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

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.

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

10.1101/2021.10.20.465103 ◽

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.

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In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

Genome Medicine ◽

10.1186/s13073-021-00876-0 ◽

2021 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Menglong Chen ◽

Hui Shi ◽

Shixue Gou ◽

Xiaomin Wang ◽

Lei Li ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Genome Editing ◽

Large Scale ◽

Gene Editing ◽

High Efficiency ◽

Muscle Fibers ◽

Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

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

10.1101/734442 ◽

2019 ◽

Cited By ~ 2

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.

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Repurposing of Anthocyanin Biosynthesis for Plant Transformation and Genome Editing

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.607982 ◽

2020 ◽

Vol 2 ◽

Author(s):

Yubing He ◽

Min Zhu ◽

Junhua Wu ◽

Lejun Ouyang ◽

Rongchen Wang ◽

...

Keyword(s):

Genome Editing ◽

Plant Species ◽

Plant Transformation ◽

Gene Editing ◽

Target Gene ◽

Target Genes ◽

Anthocyanin Biosynthesis ◽

Labor Time ◽

Visible Marker

CRISPR/Cas9 gene editing technology has been very effective in editing genes in many plant species including rice. Here we further improve the current CRISPR/Cas9 gene editing technology in both efficiency and time needed for isolation of transgene-free and target gene-edited plants. We coupled the CRISPR/Cas9 cassette with a unit that activates anthocyanin biosynthesis, providing a visible marker for detecting the presence of transgenes. The anthocyanin-marker assisted CRISPR (AAC) technology enables us to identify transgenic events even at calli stage, to select transformants with elevated Cas9 expression, and to identify transgene-free plants in the field. We used the AAC technology to edit LAZY1 and G1 and successfully generated many transgene-free and target gene-edited plants at T1 generation. The AAC technology greatly reduced the labor, time, and costs needed for editing target genes in rice.

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

International Journal of Molecular Sciences ◽

10.3390/ijms19092716 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2716 ◽

Cited By ~ 19

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.

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Precise and Efficient Crispr/Cas9 Mediated Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v128.22.2312.2312 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2312-2312

Author(s):

Jack M Heath ◽

Aditi Chalishazar ◽

Christina S Lee ◽

William Selleck ◽

Cecilia Cotta-Ramusino ◽

...

Keyword(s):

Progenitor Cells ◽

Gene Editing ◽

Target Genes ◽

High Efficiency ◽

Hematopoietic Stem ◽

Cell Therapies ◽

Immunodeficient Mice ◽

Cd34 Cells

Abstract Transplantation of gene-modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for several hematologic diseases. However, a number of blood disorders may not be amenable to gene augmentation-based therapeutics. Targeted genome editing in human HSPCs could provide a therapeutic approach for these otherwise untreatable diseases. Here we demonstrate that CRISPR/Cas9 ribonucleoprotein (RNP) edits target genes in human HSPCs with high efficiency and precision. Human adult and umbilical cord blood (CB) CD34+ cells from 20 donors were electroporated with S. pyogenes or S. aureus Cas9 RNP targeting HBB, AAVS1, or CXCR4. Sequence analysis demonstrated up to 80% editing in CB CD34+ cells (mean±s.d: 61%±9%) and up to 57% in adult CD34+ cells (39%±13%). Delivery of Cas9 RNP and a single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% ssODN-mediated homology directed repair (HDR) and also led to a 20% increase in total gene editing (HDR+NHEJ)(RNP: 48%±15%; RNP+ssODN: 69%±8%). Both Cas9 RNP gene-edited CD34+ cells and donor-matched untreated control CD34+ cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ~85% human CD45+ cell peripheral blood reconstitution 4 months after primary transplantation. Human T and B lymphoid, erythroid, and myeloid cells were detected in the spleen, thymus, and bone marrow with 20% CD34+ cell engraftment in the marrow of mice transplanted with RNP gene-edited or control CD34+ cells. The level of targeted gene editing in human erythroid, myeloid, and CD34+ cells that were recovered and enriched from the hematopoietic organs of primary recipients (~50%) was similar to the level of gene editing detected in the pre-infusion product (~60%). In summary, these results indicate that Cas9 gene-edited human HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo, thus supporting further investigation of CRISPR/Cas9 mediated gene-edited hematopoietic stem/progenitor cell therapies. Disclosures Heath: Editas Medicine: Employment. Chalishazar:Editas Medicine: Employment. Lee:Editas Medicine: Employment. Selleck:Editas Medicine: Employment. Cotta-Ramusino:Editas Medicine: Employment. Bumcrot:Editas Medicine: Employment. Gori:Editas Medicine: Employment.

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Various Aspects of a Gene Editing System—CRISPR–Cas9

International Journal of Molecular Sciences ◽

10.3390/ijms21249604 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9604

Author(s):

Edyta Janik ◽

Marcin Niemcewicz ◽

Michal Ceremuga ◽

Lukasz Krzowski ◽

Joanna Saluk-Bijak ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Genome Engineering ◽

High Efficiency ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Guide Rna ◽

Adaptive Immune ◽

Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

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Development of a gRNA-tRNA Array of CRISPR/Cas9 in Combination with Grafting Technique to Improve Gene Editing Efficiency of Sweet Orange

10.21203/rs.3.rs-765771/v1 ◽

2021 ◽

Author(s):

Xiaomei Tang ◽

Shulin Chen ◽

Huiwen Yu ◽

Xiongjie Zheng ◽

Fei Zhang ◽

...

Keyword(s):

Genetic Improvement ◽

Gene Editing ◽

Genome Engineering ◽

Target Genes ◽

Sweet Orange ◽

Fruit Crops ◽

Grafting Technique ◽

Efficient Gene ◽

Traditional Breeding ◽

Selection Of

Abstract Sweet orange is one of the most popular fruit crops worldwide. Traditional breeding approaches in sweet orange is impracticable due to the apomixis and long juvenility, making it difficult to obtain hybrids and selection of ideal genotypes. The development of targeted genome engineering technologies made it possible for the precise modification of target genes. Recently, a more efficient gene editing tool has been emerged based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system (Bhaya et al. 2011). The development of CRISPR/Cas9 technology is promising to accelerate the process of genetic improvement in perennial crops.

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CRISPR/Cas9-mediated gene-editing technology in fruit quality improvement

Food Quality and Safety ◽

10.1093/fqsafe/fyaa028 ◽

2020 ◽

Vol 4 (4) ◽

pp. 159-166

Author(s):

Xin Xu ◽

Yujin Yuan ◽

Bihong Feng ◽

Wei Deng

Keyword(s):

Quality Improvement ◽

Fruit Quality ◽

Gene Editing ◽

Target Genes ◽

High Efficiency ◽

Low Cost ◽

Fruit Size ◽

Targeted Mutagenesis ◽

Balanced Diet ◽

Low Efficiency

Abstract Fruits are an essential part of a healthy, balanced diet and it is particularly important for fibre, essential vitamins, and trace elements. Improvement in the quality of fruit and elongation of shelf life are crucial goals for researchers. However, traditional techniques have some drawbacks, such as long period, low efficiency, and difficulty in the modification of target genes, which limit the progress of the study. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique was developed and has become the most popular gene-editing technology with high efficiency, simplicity, and low cost. CRISPR/Cas9 technique is widely accepted to analyse gene function and complete genetic modification. This review introduces the latest progress of CRISPR/Cas9 technology in fruit quality improvement. For example, CRISPR/Cas9-mediated targeted mutagenesis of RIPENING INHIBITOR gene (RIN), Lycopene desaturase (PDS), Pectate lyases (PL), SlMYB12, and CLAVATA3 (CLV3) can affect fruit ripening, fruit bioactive compounds, fruit texture, fruit colouration, and fruit size. CRISPR/Cas9-mediated mutagenesis has become an efficient method to modify target genes and improve fruit quality.

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