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Chaturvedi, Sarika, and Jinny Tomar. "CRISPR/CAS 9 Mediated Treatment for UTIs." International Journal for Modern Trends in Science and Technology 6, no. 5 (May 31, 2020): 82–94. http://dx.doi.org/10.46501/ijmtst060515.
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“CRISPR" is short and used for "CRISPR-Cas9. CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA and can be used in conjunction with engineered CRISPR sequences to hunt down codes and slice into them like a molecular scalpel, allowing geneticists to cut out a target gene, either to remove it or replace it with a new sequence. Therefore it is a simple and powerful tool for editing genomes to easily alter DNA sequences and amend gene function. In 1987, The CRISPR locus was first identified in Escherichia coli and discovered when a genetic structure containing 5 highly homologous repeats of 29 nucleotides separated by 32-nucleotide spacers (Ishino Y 1987).
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Gong, Yan, Siyu Tian, Yang Xuan, and Shubiao Zhang. "Lipid and polymer mediated CRISPR/Cas9 gene editing." Journal of Materials Chemistry B 8, no. 20 (2020): 4369–86. http://dx.doi.org/10.1039/d0tb00207k.
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Nomura, Toshihisa, Mizuki Yoshikawa, Kengo Suzuki, and Keiichi Mochida. "Highly Efficient CRISPR-Associated Protein 9 Ribonucleoprotein-Based Genome Editing in Euglena gracilis." STAR Protocols 1, no. 1 (June 2020): 100023. http://dx.doi.org/10.1016/j.xpro.2020.100023.
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Aalipour, Amin, Jonathan C. Dudley, Seung-min Park, Surya Murty, Jacob J. Chabon, Evan A. Boyle, Maximilian Diehn, and Sanjiv S. Gambhir. "Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA." Clinical Chemistry 64, no. 2 (February 1, 2018): 307–16. http://dx.doi.org/10.1373/clinchem.2017.278911.
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Abstract BACKGROUND Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations. METHODS Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use. RESULTS Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in non-small cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR. CONCLUSIONS The dCas9 method provides an important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.
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Kato-Inui, Tomoko, Gou Takahashi, Szuyin Hsu, and Yuichiro Miyaoka. "Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair." Nucleic Acids Research 46, no. 9 (April 17, 2018): 4677–88. http://dx.doi.org/10.1093/nar/gky264.
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Lentsch, Eva, Lifei Li, Susanne Pfeffer, Arif B. Ekici, Leila Taher, Christian Pilarsky, and Robert Grützmann. "CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines." International Journal of Molecular Sciences 20, no. 22 (November 14, 2019): 5706. http://dx.doi.org/10.3390/ijms20225706.
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In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.
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Lone, Bilal Ahmad, Shibendra Kumar Lal Karna, Faiz Ahmad, Nerina Shahi, and Yuba Raj Pokharel. "CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering." Genetics Research International 2018 (September 18, 2018): 1–17. http://dx.doi.org/10.1155/2018/3797214.
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Microbes use diverse defence strategies that allow them to withstand exposure to a variety of genome invaders such as bacteriophages and plasmids. One such defence strategy is the use of RNA guided endonuclease called CRISPR-associated (Cas) 9 protein. The Cas9 protein, derived from type II CRISPR/Cas system, has been adapted as a versatile tool for genome targeting and engineering due to its simplicity and high efficiency over the earlier tools such as ZFNs and TALENs. With recent advancements, CRISPR/Cas9 technology has emerged as a revolutionary tool for modulating the genome in living cells and inspires innovative translational applications in different fields. In this paper we review the developments and its potential uses in the CRISPR/Cas9 technology as well as recent advancements in genome engineering using CRISPR/Cas9.
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Xia, An-Liang, Qi-Feng He, Jin-Cheng Wang, Jing Zhu, Ye-Qin Sha, Beicheng Sun, and Xiao-Jie Lu. "Applications and advances of CRISPR-Cas9 in cancer immunotherapy." Journal of Medical Genetics 56, no. 1 (July 3, 2018): 4–9. http://dx.doi.org/10.1136/jmedgenet-2018-105422.
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Immunotherapy has emerged as one of the most promising therapeutic strategies in cancer. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9) system, as an RNA-guided genome editing technology, is triggering a revolutionary change in cancer immunotherapy. With its versatility and ease of use, CRISPR-Cas9 can be implemented to fuel the production of therapeutic immune cells, such as construction of chimeric antigen receptor T (CAR-T) cells and programmed cell death protein 1 knockout. Therefore, CRISPR-Cas9 technology holds great promise in cancer immunotherapy. In this review, we will introduce the origin, development and mechanism of CRISPR-Cas9. Also, we will focus on its various applications in cancer immunotherapy, especially CAR-T cell-based immunotherapy, and discuss the potential challenges it faces.
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Cao, Hieu X., Wenqin Wang, Hien T. T. Le, and Giang T. H. Vu. "The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding." International Journal of Genomics 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/5078796.
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Genome editing with engineered nucleases enabling site-directed sequence modifications bears a great potential for advanced plant breeding and crop protection. Remarkably, the RNA-guided endonuclease technology (RGEN) based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) is an extremely powerful and easy tool that revolutionizes both basic research and plant breeding. Here, we review the major technical advances and recent applications of the CRISPR-Cas9 system for manipulation of model and crop plant genomes. We also discuss the future prospects of this technology in molecular plant breeding.
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Horodecka, Katarzyna, and Markus Düchler. "CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles." International Journal of Molecular Sciences 22, no. 11 (June 4, 2021): 6072. http://dx.doi.org/10.3390/ijms22116072.
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The establishment of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology for eukaryotic gene editing opened up new avenues not only for the analysis of gene function but also for therapeutic interventions. While the original methodology allowed for targeted gene disruption, recent technological advancements yielded a rich assortment of tools to modify genes and gene expression in various ways. Currently, clinical applications of this technology fell short of expectations mainly due to problems with the efficient and safe delivery of CRISPR/Cas9 components to living organisms. The targeted in vivo delivery of therapeutic nucleic acids and proteins remain technically challenging and further limitations emerge, for instance, by unwanted off-target effects, immune reactions, toxicity, or rapid degradation of the transfer vehicles. One approach that might overcome many of these limitations employs extracellular vesicles as intercellular delivery devices. In this review, we first introduce the CRISPR/Cas9 system and its latest advancements, outline major applications, and summarize the current state of the art technology using exosomes or microvesicles for transporting CRISPR/Cas9 constituents into eukaryotic cells.
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Chu, Van Trung, Robin Graf, Tristan Wirtz, Timm Weber, Jeremy Favret, Xun Li, Kerstin Petsch, et al. "Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line." Proceedings of the National Academy of Sciences 113, no. 44 (October 11, 2016): 12514–19. http://dx.doi.org/10.1073/pnas.1613884113.
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Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.
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Siew, Wei Sheng, Yin Quan Tang, Chee Kei Kong, Bey-Hing Goh, Serena Zacchigna, Kamal Dua, Dinesh Kumar Chellappan, et al. "Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications." International Journal of Molecular Sciences 22, no. 16 (August 5, 2021): 8422. http://dx.doi.org/10.3390/ijms22168422.
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Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.
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Ahmed, Temoor, Muhammad Noman, Muhammad Shahid, Sher Muhammad, Muhammad Tahir ul Qamar, Md Arshad Ali, Awais Maqsood, Rahila Hafeez, Solabomi Olaitan Ogunyemi, and Bin Li. "Potential Application of CRISPR/Cas9 System to Engineer Abiotic Stress Tolerance in Plants." Protein & Peptide Letters 28, no. 8 (September 10, 2021): 861–77. http://dx.doi.org/10.2174/0929866528666210218220138.
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Abiotic stresses in plants such as salinity, drought, heavy metal toxicity, heat, and nutrients limitations significantly reduce agricultural production worldwide. The genome editing techniques such as transcriptional activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have been used for genome manipulations in plants. However, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technique has recently emerged as a promising tool for genome editing in plants to acquire desirable traits. The CRISPR/Cas9 system has a great potential to develop crop varieties with improved tolerance against abiotic stresses. This review is centered on the biology and potential application of the CRISPR/Cas9 system to improve abiotic stress tolerance in plants. Furthermore, this review highlighted the recent advancements of CRISPR/Cas9-mediated genome editing for sustainable agriculture.
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Ribeiro, Lucas F., Liliane F. C. Ribeiro, Matheus Q. Barreto, and Richard J. Ward. "Protein Engineering Strategies to Expand CRISPR-Cas9 Applications." International Journal of Genomics 2018 (August 2, 2018): 1–12. http://dx.doi.org/10.1155/2018/1652567.
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The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
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Gong, Chongzhi, Shengchan Huang, Rentao Song, and Weiwei Qi. "Comparative Study between the CRISPR/Cpf1 (Cas12a) and CRISPR/Cas9 Systems for Multiplex Gene Editing in Maize." Agriculture 11, no. 5 (May 10, 2021): 429. http://dx.doi.org/10.3390/agriculture11050429.
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Although the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been proved to be an efficient multiplex gene editing system in maize, it was still unclear how CRISPR/Cpf1 (Cas12a) system would perform for multiplex gene editing in maize. To this end, this study compared the CRISPR/Cpf1 system and CRISPR/Cas9 system for multiplex gene editing in maize. The bZIP transcription factor Opaque2 (O2) was used as the target gene in both systems. We found that in the T0 and T1 generations, the CRISPR/Cpf1 system 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. In conclusion, the CRISPR/Cpf1 system offers alternative choices for target-site selection for multiplex gene editing and has acceptable editing efficiency in maize and is a valuable alternative choice for gene editing in crops.
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Ahmad, Shakeel, Xiangjin Wei, Zhonghua Sheng, Peisong Hu, and Shaoqing Tang. "CRISPR/Cas9 for development of disease resistance in plants: recent progress, limitations and future prospects." Briefings in Functional Genomics 19, no. 1 (January 2020): 26–39. http://dx.doi.org/10.1093/bfgp/elz041.
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Abstract Several plant pathogens severely affect crop yield and quality, thereby threatening global food security. In order to cope with this challenge, genetic improvement of plant disease resistance is required for sustainable agricultural production, for which conventional breeding is unlikely to do enough. Luckily, genome editing systems that particularly clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has revolutionized crop improvement by enabling robust and precise targeted genome modifications. It paves the way towards new methods for genetic improvement of plant disease resistance and accelerates resistance breeding. In this review, the challenges, limitations and prospects for conventional breeding and the applications of CRISPR/Cas9 system for the development of transgene-free disease-resistant crops are discussed.
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Ge, Huanhuan, and Mario Andrea Marchisio. "Aptamers, Riboswitches, and Ribozymes in S. cerevisiae Synthetic Biology." Life 11, no. 3 (March 17, 2021): 248. http://dx.doi.org/10.3390/life11030248.
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Among noncoding RNA sequences, riboswitches and ribozymes have attracted the attention of the synthetic biology community as circuit components for translation regulation. When fused to aptamer sequences, ribozymes and riboswitches are enabled to interact with chemicals. Therefore, protein synthesis can be controlled at the mRNA level without the need for transcription factors. Potentially, the use of chemical-responsive ribozymes/riboswitches would drastically simplify the design of genetic circuits. In this review, we describe synthetic RNA structures that have been used so far in the yeast Saccharomyces cerevisiae. We present their interaction mode with different chemicals (e.g., theophylline and antibiotics) or proteins (such as the RNase III) and their recent employment into clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas) systems. Particular attention is paid, throughout the whole paper, to their usage and performance into synthetic gene circuits.
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Li, Chenggong, Heng Mei, and Yu Hu. "Applications and explorations of CRISPR/Cas9 in CAR T-cell therapy." Briefings in Functional Genomics 19, no. 3 (January 17, 2020): 175–82. http://dx.doi.org/10.1093/bfgp/elz042.
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Abstract Chimeric antigen receptor(CAR) T-cell therapy has shown remarkable effects and promising prospects in patients with refractory or relapsed malignancies, pending further progress in the next-generation CAR T cells with more optimized structure, enhanced efficacy and reduced toxicities. The clustered regulatory interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology holds immense promise for advancing the field owing to its flexibility, simplicity, high efficiency and multiplexing in precise genome editing. Herein, we review the applications and explorations of CRISPR/Cas9 technology in constructing allogenic universal CAR T cells, disrupting inhibitory signaling to enhance potency and exploration of safer and more controllable novel CAR T cells.
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Sun, Jinyu, Jianchu Wang, Donghui Zheng, and Xiaorong Hu. "Advances in therapeutic application of CRISPR-Cas9." Briefings in Functional Genomics 19, no. 3 (November 26, 2019): 164–74. http://dx.doi.org/10.1093/bfgp/elz031.
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Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.
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Bhatta, Bed Prakash, and Subas Malla. "Improving Horticultural Crops via CRISPR/Cas9: Current Successes and Prospects." Plants 9, no. 10 (October 14, 2020): 1360. http://dx.doi.org/10.3390/plants9101360.
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Horticultural crops include a diverse array of crops comprising fruits, vegetables, nuts, flowers, aromatic and medicinal plants. They provide nutritional, medicinal, and aesthetic benefits to mankind. However, these crops undergo many biotic (e.g., diseases, pests) and abiotic stresses (e.g., drought, salinity). Conventional breeding strategies to improve traits in crops involve the use of a series of backcrossing and selection for introgression of a beneficial trait into elite germplasm, which is time and resource consuming. Recent new plant breeding tools such as clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein-9 (Cas9) technique have the potential to be rapid, cost-effective, and precise tools for crop improvement. In this review article, we explore the CRISPR/Cas9 technology, its history, classification, general applications, specific uses in horticultural crops, challenges, existing resources, associated regulatory aspects, and the way forward.
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Hu, S., M. Yang, and I. Polejaeva. "360 DOUBLE KNOCKOUT OF GOAT MYOSTATIN AND PRION PROTEIN GENE USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT (CRISPR)/Cas9 SYSTEMS." Reproduction, Fertility and Development 27, no. 1 (2015): 268. http://dx.doi.org/10.1071/rdv27n1ab360.
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Myostatin (MSTN) acts as a negative regulator of skeletal muscle development and growth. Inhibition of MSTN expression may be applied to enhance animal growth performance in livestock production. Prion protein (PrPc) is associated directly with the pathogenesis of the transmissible spongiform encephalopathies occurring in variety of species including human, cattle, sheep, goats and deer. Prion protein-deficient livestock may be a useful model for prion research and producing animal conferring potential disease resistance. The goal of this study was to generate MSTN/PrPc double knockout goat by using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. We generated 2 CRISPR/Cas9 plasmids targeting MSTN and PrPc genes, respectively. The CRISPR/Cas9 plasmids targeting each gene were respectively transfected into goat fibroblasts, and the efficiency of gene modification was determined at Day 3 using restriction fragment length polymorphism (RFLP) assay. The RFLP assay showed that CRISPR/Cas9 plasmids targeting MSTN and PrPc induced precise gene mutations with efficiency of 59 and 70%, respectively. Single cell-derived colonies were further isolated by limiting dilution after co-transfection of 2 CRISPR/Cas9 plasmids targeting MSTN and PrPc. The RFLP assay and DNA sequence analysis indicated that 9 out of 45 colonies (20%) carried simultaneous disruption of both target genes. Moreover, 5 of 9 mutant colonies (55%) had mutations in all 4 alleles of 2 genes. These double-gene knockout fibroblast cells will be used as nuclear donors for developing double knockout goat deficient in MSTN and PrPc. The CRISPR/Cas9 system represents a highly effective and facile platform for multiplex editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.This work was supported by the Utah Science Technology and Research Initiative and Utah Agricultural Experiment Station project #31294.
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Seok, Heeyoung, Rui Deng, Douglas B. Cowan, and Da-Zhi Wang. "Application of CRISPR-Cas9 gene editing for congenital heart disease." Clinical and Experimental Pediatrics 64, no. 6 (June 15, 2021): 269–79. http://dx.doi.org/10.3345/cep.2020.02096.
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Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is an ancient prokaryotic defense system that precisely cuts foreign genomic DNA under the control of a small number of guide RNAs. The CRISPR-Cas9 system facilitates efficient double-stranded DNA cleavage that has been recently adopted for genome editing to create or correct inherited genetic mutations causing disease. Congenital heart disease (CHD) is generally caused by genetic mutations such as base substitutions, deletions, and insertions, which result in diverse developmental defects and remains a leading cause of birth defects. Pediatric CHD patients exhibit a spectrum of cardiac abnormalities such as septal defects, valvular defects, and abnormal chamber development. CHD onset occurs during the prenatal period and often results in early lethality during childhood. Because CRISPR-Cas9-based genome editing technology has gained considerable attention for its potential to prevent and treat diseases, we will review the CRISPR-Cas9 system as a genome editing tool and focus on its therapeutic application for CHD.
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Liu, Wenlou, Chunsheng Yang, Yanqun Liu, and Guan Jiang. "CRISPR/Cas9 System and its Research Progress in Gene Therapy." Anti-Cancer Agents in Medicinal Chemistry 19, no. 16 (January 23, 2020): 1912–19. http://dx.doi.org/10.2174/1871520619666191014103711.
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Genome editing refers to changing the genome sequence of an organism by knockout, insertion, and site mutation, resulting in changes in the genetic information of the organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein-9 nuclease (Cas9) system is a genome editing technique developed by the acquired immune system in the microbes, such as bacteria and archaebacteria, which targets and edits genome sequences according to the principle of complementary base pairing. This technique can be used to edit endogenous genomic DNA sequences in organisms accurately and has been widely used in fields, such as biotechnology, cancer gene therapy, and dermatology. In this review, we summarize the history, structure, mechanism, and application of CRISPR/Cas9 in gene therapy and dermatological diseases.
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Beneke, Tom, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter, and Eva Gluenz. "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids." Royal Society Open Science 4, no. 5 (May 2017): 170095. http://dx.doi.org/10.1098/rsos.170095.
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Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.
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Ratan, Zubair Ahmed, Young-Jin Son, Mohammad Faisal Haidere, Bhuiyan Mohammad Mahtab Uddin, Md Abdullah Yusuf, Sojib Bin Zaman, Jong-Hoon Kim, Laila Anjuman Banu, and Jae Youl Cho. "CRISPR-Cas9: a promising genetic engineering approach in cancer research." Therapeutic Advances in Medical Oncology 10 (January 1, 2018): 175883401875508. http://dx.doi.org/10.1177/1758834018755089.
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Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions in in vitro and in vivo environments. The accuracy and versatility of CRISPR-Cas have been capitalized upon in biological and medical research and bring new hope to cancer research. Cancer involves complex alterations and multiple mutations, translocations and chromosomal losses and gains. The ability to identify and correct such mutations is an important goal in cancer treatment. In the context of this complex cancer genomic landscape, there is a need for a simple and flexible genetic tool that can easily identify functional cancer driver genes within a comparatively short time. The CRISPR-Cas system shows promising potential for modeling, repairing and correcting genetic events in different types of cancer. This article reviews the concept of CRISPR-Cas, its application and related advantages in oncology.
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Korablev, Alexey, Varvara Lukyanchikova, Irina Serova, and Nariman Battulin. "On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes." International Journal of Molecular Sciences 21, no. 10 (May 20, 2020): 3604. http://dx.doi.org/10.3390/ijms21103604.
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Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.
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Zhu, Shouhong, Xiuli Yu, Yanjun Li, Yuqiang Sun, Qianhao Zhu, and Jie Sun. "Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System." International Journal of Molecular Sciences 19, no. 10 (October 1, 2018): 3000. http://dx.doi.org/10.3390/ijms19103000.
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The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4–100% and 92.9–100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton.
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Xiao, Bo, Shigang Yin, Yang Hu, Maoxin Sun, Jieqiong Wei, Zhenghui Huang, Yuhao Wen, et al. "Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum." Proceedings of the National Academy of Sciences 116, no. 1 (December 24, 2018): 255–60. http://dx.doi.org/10.1073/pnas.1813542116.
Full textAbstract:
Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum. Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum. By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum. This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.
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Amoasii, Leonela, Chengzu Long, Hui Li, Alex A. Mireault, John M. Shelton, Efrain Sanchez-Ortiz, John R. McAnally, et al. "Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy." Science Translational Medicine 9, no. 418 (November 29, 2017): eaan8081. http://dx.doi.org/10.1126/scitranslmed.aan8081.
Full textAbstract:
Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.
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Ma, Hanhui, Ardalan Naseri, Pablo Reyes-Gutierrez, Scot A. Wolfe, Shaojie Zhang, and Thoru Pederson. "Multicolor CRISPR labeling of chromosomal loci in human cells." Proceedings of the National Academy of Sciences 112, no. 10 (February 23, 2015): 3002–7. http://dx.doi.org/10.1073/pnas.1420024112.
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The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome’s physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.
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Wang, Xing, Yu Wu, Zijin Liu, Tong Liu, Lamei Zheng, and Genfa Zhang. "The pip1s Quintuple Mutants Demonstrate the Essential Roles of PIP1s in the Plant Growth and Development of Arabidopsis." International Journal of Molecular Sciences 22, no. 4 (February 7, 2021): 1669. http://dx.doi.org/10.3390/ijms22041669.
Full textAbstract:
Plasma membrane intrinsic proteins (PIPs) transport water, CO2 and small neutral solutes across the plasma membranes. In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 system (CRISPR/Cas9) to mutate PIP1;4 and PIP1;5 in a pip1;1,2,3 triple mutant to generate a pip1;1,2,3,4,5 (pip1s−) quintuple mutant. Compared to the wild-type (WT) plant, the pip1s− mutants had smaller sized rosette leaves and flowers, less rosette leaf number, more undeveloped siliques, shorter silique and less seeds. The pollen germination rate of the pip1s− mutant was significantly lower than that of the WT and the outer wall of the pip1s− mutant’s pollen was deformed. The transcriptomic analysis showed significant alterations in the expression of many key genes and transcription factors (TFs) in the pip1s− mutant which involved in the development of leaf, flower and pollen, suggesting that the mutant of PIP1s not only directly affects hydraulics and carbon fixation, but also regulates the expression of related genes to affect plant growth and development.
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Ding, Fei, Meiling Wang, and Shuoxin Zhang. "Sedoheptulose-1,7-Bisphosphatase is Involved in Methyl Jasmonate- and Dark-Induced Leaf Senescence in Tomato Plants." International Journal of Molecular Sciences 19, no. 11 (November 20, 2018): 3673. http://dx.doi.org/10.3390/ijms19113673.
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Leaf senescence represents the final stage of leaf development and is regulated by diverse internal and environmental factors. Jasmonates (JAs) have been demonstrated to induce leaf senescence in several species; however, the mechanisms of JA-induced leaf senescence remain largely unknown in tomato plants (Solanum lycopersicum). In the present study, we tested the hypothesis that sedoheptulose-1,7-bisphosphatase (SBPase), an enzyme functioning in the photosynthetic carbon fixation in the Calvin–Benson cycle, was involved in methyl jasmonate (MeJA)- and dark-induced leaf senescence in tomato plants. We found that MeJA and dark induced senescence in detached tomato leaves and concomitantly downregulated the expression of SlSBPASE and reduced SBPase activity. Furthermore, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9)-mediated mutagenesis of SlSBPASE led to senescence-associated characteristics in slsbpase mutant plants, including loss of chlorophyll, repressed photosynthesis, increased membrane ion leakage, and enhanced transcript abundance of senescence-associated genes. Collectively, our data suggest that repression of SBPase by MeJA and dark treatment plays a role in JA- and dark-induced leaf senescence.
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Hiranniramol, Kasidet, Yuhao Chen, Weijun Liu, and Xiaowei Wang. "Generalizable sgRNA design for improved CRISPR/Cas9 editing efficiency." Bioinformatics 36, no. 9 (January 23, 2020): 2684–89. http://dx.doi.org/10.1093/bioinformatics/btaa041.
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Abstract Motivation The development of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has provided a simple yet powerful system for targeted genome editing. In recent years, this system has been widely used for various gene editing applications. The CRISPR editing efficacy is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage. While there have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA potency and generalizability across various experimental conditions. Results We employed a unique plasmid library expressed in human cells to quantify the potency of thousands of CRISPR/Cas9 sgRNAs. Differential sequence and structural features among the most and least potent sgRNAs were then used to train a machine learning algorithm for assay design. Comparative analysis indicates that our new algorithm outperforms existing CRISPR/Cas9 sgRNA design tools. Availability and implementation The new sgRNA design tool is freely accessible as a web application, http://crispr.wustl.edu. Supplementary information Supplementary data are available at Bioinformatics online.
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Wardhani, Bantari W. K., Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy, and Mitsuyasu Kato. "TMEPAI genome editing in triple negative breast cancer cells." Medical Journal of Indonesia 26, no. 1 (May 16, 2017): 14–8. http://dx.doi.org/10.13181/mji.v26i1.1871.
Full textAbstract:
Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.
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Nakamura, Watanabe, Ando, Ishihara, and Sato. "Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice." International Journal of Molecular Sciences 20, no. 23 (November 25, 2019): 5926. http://dx.doi.org/10.3390/ijms20235926.
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Transplacental gene delivery (TPGD) is a technique for delivering nucleic acids to fetal tissues via tail-vein injections in pregnant mice. After transplacental transport, administered nucleic acids enter fetal circulation and are distributed among fetal tissues. TPGD was established in 1995 by Tsukamoto et al., and its mechanisms, and potential applications have been further characterized since. Recently, discoveries of sequence specific nucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9), have revolutionized genome editing. In 2019, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells, which are comparatively amenable to transfection with exogenous DNA. In the future, this unique technique will allow manipulation of fetal cell functions in basic studies of fetal gene therapy. In this review, we describe developments of TPGD and discuss their applications to the manipulation of fetal cells.
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Ma, Hanhui, Li-Chun Tu, Ardalan Naseri, Maximiliaan Huisman, Shaojie Zhang, David Grunwald, and Thoru Pederson. "CRISPR-Cas9 nuclear dynamics and target recognition in living cells." Journal of Cell Biology 214, no. 5 (August 22, 2016): 529–37. http://dx.doi.org/10.1083/jcb.201604115.
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The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)–associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9–guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time.
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Wang, Yuanzheng, Yansha Li, Tabata Rosas-Diaz, Carlos Caceres-Moreno, Rosa Lozano-Duran, and Alberto P. Macho. "The IMMUNE-ASSOCIATED NUCLEOTIDE-BINDING 9 Protein Is a Regulator of Basal Immunity in Arabidopsis thaliana." Molecular Plant-Microbe Interactions® 32, no. 1 (January 2019): 65–75. http://dx.doi.org/10.1094/mpmi-03-18-0062-r.
Full textAbstract:
A robust regulation of plant immune responses requires a multitude of positive and negative regulators that act in concert. The immune-associated nucleotide-binding (IAN) gene family members are associated with immunity in different organisms, although no characterization of their function has been carried out to date in plants. In this work, we analyzed the expression patterns of IAN genes and found that IAN9 is repressed upon pathogen infection or treatment with immune elicitors. IAN9 encodes a plasma membrane-localized protein that genetically behaves as a negative regulator of immunity. A novel ian9 mutant generated by CRISPR/Cas9 shows increased resistance to Pseudomonas syringae, while transgenic plants overexpressing IAN9 show a slight increase in susceptibility. In vivo immunoprecipitation of IAN9-green fluorescent protein followed by mass spectrometry analysis revealed that IAN9 associates with a previously uncharacterized C3HC4-type RING-finger domain-containing protein that we named IAN9-associated protein 1 (IAP1), which also acts as a negative regulator of basal immunity. Interestingly, neither ian9 or iap1 mutant plants show any obvious developmental phenotype, suggesting that they display enhanced inducible immunity rather than constitutive immune responses. Because both IAN9 and IAP1 have orthologs in important crop species, they could be suitable targets to generate plants more resistant to diseases caused by bacterial pathogens without yield penalty.
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Jing, Weixia, Xuewu Zhang, Wenyan Sun, Xiujuan Hou, Zhongqiang Yao, and Yuelan Zhu. "CRISPR/CAS9-Mediated Genome Editing of miRNA-155 Inhibits Proinflammatory Cytokine Production by RAW264.7 Cells." BioMed Research International 2015 (2015): 1–7. http://dx.doi.org/10.1155/2015/326042.
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MicroRNA 155 (miR-155) is a key proinflammatory regulator in clinical and experimental rheumatoid arthritis (RA). Here we generated a miR-155 genome knockout (GKO) RAW264.7 macrophage cell line using the clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CAS9) technology. While upregulating the Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1), the miR-155 GKO line is severely impaired in producing proinflammatory cytokines but slightly increased in osteoclastogenesis upon treatment with receptor activator of nuclear factor-κB ligand (RANKL). Taken together, our results suggest that genome editing of miR-155 holds the potential as a therapeutic strategy in RA.
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Yang Zhou, Jordi, Keittisak Suwan, and Amin Hajitou. "Initial Steps for the Development of a Phage-Mediated Gene Replacement Therapy Using CRISPR-Cas9 Technology." Journal of Clinical Medicine 9, no. 5 (May 16, 2020): 1498. http://dx.doi.org/10.3390/jcm9051498.
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p53 gene (TP53) replacement therapy has shown promising results in cancer gene therapy. However, it has been hampered, mostly because of the gene delivery vector of choice. CRISPR-Cas9 technology (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) can knock out the mutated TP53 (mutTP53), but due to its large size, many viral vectors are not suitable or require implemented strategies that lower the therapeutic efficiency. Here, we introduced a bacteriophage or phage-based vector with the ability to target cancer cells and aimed to investigate the feasibility of using this vector to deliver CRISPR-Cas9 transgene in human lung adenocarcinoma cells. First, we produced a tumour-targeted bacteriophage carrying a CRISPR-Cas9 transgene cassette. Next, we investigated any negative impact on vector titers via quantitative polymerase chain reaction (qPCR) and colony-forming agar plate. Last, we combined Western blot analysis and immunofluorescence staining to prove cell transduction in vitro. We showed that the tumour-targeted bacteriophage can package a large-size vector genome, ~10 kb, containing the CRISPR-Cas9 sequence without any negative impact on the active or total number of bacteriophage particles. Then, we detected expression of the Cas9 in human lung adenocarcinoma cells in a targeted and efficient manner. Finally, we proved loss of p53 protein expression when a p53 gRNA was incorporated into the CRISPR-Cas9 phage DNA construct. These proof-of-concept findings support the use of engineered bacteriophage for TP53 replacement therapy in lung cancer.
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Campbell, Amy E., and Daimark Bennett. "Targeting protein function: the expanding toolkit for conditional disruption." Biochemical Journal 473, no. 17 (August 30, 2016): 2573–89. http://dx.doi.org/10.1042/bcj20160240.
Full textAbstract:
A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.
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Yu, Hui-Chun, Chien-Hsueh Tung, Kuang-Yung Huang, Hsien-Bin Huang, and Ming-Chi Lu. "The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage." International Journal of Molecular Sciences 21, no. 16 (August 10, 2020): 5720. http://dx.doi.org/10.3390/ijms21165720.
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Objective: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. Methods: The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated plate. Results: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 knockout decreased the protein levels of citrullinated nuclear factor κB (NF-κB) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1β and TNF-α in the U937 cells and IL-1β and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1β, IL-6, and TNF-α were all dramatically decreased in the PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1. Conclusion: This study showed that PADI2 could promote IL-1β, IL-6, and TNF-α production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages.
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Niu, Fengjuan, Qiyan Jiang, Rui Cheng, Xianjun Sun, Zheng Hu, Lixia Wang, and Hui Zhang. "CRISPR/Cas9-Mediated Targeted Mutagenesis of Wild Soybean (Glycine soja) Hairy Roots Altered the Transcription Profile of the Mutant." Journal of Agricultural Science 12, no. 9 (August 15, 2020): 14. http://dx.doi.org/10.5539/jas.v12n9p14.
Full textAbstract:
Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system has been regularly applied for genome editing and gene function identification in wild soybean (Glycine max) cultivars. However, till date no studies have demonstrated successful mutagenesis in wild soybean (Glycine soja) which is the ancestor of Glycine max and rich in stress tolerance genes. In the current study, we report the successful creation of mutations in the loci encoding plasma membrane Na+/H+ antiporter (SOS1) and nonselective cation channels (NSCC) in wild soybean hairy roots using the CRISPR/Cas9 system. Two genes, GsSOS1 and GsNSCC, were mutagenized with frequencies of 28.5% and 39.9%, respectively. Biallelic mutations in GsSOS1 were detected in transgenic hairy roots. GsSOS1 mutants exhibited altered Na+/K+ ratios in the roots under both control and salt-treated conditions. However, no significant effects of GsNSCC mutation on Na+/K+ ratios were observed. RNA-Seq analysis revealed that both GsSOS1 and GsNSCC mutation altered the transcription profiles in mutant roots. Many differentially expressed gene sets that are associated with various cellular functions were identified. Our results demonstrated that CRISPR/Cas9 systems as powerful tools for wild soybean genome editing and would significantly advance the gene mining and functional identification in wild soybean.
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Zhou, Shiwei, Honghao Yu, Xiaoe Zhao, Bei Cai, Qiang Ding, Yu Huang, Yaxin Li, et al. "Generation of gene-edited sheep with a defined Booroola fecundity gene (FecBB) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9." Reproduction, Fertility and Development 30, no. 12 (2018): 1616. http://dx.doi.org/10.1071/rd18086.
Full textAbstract:
Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q > R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.
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Long, Shaojun, Qiuling Wang, and L. David Sibley. "Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9." Infection and Immunity 84, no. 5 (January 11, 2016): 1262–73. http://dx.doi.org/10.1128/iai.01173-15.
Full textAbstract:
Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially inToxoplasma gondiiwhere 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division inT. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growthin vitroor infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes inT. gondii.
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Monsur, Mahmuda Binte, Gaoneng Shao, Yusong Lv, Shakeel Ahmad, Xiangjin Wei, Peisong Hu, and Shaoqing Tang. "Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants." Genes 11, no. 4 (April 24, 2020): 466. http://dx.doi.org/10.3390/genes11040466.
Full textAbstract:
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach.
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Zhu, Yu, Yu Lin, Yue He, Hanshu Wang, Shitao Chen, Zhenhua Li, Ning Song, and Fei Sun. "Deletion of lncRNA5512 has no effect on spermatogenesis and reproduction in mice." Reproduction, Fertility and Development 32, no. 7 (2020): 706. http://dx.doi.org/10.1071/rd19246.
Full textAbstract:
Long non-coding (lnc) RNAs are a series of RNAs longer than 200 nucleotides that do not code for protein products. Whole-genome expression profiles of lncRNAs suggest that they play important roles in spermatogenesis because they are particularly abundant in testes. However, most of their characteristics and functions remain unclear. The aim of this study was to define the function of lncRNA5512, which is abundant in spermatocytes and round spermatids, in mouse fertility invivo. To investigate this we generated lncRNA5512-knockout mice by clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 technology. Knockout mice showed normal spermatogenesis and fertility, and had no detectable abnormalities. This indicates that lncRNA5512 does not affect mouse fertility despite its high expression in the testes. Its specific localisation in spermatocytes and round spermatids suggests that it could be a useful marker for the identification of spermatocytes and round spermatids in mouse testes.
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Busatto, Sara, Dalila Iannotta, Sierra A. Walker, Luisa Di Marzio, and Joy Wolfram. "A Simple and Quick Method for Loading Proteins in Extracellular Vesicles." Pharmaceuticals 14, no. 4 (April 13, 2021): 356. http://dx.doi.org/10.3390/ph14040356.
Full textAbstract:
Extracellular vesicles (EVs) mediate intercellular transport of biomolecular cargo in the body, making them promising delivery vehicles for bioactive compounds. Genetic engineering of producer cells has enabled encapsulation of therapeutic proteins in EVs. However, genetic engineering approaches can be expensive, time-consuming, and incompatible with certain EV sources, such as human plasma and bovine milk. The goal of this study was to develop a quick, versatile, and simple method for loading proteins in EVs post-isolation. Proteins, including CRISPR associated protein 9 (Cas9), were bound to cationic lipids that were further complexed with MDA-MB-231 cell-derived EVs through passive incubation. Size-exclusion chromatography was used to remove components that were not complexed with EVs. The ability of EVs to mediate intracellular delivery of proteins was compared to conventional methods, such as electroporation and commercial protein transfection reagents. The results indicate that EVs retain native features following protein-loading and obtain similar levels of intracellular protein delivery as conventional methods, but display less toxicity. This method opens up opportunities for rapid exploration of EVs for protein delivery.
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Kim, Brian H., and GuangJun Zhang. "Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs." G3: Genes|Genomes|Genetics 10, no. 3 (January 8, 2020): 1029–37. http://dx.doi.org/10.1534/g3.119.401035.
Full textAbstract:
The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes (rnf185 and rnf215) were successfully mutated in F1 fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear.
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Kolb, Alexander L., Marinaliz Reynoso, and Ronald W. Matheny. "Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation." Journal of Biological Methods 8, no. 3 (July 15, 2021): e151. http://dx.doi.org/10.14440/jbm.2021.359.
Full textAbstract:
Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability togenetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.
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Shahriar, Saleh Ahmed, M. Nazrul Islam, Charles Ng Wai Chun, Md Abdur Rahim, Narayan Chandra Paul, Jasim Uddain, and Shafiquzzaman Siddiquee. "Control of Plant Viral Diseases by CRISPR/Cas9: Resistance Mechanisms, Strategies and Challenges in Food Crops." Plants 10, no. 7 (June 22, 2021): 1264. http://dx.doi.org/10.3390/plants10071264.
Full textAbstract:
Protecting food crops from viral pathogens is a significant challenge for agriculture. An integral approach to genome-editing, known as CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9), is used to produce virus-resistant cultivars. The CRISPR/Cas9 tool is an essential part of modern plant breeding due to its attractive features. Advances in plant breeding programs due to the incorporation of Cas9 have enabled the development of cultivars with heritable resistance to plant viruses. The resistance to viral DNA and RNA is generally provided using the Cas9 endonuclease and sgRNAs (single-guide RNAs) complex, targeting particular virus and host plant genomes by interrupting the viral cleavage or altering the plant host genome, thus reducing the replication ability of the virus. In this review, the CRISPR/Cas9 system and its application to staple food crops resistance against several destructive plant viruses are briefly described. We outline the key findings of recent Cas9 applications, including enhanced virus resistance, genetic mechanisms, research strategies, and challenges in economically important and globally cultivated food crop species. The research outcome of this emerging molecular technology can extend the development of agriculture and food security. We also describe the information gaps and address the unanswered concerns relating to plant viral resistance mediated by CRISPR/Cas9.