Improving the efficiency for generation of genome-edited zebrafish by labeling primordial germ cells

Although CRISPR/Cas, a new versatile genome-editing tool, has been widely used in a variety of species including zebrafish, an important vertebrate model animal for biomedical research, the low efficiency of germline transmission of induced mutations and particularly knockin alleles made subsequent screening for heritable offspring tedious, time-consuming, expensive and at times impossible. In this study, we reported a method for improving the efficiency of germline transmission screening for generation of genome-edited zebrafish mutants. Co-microinjecting yfp-nanos3 mRNA with Cas9 mRNA, sgRNA and single strand DNA donor to label the distribution of microinjected nucleotides in PGCs (primordial germ cells), we demonstrated that founders carrying labeled PGCs produced much higher numbers of knockin and knockout progeny. In comparison with the common practice of selecting founders by genotyping fin clips, our new strategy of selecting founders with tentatively fluorescent-labeled PGCs significantly increase the ease and speed of generating heritable knocking and knockout animals with CRISPR/Cas9.     

Primordial germ cell‐specific expression of eGFP in transgenic chickens

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked‐in (gene‐trapped) PGCs into the blood vessels of Hamburger–Hamilton stages (HH‐stages) 13–16 chicken embryos. Gene‐trapped chickens were established by crossing a chimeric chicken with a wild‐type hen with very high efficiency. Heterozygous gene‐trapped chickens grew normally and SSEA‐1‐positive cells expressed eGFP during HH‐stages 13–30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene‐trapped embryos obtained by crossing heterozygous gene‐trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.     

Recent advances in CRISPR/Cas9 mediated genome editing in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.     

High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50–100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.     

Efficient genome editing in cultured cells and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system

Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat.     

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。     

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 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.     

CRISPR/Cas9 system in Plasmodium falciparum using the centromere plasmid

The CRISPR/Cas9 nuclease system is a powerful method to genetically modify the human malarial parasite, Plasmodium falciparum. Currently, this method is carried out by co-transfection with two plasmids, one containing the Cas9 nuclease gene, and another encoding the sgRNA and the donor template DNA. However, the efficiency of modification is currently low owing to the low frequency of these plasmids in the parasites. To improve the CRISPR/Cas9 nuclease system for P. falciparum, we developed a novel method using the transgenic parasite, PfCAS9, which stably expresses the Cas9 nuclease using the centromere plasmid. To examine the efficiency of genetic modification using the PfCAS9 parasite, we performed site-directed mutagenesis of kelch13 gene, which is considered to be involved in artemisinin resistance. Our results demonstrated that the targeted mutation could be introduced with almost 100% efficiency when the transfected PfCAS9 parasites were treated with two drugs to maintain both the centromere plasmid containing the Cas9 nuclease and the plasmid having the sgRNA. Therefore, the PfCAS9 parasite is a useful parasite line for the genetic modification of P. falciparum.     

利用CRISPR/Cas9技术对人多能干细胞进行高效基因组编辑

CRISPR/Cas9技术提供了一个全新的基因组编辑体系。本文利用CRISPR/Cas9平台,在人胚胎干细胞株中对选取的一段特定基因组区域进行了多种基因组编辑：通过在基因编码框中引入移码突变进行基因敲除;通过单链DNA提供外源模板经由同源重组定点敲入FLAG序列;通过同时靶向多个位点诱导基因组大片段删除。研究结果表明CRISPR/Cas9可以对多能干细胞进行高效基因编辑,获得的突变干细胞株有助于对基因和基因组区域的功能进行分析和干细胞疾病模型的建立。     

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-Mediated Gene Knock-Down in Post-Mitotic Neurons

The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.     

Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.     

Mosaicism in CRISPR/Cas9-mediated genome editing

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue.     

Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes

Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.     

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.     

<Emphasis Type="Italic">CAR1</Emphasis> deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98 % decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.