scholarly journals Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

2019 ◽  
Author(s):  
Remi L. Gratacap ◽  
Tim Regan ◽  
Carola E. Dehler ◽  
Samuel A.M. Martin ◽  
Pierre Boudinot ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Cell Line ◽  
Genome Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Genetic Resistance ◽  
Model Organisms ◽  
Fish Cell ◽  
Genome Wide ◽  
Lentivirus Transduction

1AbstractGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. In the current study, an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214) was developed. As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

scholarly journals Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

2021 ◽  
Author(s):  
Xiaoen Huang ◽  
Nian Wang
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Sweet Orange ◽  
Susceptibility Gene ◽  
Transformation Rate ◽  
Biallelic Mutation ◽  
Important Species ◽  
Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

scholarly journals Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Remi L. Gratacap ◽  
Tim Regan ◽  
Carola E. Dehler ◽  
Samuel A. M. Martin ◽  
Pierre Boudinot ◽  
...  
Keyword(s):  
Cell Line ◽  
Genome Editing ◽  
Delivery System ◽  
Salmonid Fish ◽  
Fish Cell Line ◽  
Fish Cell

scholarly journals Genome-wide identification and characterization of long non-coding RNAs conferring resistance to Colletotrichum gloeosporioides in walnut (Juglans regia)

2020 ◽  
Author(s):  
Shan Feng ◽  
Hongcheng Fang ◽  
Xia Liu ◽  
Yuhui Dong ◽  
Qingpeng Wang ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Plant Disease Resistance ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Colletotrichum Gloeosporioides ◽  
Juglans Regia ◽  
Bioinformatic Analysis ◽  
Genome Wide ◽  
Phenylpropanoid Biosynthesis ◽  
Non Coding Rnas

Abstract Background: Walnut anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. is an important walnut production problem in China. Although the long non-coding RNAs (lncRNAs) are important for plant disease resistance , the molecular mechanisms underlying resistance to C. gloeosporioides in walnut remain poorly understood.Results: The anthracnose-resistant F26 fruits from the B26 clone and the anthracnose-susceptible F423 fruits from the 4-23 clone of walnut were used as the test materials. Specifically, we performed a comparative transcriptome analysis of F26 and F423 fruit bracts to identify differentially expressed LncRNAs (DELs) at five time-points (tissues at 0 hpi, pathological tissues at 24 hpi, 48 hpi, 72 hpi, and distal uninoculated tissues at 120 hpi). Compared with F423, a total of 14525 DELs were identified, including 10645 upregulated lncRNAs and 3846 downregulated lncRNAs in F26. The number of upregulated lncRNAs in F26 compared to in F423 was significantly higher at the early stages of C. gloeosporioides infection. A total of 5 modules related to disease resistance were screened by WGCNA and the target genes of lncRNAs were obtained. Bioinformatic analysis showed that the target genes of upregulated lncRNAs were enriched in immune-related processes during the infection of C. gloeosporioides, such as activation of innate immune response, defense response to bacterium, incompatible interaction and immune system process, and enriched in plant hormone signal transduction, phenylpropanoid biosynthesis and other pathways. And 124 known target genes for 96 hub lncRNAs were predicted, including 10 known resistance genes. The expression of 5 lncRNAs and 5 target genes was confirmed by qPCR, which was consistent with the RNA-seq data.Conclusions: The results of this study provide the basis for future functional characterizations of lncRNAs regarding the C. gloeosporioides resistance of walnut fruit bracts.

scholarly journals Improvement of Disease Resistance in Livestock: Application of Immunogenomics and CRISPR/Cas9 Technology

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2236
Author(s):  
Md. Aminul Islam ◽  
Sharmin Aqter Rony ◽  
Mohammad Bozlur Rahman ◽  
Mehmet Ulas Cinar ◽  
Julio Villena ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Genome Editing ◽  
Public Perception ◽  
Genetic Resistance ◽  
Potential Candidate ◽  
Technological Advances ◽  
Disease Occurrence ◽  
Host Genetic ◽  
Safe Food ◽  
Genomic Locations

Disease occurrence adversely affects livestock production and animal welfare, and have an impact on both human health and public perception of food–animals production. Combined efforts from farmers, animal scientists, and veterinarians have been continuing to explore the effective disease control approaches for the production of safe animal-originated food. Implementing the immunogenomics, along with genome editing technology, has been considering as the key approach for safe food–animal production through the improvement of the host genetic resistance. Next-generation sequencing, as a cutting-edge technique, enables the production of high throughput transcriptomic and genomic profiles resulted from host-pathogen interactions. Immunogenomics combine the transcriptomic and genomic data that links to host resistance to disease, and predict the potential candidate genes and their genomic locations. Genome editing, which involves insertion, deletion, or modification of one or more genes in the DNA sequence, is advancing rapidly and may be poised to become a commercial reality faster than it has thought. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) [CRISPR/Cas9] system has recently emerged as a powerful tool for genome editing in agricultural food production including livestock disease management. CRISPR/Cas9 mediated insertion of NRAMP1 gene for producing tuberculosis resistant cattle, and deletion of CD163 gene for producing porcine reproductive and respiratory syndrome (PRRS) resistant pigs are two groundbreaking applications of genome editing in livestock. In this review, we have highlighted the technological advances of livestock immunogenomics and the principles and scopes of application of CRISPR/Cas9-mediated targeted genome editing in animal breeding for disease resistance.

scholarly journals CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.

2018 ◽  
Vol 19 (9) ◽  
pp. 2716 ◽  
Author(s):  
Qinfu Sun ◽  
Li Lin ◽  
Dongxiao Liu ◽  
Dewei Wu ◽  
Yujie Fang ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Transgenic Plants ◽  
Genome Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Pathogenic Fungus ◽  
Crop Improvement ◽  
Brassica Napus L ◽  
Significant Difference ◽  
Genomic Editing

Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T0) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T1 generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T1 plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.

scholarly journals Genome-wide identification and characterization of long non-coding RNAs conferring resistance to Colletotrichum gloeosporioides in walnut (Juglans regia)

2020 ◽  
Author(s):  
Shan Feng ◽  
Hongcheng Fang ◽  
Xia Liu ◽  
Yuhui Dong ◽  
Qingpeng Wang ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Plant Disease Resistance ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Colletotrichum Gloeosporioides ◽  
Juglans Regia ◽  
Bioinformatic Analysis ◽  
Genome Wide ◽  
Phenylpropanoid Biosynthesis ◽  
Non Coding Rnas

Abstract Background: Walnut anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. is an important walnut production problem in China. Although the long non-coding RNAs (lncRNAs) are important for plant disease resistance , the molecular mechanisms underlying resistance to C. gloeosporioides in walnut remain poorly understood.Results: The anthracnose-resistant F26 fruits from the B26 clone and the anthracnose-susceptible F423 fruits from the 4-23 clone of walnut were used as the test materials. Specifically, we performed a comparative transcriptome analysis of F26 and F423 fruit bracts to identify differentially expressed LncRNAs (DELs) at five time-points (tissues at 0 hpi, pathological tissues at 24 hpi, 48 hpi, 72 hpi, and distal uninoculated tissues at 120 hpi). Compared with F423, a total of 14525 DELs were identified, including 10645 upregulated lncRNAs and 3846 downregulated lncRNAs in F26. The number of upregulated lncRNAs in F26 compared to in F423 was significantly higher at the early stages of C. gloeosporioides infection. A total of 5 modules related to disease resistance were screened by WGCNA and the target genes of lncRNAs were obtained. Bioinformatic analysis showed that the target genes of upregulated lncRNAs were enriched in immune-related processes during the infection of C. gloeosporioides, such as activation of innate immune response, defense response to bacterium, incompatible interaction and immune system process, and enriched in plant hormone signal transduction, phenylpropanoid biosynthesis and other pathways. And 124 known target genes for 96 hub lncRNAs were predicted, including 10 known resistance genes. The expression of 5 lncRNAs and 5 target genes was confirmed by qPCR, which was consistent with the RNA-seq data.Conclusions: The results of this study provide the basis for future functional characterizations of lncRNAs regarding the C. gloeosporioides resistance of walnut fruit bracts.

scholarly journals Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

2016 ◽  
Vol 18 (4) ◽  
pp. 449-452 ◽  
Author(s):  
Carola E. Dehler ◽  
Pierre Boudinot ◽  
Samuel A. M. Martin ◽  
Bertrand Collet
Keyword(s):  
Cell Line ◽  
Genome Editing ◽  
Fish Cell Line ◽  
Fish Cell

scholarly journals Highly Efficient Generation of Canker-Resistant Sweet Orange Enabled by an Improved CRISPR/Cas9 System

2022 ◽  
Vol 12 ◽  
Author(s):  
Xiaoen Huang ◽  
Yuanchun Wang ◽  
Nian Wang
Keyword(s):  
Genome Editing ◽  
Citrus Sinensis ◽  
Gene Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Sweet Orange ◽  
Susceptibility Gene ◽  
Transformation Rate ◽  
Biallelic Mutation ◽  
Important Species

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes, and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously [dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, citrus U6 (CsU6) to drive sgRNA expression], we further improved our CRISPR/Cas9 system by choosing superior promoters [Cestrum yellow leaf curling virus (CmYLCV) or Citrus sinensis ubiquitin (CsUbi) promoter] to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker-resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease-resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

scholarly journals Genome-wide identification and characterization of long non-coding RNAs conferring resistance to Colletotrichum gloeosporioides in walnut (Juglans regia)

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shan Feng ◽  
Hongcheng Fang ◽  
Xia Liu ◽  
Yuhui Dong ◽  
Qingpeng Wang ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Plant Disease Resistance ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Colletotrichum Gloeosporioides ◽  
Juglans Regia ◽  
Bioinformatic Analysis ◽  
Genome Wide ◽  
Phenylpropanoid Biosynthesis ◽  
Non Coding Rnas

Abstract Background Walnut anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. is an important walnut production problem in China. Although the long non-coding RNAs (lncRNAs) are important for plant disease resistance, the molecular mechanisms underlying resistance to C. gloeosporioides in walnut remain poorly understood. Results The anthracnose-resistant F26 fruits from the B26 clone and the anthracnose-susceptible F423 fruits from the 4–23 clone of walnut were used as the test materials. Specifically, we performed a comparative transcriptome analysis of F26 and F423 fruit bracts to identify differentially expressed LncRNAs (DELs) at five time-points (tissues at 0 hpi, pathological tissues at 24 hpi, 48 hpi, 72 hpi, and distal uninoculated tissues at 120 hpi). Compared with F423, a total of 14,525 DELs were identified, including 10,645 upregulated lncRNAs and 3846 downregulated lncRNAs in F26. The number of upregulated lncRNAs in F26 compared to in F423 was significantly higher at the early stages of C. gloeosporioides infection. A total of 5 modules related to disease resistance were screened by WGCNA and the target genes of lncRNAs were obtained. Bioinformatic analysis showed that the target genes of upregulated lncRNAs were enriched in immune-related processes during the infection of C. gloeosporioides, such as activation of innate immune response, defense response to bacterium, incompatible interaction and immune system process, and enriched in plant hormone signal transduction, phenylpropanoid biosynthesis and other pathways. And 124 known target genes for 96 hub lncRNAs were predicted, including 10 known resistance genes. The expression of 5 lncRNAs and 5 target genes was confirmed by qPCR, which was consistent with the RNA-seq data. Conclusions The results of this study provide the basis for future functional characterizations of lncRNAs regarding the C. gloeosporioides resistance of walnut fruit bracts.

Sign in / Sign up

Export Citation Format

Share Document