Development of a gRNA–tRNA array of CRISPR/Cas9 in combination with grafting technique to improve gene-editing efficiency of sweet orange

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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Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporationmediated delivery and embryo culture

MethodsX ◽

10.1016/j.mex.2021.101419 ◽

2021 ◽

pp. 101419

Author(s):

Anil K Challa ◽

Denise Stanford ◽

Antonio Allen ◽

Lawrence Rasmussen ◽

Ferdinand K Amanor ◽

...

Keyword(s):

Embryo Culture ◽

Gene Editing ◽

Editing Efficiency

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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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Evaluation of site-specific homologous recombination activity of BRCA1 by direct quantitation of gene editing efficiency

Scientific Reports ◽

10.1038/s41598-018-38311-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Yuki Yoshino ◽

Shino Endo ◽

Zhenghao Chen ◽

Huicheng Qi ◽

Gou Watanabe ◽

...

Keyword(s):

Homologous Recombination ◽

Gene Editing ◽

Recombination Activity ◽

Site Specific ◽

Editing Efficiency

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Assessment of Cas12a‐mediated gene editing efficiency in plants

Plant Biotechnology Journal ◽

10.1111/pbi.13113 ◽

2019 ◽

Vol 17 (10) ◽

pp. 1971-1984 ◽

Cited By ~ 26

Author(s):

Joan Miquel Bernabé‐Orts ◽

Iván Casas‐Rodrigo ◽

Eugenio G. Minguet ◽

Viola Landolfi ◽

Victor Garcia‐Carpintero ◽

...

Keyword(s):

Gene Editing ◽

Editing Efficiency

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CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Agronomy ◽

10.3390/agronomy10071033 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1033 ◽

Cited By ~ 3

Author(s):

Jake Adolf V. Montecillo ◽

Luan Luong Chu ◽

Hanhong Bae

Keyword(s):

Genetic Engineering ◽

Genome Editing ◽

Gene Editing ◽

Fine Tuning ◽

Plant Genome ◽

Detection Methods ◽

Editing Efficiency ◽

Targeted Gene Editing ◽

Transgene Detection ◽

Selection Of

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

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A Highly Sensitive Assessment Tool for the Gene Editing Efficiency of Sickle Cell Disease Hemoglobin Beta Gene

Blood ◽

10.1182/blood.v126.23.5557.5557 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5557-5557

Author(s):

Mandula Borjigin ◽

Eric Brian Kmiec ◽

Rigumula Wu

Keyword(s):

Stem Cells ◽

Sickle Cell Disease ◽

Point Mutation ◽

Sickle Cell ◽

Gel Electrophoresis ◽

Gene Editing ◽

Cell Disease ◽

Transcription Activator ◽

Hematopoietic Stem ◽

Editing Efficiency

Abstract In sickle cell disease, a single point mutation in hemoglobin β gene (HBB) results in the substitution of valine for glutamic acid at position 6 of the β globin protein sequence, causing the deformation of red blood cells into a sickle (or crescent) shape. With the development of powerful gene editing tools, scientists are initiating the correction of the point mutation of HBB gene in CD34+ hematopoietic stem cells and induced pluripotent stem cells. Although the results are very exciting, the evaluation method of the gene editing is primitive. Currently, the modification at the mutation site is identified and quantified using Restriction Fragment Length Polymorphism (RFLP), which involves PCR amplification, restriction enzyme digestion and gel electrophoresis. The accuracy of the gene editing efficiency depends heavily on the quantification of the DNA bands in the gel images, which is inherently imprecise. We have developed a novel technique to quantify the correction efficiency of HBB gene editing using a fluorescence tagging of the edited DNA sequence. This method provides excellent sensitivity and accuracy, and saves time and labor, eliminating a process of gel electrophoresis. We demonstrate the assessment of gene editing in HBB of K562 cells, in which the wild type HBB (βA gene) is converted to mutant βs using the gene editing tools (i.e. Transcription Activator-Like Effector Nucleases (TALENs) and single-stranded oligo deoxynucleotides (ssODNs)). We present limited information here due to the sensitivity of the intellectual property, but will discuss in detail the experimental procedures and data at the American Society of Hematology meeting. Disclosures No relevant conflicts of interest to declare.

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Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

10.1101/2020.05.20.103614 ◽

2020 ◽

Author(s):

Xi Xiang ◽

Kunli Qu ◽

Xue Liang ◽

Xiaoguang Pan ◽

Jun Wang ◽

...

Keyword(s):

High Throughput ◽

Gene Editing ◽

High Efficiency ◽

Outcome Data ◽

Massively Parallel ◽

Human Embryonic Kidney Cells ◽

Editing Efficiency ◽

Base Editing ◽

Target Sites ◽

In Cells

AbstractThe CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

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Comparative Study Between the CRISPR/Cpf1 and CRISPR/Cas9 systems for Multiplex Gene Editing in Maize

10.21203/rs.3.rs-244759/v1 ◽

2021 ◽

Author(s):

Chongzhi Gong ◽

Shengchan Huang ◽

Rentao Song ◽

Weiwei Qi

Keyword(s):

Gene Editing ◽

Direct Repeat ◽

Bzip Transcription Factor ◽

Target Range ◽

Editing Efficiency ◽

Target Site Selection ◽

Different Types ◽

Selection For ◽

Alternative Choice ◽

New Mutations

Abstract Background: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully used for multiplex gene editing in crops. Although CRISPR/Cas9 system has been proved to be an efficient multiplex gene editing system in crops, it was still unclear how CRISPR/Cpf1, a natural direct repeat (DR)-based multiplex gene editing system, performed in crops. To this end, this study compared the CRISPR/Cpf1 system and CRISPR/Cas9 system for multiplex gene editing in maize. Results: The bZIP transcription factor Opaque2 (O2) was used as the target gene to evaluate the editing efficient of both systems. We found that in the T0 generation, the CRISPR/Cpf1 system showed low editing efficiency with only one mutation, while the CRISPR/Cas9 system generated many different types of on-target mutations. In the T1 generation, the CRISPR/Cpf1 system still showed lower editing efficiency than the CRISPR/Cas9 system. However, in the T2 generation, the CRISPR/Cpf1 system generated more types of new mutations. While the CRISPR/Cas9 system tended to edit within the on-target range, the CRISPR/Cpf1 system preferred to edit in between the targets. We also found that in the CRISPR/Cpf1 system, the editing efficiency positively correlated with the expression level of Cpf1. Conclusions: In conclusion, the CRISPR/Cpf1 system offers alternative choices for target-site selection for multiplex gene editing and has acceptable editing efficiency in maize. Thus, the CRISPR/Cpf1 system is a valuable alternative choice for gene editing in crops.

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Construction of a rAAV-SaCas9 system expressing eGFP and its application to improve muscle mass

Biotechnology Letters ◽

10.1007/s10529-021-03183-1 ◽

2021 ◽

Author(s):

Shaoting Weng ◽

Yitian Zhao ◽

Changhong Yu ◽

Xiaofan Wang ◽

Xuehan Xiao ◽

...

Keyword(s):

Muscle Mass ◽

Gene Editing ◽

Fluorescent Protein ◽

Specific Site ◽

Target Tissue ◽

Myostatin Gene ◽

Editing Efficiency ◽

Green Fluorescent

AbstractAn ideal rAAV gene editing system not only effectively edits genes at specific site, but also prevents the spread of the virus from occurring off-target or carcinogenic risks. This is important for gene editing research at specific site in vivo. We report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and the editing efficiency was similar to that of the classical Cas9 system in vitro and in vivo. And we inserted the sequence of the green fluorescent protein eGFP into rAAV. The number of cells infected with the rAAV and the region in which the rAAV spreads were known by the fluorescent expression of eGFP in cells. In addition, we demonstrated that myostatin gene in the thigh muscles of C57BL/10 mice was knocked out by the rAAV9-SaCas9 system to make muscle mass increased obviously. The protein eGFP into rAAV has significant implications for our indirect analysis of the editing efficiency of SaCas9 in the genome of the target tissue and reduces the harm caused by off-target editing and prevents other tissue mutations. The rAAV system has substantial potential in improving muscle mass and preventing muscle atrophy.

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