一种新型的CRISPR/Cas9-hLacI双链DNA供体适配基因编辑系统

在CRISPR/Cas9系统介导的基因编辑中,借助于双链DNA （double-stranded DNA,dsDNA）供体模板的重组效应能够实现对目标基因组靶位点的精确编辑和基因敲入,然而高等真核生物细胞中同源重组的低效性限制了该基因编辑策略的发展和应用。为提高CRISPR/Cas9系统介导dsDNA供体模板的同源重组效率,本研究利用大肠杆菌（Escherichia coli）乳糖操纵子阻遏蛋白LacI与操纵序列LacO特异性结合的特点,通过重组DNA技术将密码子人源化优化的阻遏蛋白基因LacI分别与脓链球菌（Streptococcus pyogenes）源的SpCas9和路邓葡萄球菌（Staphylococcus lugdunensis）源的SlugCas9-HF融合表达,通过PCR将操纵序列LacO与dsDNA供体嵌合,构建了新型的CRISPR/Cas9-hLacI供体适配系统（donor adapting system,DAS）。首先在报告载体水平上对Cas9核酸酶活性、DAS介导的同源引导修复（homology-directed repair,HDR）效率进行了验证和优化,其次在基因组水平对其介导的基因精确编辑进行了检测,并最终利用CRISPR/SlugCas9-hLacI DAS在HEK293T细胞中实现了VEGFA位点的精确编辑,效率高达30.5%,显著高于野生型。综上所述,本研究开发了新型的CRISPR/Cas9-hLacI供体适配基因编辑系统,丰富了CRISPR/Cas9基因编辑技术种类,为以后的基因编辑及分子设计育种研究提供了新的工具。     

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.     

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T₀ transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium‐delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, TaCKX2‐1, TaGLW7, TaGW2, and TaGW8, in T₀, T₁, and T₂ generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in TaCKX2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium‐delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.     

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系统纳米递送及其在肿瘤治疗中应用

新兴的CRISPR/Cas9基因编辑技术可实现在分子水平上对基因进行操作,具有设计简单、易于操作、特异性好、效率高等优点,广泛应用于肿瘤发生、发展和转移的潜在机制以及临床治疗的研究.利用纳米技术研发的非病毒纳米载体可以将CRISPR/Cas9系统高效递送到体内,为CRISPR/Cas9技术在临床领域的应用提供新途径.本文介绍CRISPR/Cas9的作用原理,简要概括目前CRISPR/Cas9系统的递送形式和常用的纳米递送载体,总结在部分肿瘤治疗中应用该技术的研究进展,并进一步对此进行展望.     

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.     

Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system

CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species.

     

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in <Emphasis Type="Italic">Arabidopsis</Emphasis> and its prevention

Key message

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations.

Abstract

Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

     

The CRISPR/Cas9 system: Their delivery,in vivo and ex vivo applications and clinical development by startups

The CRISPR/Cas9 gene editing system was originally derived from the prokaryotic adaptive immune system mediated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‐associated proteins (Cas). The system has been successfully applied to genome editing in eukaryotes and has contributed to remarkable advances in the life sciences, in areas ranging from agriculture to genetic disease therapies. For efficient editing and extending the influence of this system, proper delivery of its components is crucial. Both viral and nonviral delivery methods are reviewed here, along with the advantages and disadvantages of each. In addition, we review ex vivo and in vivo CRISPR/Cas9 applications for disease therapies. Related remarkable studies are highlighted and relevant startup companies and their drug development pipelines are described. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1035–1045, 2017     

Increasing the efficiency of CRISPR‐Cas9‐VQR precise genome editing in rice

Clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR‐Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR‐Cas9‐VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.     

Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae

Large chromosomal modifications have been performed in natural and laboratory evolution studies and hold tremendous potential for use in foundational research, medicine, and biotechnology applications. Recently, the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR/Cas9) system has emerged as a powerful tool for genome editing in various organisms. In this study, we applied the CRISPR/Cas9 system to preform large fragment deletions in Saccharomyces cerevisiae and compared the performance activity to that of a traditional method that uses the Latour system. Here we report in S. Cerevisiae the CRIPR/Cas9 system has been used to delete fragments exceeding 30 kb. The use of the CRISPR/Cas9 system for generating chromosomal segment excision showed some potential advantages over the Latour system. All the results indicated that CRISPR/Cas9 system was a rapid, efficient, low-cost, and versatile method for genome editing and that it can be applied in further studies in the fields of biology, agriculture, and medicine.     

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol) Ⅲ promoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p) and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.     

An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T₀ transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T₁ progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.     

嗜热厌氧杆菌热稳定CRISPR/Cas9基因组编辑技术及在细胞工厂构建中的应用研究进展

嗜热厌氧杆菌属(Thermoanaerobacter)来源的菌株作为一个高温发酵的细胞工厂,具有生产生物燃料和化学品的潜力,已引起了研究者的广泛兴趣.随着嗜热厌氧杆菌属来源的多个菌株全基因组测序的完成以及相关的生理生化实验的开展,建立一个简便、快捷的基因操作技术将有助于进一步深入理解和认识嗜热厌氧杆菌有关的代谢途径.最...     

High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.