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.     

Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana

Engineered nucleases can be used to induce site‐specific double‐strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error‐prone non‐homologous end‐joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single‐strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ‐mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease‐induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ‐mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740‐fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I–SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB‐inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR‐mediated gene targeting systems but also by the combined action of two nickases as DSB‐inducing agents excluding off‐target effects in homologous genomic regions.     

Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T₁ progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.     

Multiplex Gene Disruption by Targeted Base Editing of Yarrowia lipolytica Genome Using Cytidine Deaminase Combined with the CRISPR/Cas9 System

The oleaginous yeast Yarrowia lipolytica has a tendency to use the non‐homologous end joining repair (NHEJ) over the homology directed recombination as double‐strand breaks (DSB) repair system, making it difficult to edit the genome using homologous recombination. A recently developed Target‐AID (activation‐induced cytidine deaminase) base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without DSB and donor DNA. In this study, this system is adopted in Y. lipolytica for multiplex gene disruption. Target‐specific gRNA(s) and a fusion protein consisting of a nickase Cas9, pmCDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Deletion of the KU70 gene involved in the NHEJ prevents the generation of indels by base excision repair following cytidine deamination, increasing the accuracy of genome editing. Using this Target‐AID system with optimized expression levels of the base editor, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively, demonstrating this base editing system as a convenient genome editing tool in Y. lipolytica.     

Multiplex gene editing of the <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> genome using the CRISPR-Cas9 system

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.     

一种高效无选择标记的黑曲霉基因组编辑方法

黑曲霉(Aspergillus niger)是一种重要的工业生产菌株,被广泛地应用于生产酶制剂和有机酸,但仍需要进行基因组改造提高它的应用潜力。CRISPR/Cas9技术是一种被广泛采用的黑曲霉基因组编辑技术,但由于需要在基因组中整合选择标记或基因编辑效率还有待提高,影响了其在工业菌株改造中的应用。本研究建立了一种基于CRISPR/Cas9技术的高效无选择标记的基因编辑方法。首先,利用5S rRNA启动子启动sgRNA的表达,构建了一个含有AMA1(autonomously maintained in Aspergillus)复制起始片段的sgRNA和Cas9共表达质粒;同时通过敲除kusA基因构建非同源末端连接(non-homologous end joining pathway,NHEJ)修复缺陷的高效同源重组菌株;最后利用含有AMA1片段质粒的不稳定性,通过无抗平板传代丢失含有sgRNA和Cas9共表达质粒。利用该方法,在采用同源臂长度仅为20bp的无选择标记供体DNA进行基因编辑时,基因编辑效率可达到100%。该方法为黑曲霉基因功能的研究和细胞工厂的构建奠定了基础。     

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.     

A highly efficient gene-targeting system for Aspergillus parasiticus

Aims: To establish a system that greatly increases the gene‐targeting frequency in Aspergillus parasiticus. Methods and Results: The ku70 gene, a gene of the nonhomologous end‐joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O‐methylsterigmatocystin (OMST). The NHEJ‐deficient strain, RHΔku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA‐topoisomerase I complex inhibitor, camptothecin, and the DNA‐damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA‐targeting frequency in RHΔku70 reached 96%. Conclusions: The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ‐deficient RHΔku70, easy creation of homologous ends for integration, and the ptr‐based selection form a highly efficient gene‐targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study: The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus.     

Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains

In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1 kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70? HR of URA3 marker at the ADE2 locus occurred with 43 % frequency with as little as 50 bp long flanking regions. The number of Ura⁺ transformants was reduced to 1 % of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80?, Ura⁺ transformants was 60 % lower compared to the wild type.     

动物基因组编辑中提升CRISPR/Cas9介导的同源重组效率研究进展*

基因组编辑技术可以对DNA或RNA进行精准改造,极大地促进了生命科学的发展。CRISPR/Cas9系统在靶位点诱导DNA发生双链或单链损伤,细胞对损伤部位采用无供体模板的非同源末端连接(non-homologous end joining,NHEJ)或有供体模板的同源重组(homologous recombination,HR)修复。基于HR的基因组编辑策略通常被用于获得DNA的精准改造,而NHEJ在动物DNA损伤修复中起主导作用。为了提升HR效率,研究人员设计了多种方案,包括CRISPR/Cas9系统优化和DNA修复通路调控等。从DNA损伤修复途径、Cas9变体选择、sgRNA设计、供体模板设计、DNA修复途径相关蛋白功能调控、供体模板募集效率提升、细胞周期调控及编辑细胞生存效率提升等方面详细综述了相关研究成果,发现尚未开发出放之四海而皆准的HR提升策略,基于HR的基因组编辑需要针对具体案例制定个体化策略。旨在为动物基因组编辑中提升CRISPR/Cas9介导的HR效率研究提供理论参考,为动物基因功能分析、基因治疗和经济动物基因编辑育种提供帮助。     

EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.     

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR–Cas9 cassette or a transient CRISPR–Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.     

CRISPR/Cas9‐mediated genome modification in the mollusc,Crepidula fornicata

The discovery and application of the CRISPR/Cas9 genome editing method has greatly enhanced the ease with which transgenic manipulation can occur. We applied this technology to the mollusc, Crepidula fornicata, and have successfully created transgenic embryos expressing mCherry fused to endogenous β‐catenin. Specific integration of the fluorescent reporter was achieved by homologous recombination with a β‐catenin‐specific donor DNA containing the mCherry coding sequence. This fluorescent gene knock‐in strategy permits in vivo observations of β‐catenin expression during embryonic development and represents the first demonstration of CRISPR/Cas9‐mediated transgenesis in the Lophotrochozoa superphylum. The CRISPR/Cas9 method is a powerful and economical tool for genome modification and presents an option for analysis of gene expression in not only major model systems, but also in those more diverse species that may not have been amenable to the classic methods of transgenesis. This approach will allow one to generate transgenic lines of snails for future studies. genesis 53:237–244, 2015. © 2014 Wiley Periodicals, Inc.     

CRISPR/Cas13系统敲减HEK293T细胞ku70和lig4基因表达

成簇的规律间隔的短回文重复序列(Clusteredregularlyinterspacedshortpalindromicrepeats,CRISPR)/CRISPR相关蛋白(CRISPR-associatedproteins,Cas)系统是目前基因编辑、基因表达研究的热点,其中靶向RNA的CRISPR/Cas13系统的开发为RNA的干扰和编辑提供了新的方向。文中针对HEK293T细胞非同源末端连接(Nonhomologousendjoining,NHEJ)通路修复关键因子Ku70和Lig4的编码序列,设计并合成CRISPR/Cas13a、b系统相应的gRNA,检测其对ku70和lig4基因表达的影响。结果显示,Cas13a对ku70和lig4的RNA敲减效果可以达到50%以上,Cas13b对ku70和lig4的RNA敲减效果分别达到92%和76%;同时Cas13a、b系统能将Ku70和Lig4蛋白水平分别下调至68%和53%。CRISPR/Cas13系统可有效敲减HEK293T细胞RNA和蛋白质表达,为基因功能和调控研究提供一种新的策略。     

Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9

Background

Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.

Results

We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17–0.45%) that was used to knock in an approximately 5-kb long DNA fragment.

Conclusion

We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.

     

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 induction of heritable inversions in plant genomes using the CRISPR/Cas system

During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild‐type and in the non‐homologous end‐joining (NHEJ) mutant ku70‐1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild‐type, most junctions are formed by microhomology‐mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg‐cell‐specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.