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。     

CRISPR/Cas9系统中sgRNA设计与脱靶效应评估

基于CRISPR/Cas9系统介导的第三代基因组编辑技术,已成功应用于动物、植物和微生物等诸多物种的基因组改造。如何提高CRISPR/Cas9技术的基因组编辑效率和最大限度降低脱靶风险一直是本领域的研究热点,而使用高效且特异的sgRNA(Small guide RNA)是基因组改造成功的关键性因素之一。目前,已有多款针对CRISPR/Cas9技术的sgRNA设计和/或脱靶效应评估软件,但不同的软件各有优缺点。本文重点对16款sgRNA 设计和脱靶效应评估在线和单机版软件的特点进行了阐述,通过制定38项评估指标对不同软件进行了比较分析,最后对11种用于检测基因组编辑效率和脱靶的实验方法,以及如何筛选高效且特异的sgRNA进行了归纳总结。     

CRISPR/Cas9基因组编辑技术在农业动物中的应用

CRISPR(Clustered regularly interspaced short palindromic repeats)/Cas(CRISPR associated proteins)是在细菌和古细菌中发现的一种用来抵御病毒或质粒入侵的获得性免疫系统.目前已发现的CRISPR/Cas系统包括Ⅰ,Ⅱ和Ⅲ型,其中Ⅱ型系统的组成较简单,由其改造成的CRISPR/Cas9技术已成为一种高效的基因组编辑工具.自2013年CRISPR/Cas9技术成功用于哺乳动物基因组定点编辑以来,应用该技术进行基因组编辑的报道呈现出爆发式的增长.农业动物不仅是重要的经济动物,也是人类疾病和生物医药研究的重要模式动物.本文综述了CRISPR/Cas9技术在农业动物中的研究和应用进展,简述了该技术的脱靶效应及减少脱靶的主要方法,并展望了该技术的应用前景.     

CRISPR/Cas9技术在非模式植物中的应用进展

基因组编辑技术的出现对植物遗传育种及作物性状的改良产生了深远意义。CRISPR/Cas(clustered regularly interspaced short palindromic repeat)是由成簇规律间隔短回文重复序列及其关联蛋白组成的免疫系统,其作用是原核生物(40%细菌和90%古细菌)用来抵抗外源遗传物质(噬菌体和病毒)的入侵。该技术实现了对基因组中多个靶基因同时进行编辑,与前两代基因编辑技术:锌指核酶(ZFNs)和转录激活因子样效应物核酶(TALENs)相比更加简单、廉价、高效。目前CRISPR/Cas9基因编辑技术已在拟南芥(Arabidopsis thaliana)、烟草(Nicotiana benthamiana)、水稻(Oryza sativa)、小麦(Triticum aestivum)、玉米(Zea mays)、番茄(tomato)等模式植物和多数大作物中实现了定点基因组编辑,其应用范围不断地向各类植物扩展。但与模式植物和一些大作物相比,CRISPR/Cas9基因编辑技术在非模式植物,尤其在一些小作物的应用中存在如载体构建、靶点设计、脱靶检测、同源重组等问题有待进一步完善。该文对CRISPR/Cas9技术在非模式植物与小作物研究的最新研究进展进行了总结,讨论了该技术目前在非模式植物、小作物应用的局限性,在此基础上提出了相关改进策略,并对CRISPR/Cas9系统在非模式植物中的研究前景进行了展望。     

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.     

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off‐target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR‐induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5‐fold in somatic tissues and up to 100‐fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double‐stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on‐target mutagenesis in plants using CRISPR/Cas9.     

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.     

靶向番茄SlACS2基因CRISPR-Cas9 sgRNA的设计和分析

番茄为呼吸跃变型果实,伴随呼吸跃变产生大量乙烯,即系统II乙烯,易使番茄果实过熟,导致腐烂变质。SlACS2是番茄系统II乙烯合成的限速酶,通过CRISPR-Cas9基因组编辑系统修饰该基因,调控系统II乙烯过量表达,将迟滞番茄过熟。本研究基于RNA-seq建立了SlACS2基因的数字表达谱,表明该基因呈果实特异性表达,在植株的根、茎、叶等部位不表达。SlACS2位于番茄1号染色体,含4个外显子和3个内含子。利用在线工具CRISPRdirect 和CRISPR-P发现第1、2、3外显子分别具有18、9和11条sgRNA。其中,sgRNA1-14和sgRNA3-8及二者的近PAM的12 nt 种子序列在番茄基因组是唯一序列,GC含量高于40%,不存在TTTT终止序列。BLAST结果表明,sgRNA1-14和sgRNA3-8与GenBank公布的8条SlACS2同源序列高度一致,位于该基因的保守区,而与SlACS4和SlACS6的同源序列存在多个SNP,预示这2条sgRNA可用于番茄不同品种SlACS2基因的靶向编辑,并可规避对SlACS家族其他同源基因的脱靶效应。