Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F₁. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species.     

CRISPR/Cas9高效敲除突触结合蛋白Ⅰ的电生理学研究

研究一种蛋白质在神经元中的功能,最有效的方法之一是在该基因敲除动物的神经元中确认其表型.传统的用胚胎干细胞建立基因敲除动物模型的方法虽然稳定,但是复杂、耗时.近几年来,一种新型基因组编辑技术——CRISPR/Cas9,能够在不分裂的神经元中高效特异地敲除目的基因.本文研究了用CRISPR/Cas9系统敲除突触结合蛋白Ⅰ(synaptotagminⅠ,Syt1)基因后的小鼠海马培养神经元的电生理学特性.我们设计并构建了Syt1单导向RNA(Syt1 sgRNA)的慢病毒载体质粒,并用编码Cas9和Syt1 sgRNA的慢病毒感染培养的小鼠海马神经元,急性敲除神经元中Syt1基因(Syt1 sgRNA组),并用不靶向任何基因的Scramble sgRNA感染神经元作为阴性对照(Scramble组).通过全细胞膜片钳的方法检测单动作电位诱发的兴奋性突触后电流(single AP-eEPSC)、微小兴奋性突触后电流(mEPSCs)、高糖反应测量的即刻可释放囊泡池(RRP)以及10 Hz串刺激测量的囊泡释放概率(P_r).结果显示,Syt1 sgRNA组神经元丧失了Syt1的功能,并且与Syt1敲除(Syt1 KO)小鼠神经元的突触传递表型相似,而Scramble组神经元的各参数和野生型(WT)小鼠神经元相比没有显著性差异.本文为CRISPR/Cas9技术应用于神经元中基因的急性修饰提供了依据.     

Gene network analysis of oxaliplatin-resistant colorectal cancer to target a crucial gene using chitosan/hyaluronic acid/protamine polyplexes containing CRISPR-Cas9

Colorectal cancer (CRC) treatment is dramatically hampered by resistance to oxaliplatin alone or in the combination of irinotecan or 5-fluorouracil and leucovorin. This study aims to design and assess Chitosan/Hyaluronic Acid/Protamine sulfate (CS/HA/PS) polyplexes loaded with CRISPR plasmid for targeting a key gene in cancer drug resistance. Here, recent findings were considered to validate oxaliplatin-resistant CRC-related genes and systems biology approaches employed to detect the critical gene. The polyplexes were characterized according to particle size, zeta potential, and stability. Moreover, carrier toxicity and transfection efficiency were assessed on oxaliplatin-resistant HT-29 cells. The post-transfection evaluations were performed to confirm gene disruption-mediated CRISPR. Eventually, excision cross complementation group 1(ERCC1), a crucial member of the nucleotide excision repair pathway, was selected to be targeted using CRISPR/Cas9 to reverse oxaliplatin resistance in HT-29 cells. CS/HA/PS polyplexes containing CRISPR/Cas9 plasmid exhibited negligible toxicity and comparable transfection efficiency with Lipofectamine™. Following the efficient gene delivery, sequences in CRISPR/Cas9 target sites were altered, ERCC1 was downregulated, and drug sensitivity was successfully restored in oxaliplatin-resistant cells. Findings indicate that CS/HA/PS/CRISPR polyplexes provide a potential strategy for delivering cargo and targeting oxaliplatin resistance-related gene to manipulate drug resistance as a rising concern in cancer therapeutic approaches.     

Creation of zebrafish knock‐in reporter lines in the nefma gene by Cas9‐mediated homologous recombination

CRISPR/Cas9‐based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)‐induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock‐ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9‐targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock‐in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.     

CRISPR/Cas9: A new tool for the study and control of helminth parasites

Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9‐targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9‐mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.     

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.     

Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.     

Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10⁹ cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.     

Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.     

植物CRISPR基因组编辑技术的新进展

在过去的4年中,CRISPR/Cas9基因组编辑技术成为生命科学领域的革命性工具,为植物学基础研究和农作物遗传改良提供了高效、快速而又廉价的遗传操作工具。利用CRISPR/Cas9系统可以实现精准的knock-out和knock-in等遗传操作,也可用于靶向激活或抑制基因的表达。在CRISPR/Cas9被广泛地用于基因组编辑的同时,它的编辑能力、效率和精确度也在不断地改进和完善,特别是CRISPR/Cpf1系统的发掘和单碱基编辑技术的创建,使CRISPR系统正逐步成为一个理想的遗传工程技术平台。此外,利用CRISPR/Cas9技术改良的农作物品种也已经涌现,这必将推动精准基因组编辑技术在农作物遗传改良中的应用和发展。     

CRISPR-Cas9基因编辑技术在病毒感染疾病治疗中的应用

CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的DNA上进行编辑。病毒通过特异的受体侵染细胞,其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中。基因治疗是病毒感染疾病治疗的新趋势。因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义。文章主要从CRISPR-Cas9作用机制以及在病毒感染疾病治疗中的应用等方面进行了综述。     

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.     

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.     

CRISPR/Cas9 in rice can induce new mutations in later generations,leading to chimerism and unpredicted segregation of the targeted mutation

CRISPR/Cas9 is a novel tool for targeted mutagenesis and is applicable to plants, including rice. Previous reports on CRISPR/Cas9 in rice have demonstrated that target mutations are transmitted to the next generation in accordance with Mendelian law, but heritability of the target mutation and the role of inherited Cas9 gene have not been fully elucidated. Here, we targeted the rice phytoene desaturase gene, mutants of which exhibit an albino phenotype, by using CRISPR/Cas9 and analyzed segregation of target mutations. Agrobacterium-mediated methods using immature embryos successfully transformed a CRISPR/Cas9 system into five rice cultivars and subsequently induced mutation. Unpredicted segregations, with more mutants than theoretically predicted, were frequently found in T₁ plants from monoallelic T₀ mutants. Chimeric plants with both biallelic and monoallelic mutated cells were also observed in the T₁. Next, we followed segregation of a target mutation in the T₂ from monoallelic T₁ mutants. When T₁ mutants possessed Cas9, unpredicted segregations of the target mutation and chimeric plants were observed again in the T₂. When T₁ mutants did not possess Cas9, segregation of the target mutations followed Mendelian law and no chimeric plants appeared in the T₂. T₂ mutants with Cas9 had mutations different from the original ones found in T₀. Our results indicated that inherited Cas9 was still active in later generations and could induce new mutations in the progeny, leading to chimerism and unpredicted segregation. We conclude that Cas9 has to be eliminated by segregation in T₁ to generate homozygous mutants without chimerism or unpredicted segregation.     

Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.     

Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system

Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18 Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.     

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.