺乳动物细胞、植物、微生物等领域相继得到开发与应用。为了进一步丰富碱基编辑系统在谷氨酸棒杆菌中的应用,将鼠源胞嘧啶脱氨酶(rAPOBEC1)与nCas9蛋白融合,实现了在谷氨酸棒杆菌中C到T的编辑,编辑比例较低(0-20%);在上述融合蛋白C端添加UGI蛋白,构建BE3型胞嘧啶碱基编辑器,抑制体内的DNA碱基切除修复机制,显著的提高了碱基编辑效率,使得C到T的碱基编辑效率高达90%;为了简化操作,将双质粒碱基编辑系统优化为单质粒碱基编辑系统,并显著提高转化效率;最后通过单质粒碱基编辑系统对基因组中其他位点的编辑测试,进一步证明了BE3型碱基编辑器在谷氨酸棒杆菌中的高效性,同时发现该碱基编辑器具有较宽的编辑窗口(PAM上游-11到-19位),有助于覆盖更多的基因组靶标位点,为谷氨酸棒杆菌的基因组改造提供了更多的工具选择。     

谷氨酸棒杆菌碱基编辑的条件优化

近年来,基于CRISPR/Cas9的碱基编辑技术因其具有不产生DNA双链断裂、无需外源DNA模板、不依赖宿主同源重组修复的优势,已经逐渐发展成为一种强大的基因组编辑工具,在动物、植物、酵母和细菌中得到了开发和应用。研究团队前期已在重要的工业模式菌株谷氨酸棒杆菌中开发了一种多元自动化的碱基编辑技术MACBETH,为进一步优化该方法,提高碱基编辑技术在谷氨酸棒杆菌中的应用效率,本研究首先在谷氨酸棒杆菌中构建了基于绿色荧光蛋白(GFP)的检测系统:将GFP基因的起始密码子ATG人工突变为ACG,GFP无法正常表达,当该密码子的C经编辑后恢复为T,即实现GFP蛋白的复活,结合流式细胞仪分析技术,可快速衡量编辑效率。然后,构建针对靶标位点的碱基编辑工具,经测试,该位点可成功被编辑,在初始编辑条件下碱基编辑效率为(13.11±0.21)%。在此基础上,通过对不同培养基类型、诱导初始OD600、诱导时间、诱导物浓度进行优化,确定最优编辑条件是:培养基为CGXII,初始OD600为0.05,诱导时间为20 h,IPTG浓度为0.01 mmol/L。经过优化,编辑效率达到(30.35±0.75)%,较初始条件提高了1.3倍。最后,选取原编辑条件下编辑效率较低的位点,进行了优化后编辑条件下的编辑效率评估,结果显示,不同的位点在最优编辑条件下的编辑效率提高了1.7–2.5倍,进一步证实该优化条件的有效性及通用性。研究结果为碱基编辑技术在谷氨酸棒杆菌中更好的应用提供了重要的参考价值。     

基于CRISPR/Cas系统的单碱基编辑技术研究进展*

基于CRISPR/Cas系统出现的单碱基编辑技术可以实现高效且简便的单个碱基的替换编辑,其原理是将胞嘧啶脱氨酶(cytosine deaminase)或腺苷脱氨酶(adenosine deaminase)与Cas9n(D10A)形成融合蛋白,通过CRISPR/Cas精准识别和定位DNA上的靶位点后,利用胞嘧啶脱氨酶或腺苷脱氨酶将靶点距离sgRNA位点基序(protospacer adjacent motif,PAM)序列端的4~7位的单个碱基发生单碱基转换或颠换。对基于CRISPR/Cas系统的单碱基编辑技术发现的历史、组成和分类、工作原理进行了概述,并总结了该系统最新进展及应用。     

基于CRISPR编辑系统的DNA片段删除技术

基于CRISPR/Cas9系统的基因组编辑技术已成为基因功能研究和遗传修饰的重要工具。在引导RNA的引导下, Cas9蛋白对基因组靶位点进行精准切割产生DNA双链断裂(DSB), 借助细胞内的DSB修复机制, 可实现基因组靶位点碱基的缺失、插入或者替换, 甚至发生片段删除。该文介绍了基于CRISPR/Cas9基因组编辑系统的DSB微同源末端连接修复方式(MMEJ)提高基因组DNA片段删除效率的方法, 主要从靶点设计和突变检测方面展开详细描述。     

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。     

基于CRISPR/Cas系统的单碱基编辑技术研究进展*

基于CRISPR/Cas系统出现的单碱基编辑技术可以实现高效且简便的单个碱基的替换编辑,其原理是将胞嘧啶脱氨酶(cytosine deaminase)或腺苷脱氨酶(adenosine deaminase)与Cas9n(D10A)形成融合蛋白,通过CRISPR/Cas精准识别和定位DNA上的靶位点后,利用胞嘧啶脱氨酶或腺苷脱氨酶将靶点距离sgRNA位点基序(protospacer adjacent motif,PAM)序列端的4~7位的单个碱基发生单碱基转换或颠换。对基于CRISPR/Cas系统的单碱基编辑技术发现的历史、组成和分类、工作原理进行了概述,并总结了该系统最新进展及应用。     

CRISPR/Cas9技术在表观基因组中应用的探讨

成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列关联蛋白9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins,CRISPR/Cas9)技术是近5年来在生物基因改造中应用最广泛的新型编辑工具,具有成本低、效率高、多位点打靶以及脱靶效应可预测等优点,正在成为不同生物体中设计基因组的有力工具。表观遗传的不断发展对分子生物学的中心法则提出挑战,包括DNA中碱基和组蛋白的修饰、非编码RNA对基因组和染色质结构的调节中都起到至关重要的作用。对CRISPR/Cas9系统的改造可以对表观基因组进行修饰。对CRISPR/Cas9系统在表观基因组中进行修饰的应用进行概述,讨论了定向修饰基因组存在的问题,以期为CRISPR/Cas9系统在基因组编辑领域的应用提供参考。     

利用CRISPR 系统介导人类mir-505基因位点的编辑

目的:通过针对人源mir-505基因,设计不同sgRNA,从而利用CRIPSR系统对mir-505基因位点进行编辑,并探讨CRISPR系统对靶点的剪切效率的影响因素。方法:针对人源mir-505基因设计两条具有不同GC%的sgRNA,随后分别将其构建入41824-sgRNA表达载体和42230-Cas9蛋白/sgRNA共表达载体,并将表达载体利用脂质体转染法转染入人类细胞,通过T7E1assay检测不同CRISPR系统对靶点剪切效率的影响。结果:对靶点的剪切与Cas9蛋白和sgRNA的剂量成正比;当sgRNA与Cas9蛋白共传递时,也能够明显提高靶点的剪切效率;而较低的sgRNA的GC%会降低其对靶点的剪切效率。结论:本研究利用CRISPR系统靶向人源细胞的mir-505基因,使得mir-505基因发生突变。CRISPR系统对靶点的剪切效率和sgRNA和Cas9蛋白的剂量、sgRNA是否和Cas9蛋白共传递以及sgRNA的GC%有关。     

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.     

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.     

Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system

The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.     

Targeted mutagenesis in Arabidopsis thaliana using CRISPR-Cas12b/C2c1

Cas12b/C2c1 is a newly identified class 2 CRISPR endonuclease that was recently engineered for targeted genome editing in mammals and rice. To explore the potential applications of the CRISPR‐Cas12b system in the dicot Arabidopsis thaliana, we selected BvCas12b and BhCas12b v4 for analysis. We successfully used both endonucleases to induce mutations, perform multiplex genome editing, and create large deletions at multiple loci. No significant mutations were detected at potential off‐target sites. Analysis of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of CRISPR‐Cas12b systems for genome editing in Arabidopsis.     

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.     

Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector

Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.