共查询到20条相似文献,搜索用时 15 毫秒
1.
Takuya Katayama Yuki Tanaka Tomoya Okabe Hidetoshi Nakamura Wataru Fujii Katsuhiko Kitamoto Jun-ichi Maruyama 《Biotechnology letters》2016,38(4):637-642
Objectives
To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins.Results
To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations.Conclusions
We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.2.
Qianqian Ji Caiping Guo Chen Xie Yingdan Wu Pei Zhang Hui Li Yongjun Lu 《Biotechnology letters》2017,39(10):1471-1476
Objectives
To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination.Results
α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines. Cell clones obtained had the AAT gene integrated at the AAVS1 locus and secreted approx. 0.04 g/l recombinant AAT into the medium. Moreover, the secreted AAT showed an inhibitory activity that is comparable to plasma AAT.Conclusions
CRISPR/Cas9-mediated engineering of human cells is a promising alternative for generating isogenic cell lines with consistent AAT production. This work sheds new light on the generation of therapeutic liver stem cells for AATD.3.
Zhe Yang Shihao Chen Songlei Xue Xinxiu Li Zhen Sun Yu Yang Xuming Hu Tuoyu Geng Hengmi Cui 《Biotechnology letters》2018,40(11-12):1507-1518
Objectives
To investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.Results
Here we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.Conclusions
The transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.4.
Nadia Amrani Xin D. Gao Pengpeng Liu Alireza Edraki Aamir Mir Raed Ibraheim Ankit Gupta Kanae E. Sasaki Tong Wu Paul D. Donohoue Alexander H. Settle Alexandra M. Lied Kyle McGovern Chris K. Fuller Peter Cameron Thomas G. Fazzio Lihua Julie Zhu Scot A. Wolfe Erik J. Sontheimer 《Genome biology》2018,19(1):214
Background
The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.Results
Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N4GATT-3′), for NmeCas9 genome editing in human cells.Conclusions
Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.5.
6.
Neftali Vazquez Lilia Sanchez Rebecca Marks Eduardo Martinez Victor Fanniel Alma Lopez Andrea Salinas Itzel Flores Jesse Hirschmann Robert Gilkerson Erin Schuenzel Robert Dearth Reginald Halaby Wendy Innis-Whitehouse Megan Keniry 《BMC molecular biology》2018,19(1):3
Background
Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.Results
We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9?=?CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.Conclusions
Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).7.
Key message
The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination.Abstract
The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination.8.
Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism 总被引:1,自引:0,他引:1
Xuelian Zheng Shixin Yang Dengwei Zhang Zhaohui Zhong Xu Tang Kejun Deng Jianping Zhou Yiping Qi Yong Zhang 《Plant cell reports》2016,35(7):1545-1554
Key message
A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice.Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.9.
10.
Zhou Hong Zhou Michael Li Daisy Manthey Joseph Lioutikova Ekaterina Wang Hong Zeng Xiao 《BMC genomics》2017,18(9):826-38
Background
The beauty and power of the genome editing mechanism, CRISPR Cas9 endonuclease system, lies in the fact that it is RNA-programmable such that Cas9 can be guided to any genomic loci complementary to a 20-nt RNA, single guide RNA (sgRNA), to cleave double stranded DNA, allowing the introduction of wanted mutations. Unfortunately, it has been reported repeatedly that the sgRNA can also guide Cas9 to off-target sites where the DNA sequence is homologous to sgRNA.Results
Using human genome and Streptococcus pyogenes Cas9 (SpCas9) as an example, this article mathematically analyzed the probabilities of off-target homologies of sgRNAs and discovered that for large genome size such as human genome, potential off-target homologies are inevitable for sgRNA selection. A highly efficient computationl algorithm was developed for whole genome sgRNA design and off-target homology searches. By means of a dynamically constructed sequence-indexed database and a simplified sequence alignment method, this algorithm achieves very high efficiency while guaranteeing the identification of all existing potential off-target homologies. Via this algorithm, 1,876,775 sgRNAs were designed for the 19,153 human mRNA genes and only two sgRNAs were found to be free of off-target homology.Conclusions
By means of the novel and efficient sgRNA homology search algorithm introduced in this article, genome wide sgRNA design and off-target analysis were conducted and the results confirmed the mathematical analysis that for a sgRNA sequence, it is almost impossible to escape potential off-target homologies. Future innovations on the CRISPR Cas9 gene editing technology need to focus on how to eliminate the Cas9 off-target activity.11.
Qiang Zhang Hui-Li Xing Zhi-Ping Wang Hai-Yan Zhang Fang Yang Xue-Chen Wang Qi-Jun Chen 《Plant molecular biology》2018,96(4-5):445-456
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.12.
13.
14.
Objective
To use HIV-1 based lentivirus components to produce gene integration and the formation of a stable cell line in the packaging cell line without viral infection.Results
A co-transfection of a Human Embryonic Kidney (HEK) 293 packaging cell line with Gag–pol (GP) and a transfer vector, without the envelope vector, produces a stable cell line after 2 weeks of selection. Furthermore, a matrix protein deficient GP in the packaging vector enhances this integration. This supports that, in theory, unexported lentiviral cores produced within the packaging cell can infect itself without requiring the release of any lentiviral particles.Conclusion
If the packaging cell is also the target cell, then gene integration leading to a stable cell line can be accomplished without viral particle infection.15.
Xin Yu Xiquan Liang Huimin Xie Shantanu Kumar Namritha Ravinder Jason Potter Xavier de Mollerat du Jeu Jonathan D. Chesnut 《Biotechnology letters》2016,38(6):919-929
Objectives
To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.Results
Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.Conclusion
Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.16.
Background
Fevers of unknown origin constitute a substantial disease burden in Southeast Asia. In majority of the cases, the cause of acute febrile illness is not identified.Methods
We used MassTag PCR, a multiplex assay platform, to test for the presence of 15 viral respiratory agents from 85 patients with unexplained respiratory illness representing six disease clusters that occurred in Cambodia between 2009 and 2012.Results
We detected a virus in 37 (44%) of the cases. Human rhinovirus, the virus detected most frequently, was found in both children and adults. The viruses most frequently detected in children and adults, respectively, were respiratory syncytial virus and enterovirus 68. Sequence analysis indicated that two distinct clades of enterovirus 68 were circulating during this time period.Conclusions
This is the first report of enterovirus 68 in Cambodia and contributes to the appreciation of this virus as an important respiratory pathogen.17.
Background
Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation.Design and methods
We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants.Results
We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo.Conclusion
The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.18.
Yu Nie Peiqing Cong Xiaofeng Liu Min Wang Yaosheng Chen Zuyong He 《Biotechnology letters》2017,39(3):351-358
Objectives
To develop an in vitro method for rapid evaluation of the capability of a designed single guide RNAs (sgRNAs) to guide Cas9 nucleases to cleave target loci in mammalian cells.Results
We constructed a Cas9/sgRNA plasmid with two SP6 promoters to simultaneously express Cas9 nuclease and the sgRNA and a negative selection plasmid harbouring a target site of the sgRNA. After co-transforming chemically competent E. coli DH5α cells with the two plasmids, the transformants were plated at a low density on two LB plates: one containing only ampicillin and the other containing both ampicillin and chloramphenicol. The colony-count on the ampicillin + chloramphenicol plate was compared with that on the ampicillin-only plate to calculate the survival percentage. The survival % was negatively correlated with the genome editing efficiency of the sgRNA in mammalian cells evaluated by a T7 endonuclease 1 (T7E1) assay (r ranged from ?0.8 to ?0.92). This system eliminates the need for cell culture, transfection, FACS sorting, PCR and T7E1 nuclease treatment, and significantly reduces the cost of screening for active sgRNAs, especially in the case of large-scale screening.Conclusions
We have developed a bacterial-based negative selection system for rapid screening of active sgRNAs in mammalian cells at a very low cost.19.
Asghar Abdoli Hoorieh Soleimanjahi Abbas Jamali Parvaneh Mehrbod Shima Gholami Zahra Kianmehr Neda Feizi Maryam Saleh Fariborz Bahrami Talat Mokhtari-Azad Mohsen Abdoli Masoumeh Tavassoti Kheiri 《Biotechnology letters》2016,38(6):941-948