Transcription Activator-like Nucleases Research Articles

HPSC gene editing for cardiac disease therapy.

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. However, the lack of human cardiomyocytes with proper genetic backgrounds limits the study of disease mechanisms. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have significantly advanced the study of these conditions. Moreover, hPSC-CMs made it easy to study CVDs using genome-editing techniques. This article discusses the applications of these techniques in hPSC for studying CVDs. Recently, several genome-editing systems have been used to modify hPSCs, including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9). We focused on the recent advancement of genome editing in hPSCs, which dramatically improved the efficiency of the cell-based mechanism study and therapy for cardiac diseases.

The role of the cd99l2 gene on leukocyte interstitial migration in zebrafish.

Leukocytes play an essential role in ontogeny, tissue regeneration, and innate and adaptive immunity. The migration of leukocytes to the infected or traumatized areas is necessary for their immune response function. As an adhesion molecule, CD99L2 is crucial in the extravasation of leukocytes, however, its role in the interstitial migration of leukocytes remains unclear. In this study, the cd99l2 gene was knock-out by TALEN (transcription activator-like effector nuclease) in zebrafish and discovered that the deletion had no effect on zebrafish development. The number of granulocytes and macrophages recruited to the wounded tissue was significantly reduced in the cd99l2 mutants following caudal fin damage. Further research revealed that the expression of mfap4 was drastically decreased in the cd99l2 mutants, which may be one of the reasons that affect the migration of macrophages to the wound site. Moreover,transgenic lines with labeled vasculature, neutrophils and macrophages demonstrated that neutrophils and macrophages migrate throughout the interstitial space to the wound tissue in both wild-type and mutant zebrafish at 60 hours post-fertilization, indicating that the cd99l2 gene is involved in the interstitial migration of leukocytes. Finally, RNA transcription, protein folding, and the P450 pathway were enriched in cd99l2 mutants by RNA-seq analysis. Previous research had demonstrated that the regulation of transcription and signal transduction could be affected by adhesion molecules, which may suggest that the cd99l2 gene is involved in the cascade signaling pathway of leukocyte migration as an adhesion molecule. In conclusion, this study uncovered a novel function of the cd99l2 gene in the process of leukocyte migration in zebrafish, which is expected to provide a theoretical foundation for inflammatory and immune-related diseases.

Rice grain yield and quality improvement via CRISPR/Cas9 system: an updated review

Rice (Oryza sativa L.) is an important staple food crop worldwide. To meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. During recent years, genome editing has played a great role in the development of superior varieties of grain crops. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Over a decade, site-directed mutagenesis tools like Zinc Finger Nucleases (ZFN), Transcriptional activator-like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) System were used and have played a great role in rice yield and quality enhancement. In addition, most recently other genome editing techniques like prime editing and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close synthetic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. Genomic alteration employing genome editing technologies (GETs) like CRISPR/Cas9 for reverse genetics has opened new avenues in agricultural sciences such as rice yield and grain quality improvement. Currently, CRISPR/Cas9 technology is widely used by researchers for genome editing to achieve the desired biological objectives, because of its simple targeting, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. Over the past few years many genes related to rice grain quality and yield enhancement have been successfully edited via CRISPR/Cas9 technology method to cater to the growing demand for food worldwide. The effectiveness of these methods is being verified by the researchers and crop scientists worldwide. In this review we focus on genome-editing tools for rice improvement to address the progress made and provide examples of genome editing in rice. We also discuss safety concerns and methods for obtaining transgene-free crops.

Engineering the plastid and mitochondrial genomes of flowering plants.

Engineering the plastid genome based on homologous recombination is well developed in a few model species. Homologous recombination is also the rule in mitochondria, but transformation of the mitochondrial genome has not been realized in the absence of selective markers. The application of transcription activator-like (TAL) effector-based tools brought about a dramatic change because they can be deployed from nuclear genes and targeted to plastids or mitochondria by an N-terminal targeting sequence. Recognition of the target site in the organellar genomes is ensured by the modular assembly of TALE repeats. In this paper, I review the applications of TAL effector nucleases and TAL effector cytidine deaminases for gene deletion, base editing and mutagenesis in plastids and mitochondria. I also review emerging technologies such as post-transcriptional RNA modification to regulate gene expression, Agrobacterium- and nanoparticle-mediated organellar genome transformation, and self-replicating organellar vectors as production platforms.

Comprehending the evolution of gene editing platforms for crop trait improvement.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base editing uses base editors to engineer precise changes for trait improvement. Newer technologies such as prime editing have now been developed as a “search-and-replace” tool to engineer all possible single-base changes. Owing to the availability of these, the field of genome editing has evolved rapidly to develop crop plants with improved traits. In this review, we present the evolution of the CRISPR/Cas system into new-age methods of genome engineering across various plant species and the impact they have had on tweaking plant genomes and associated outcomes on crop improvement initiatives.

Knockout of MITOGEN-ACTIVATED PROTEIN KINASE 3 causes barley root resistance against Fusarium graminearum.

The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are poorly understood in crops. Here, microscopic, phenotypic, proteomic, and biochemical analyses revealed that roots of independent transcription activator-like effector nuclease (TALEN)-based knockout lines of barley (Hordeum vulgare L.) MAPK 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of extracellular hyphae was considerably deregulated, mycelia development was less efficient, and number of appressoria-like structures and their penetration potential were substantially reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild-type (WT), but ROS production was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with elevated abundance of catalase (CAT) and ascorbate peroxidase (APX). Moreover, differential proteomic analysis revealed downregulation of several defense-related proteins in WT, and the upregulation of pathogenesis-related protein 1 (PR-1) and cysteine proteases in HvMPK3 KO lines. Proteins involved in suberin formation, such as peroxidases, lipid transfer proteins (LTPs), and the GDSL esterase/lipase (containing "GDSL" aminosequence motif) were differentially regulated in HvMPK3 KO lines after F. graminearum inoculation. Consistent with proteomic analysis, microscopic observations showed enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to barley root resistance against Fusarium root rot.

Genome Editing in Translational Medicine: An Inventory.

Genomic mutations are the driving force of biological diversity but they are also the cause of a plethora of human diseases ranging from heritable disorders to neurological pathologies and cancer. For most genetic disorders, there is no curative treatment available to date. The demand for precise, preferably patient-specific, treatment regimen offering cure is naturally high. Genome editing by Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas enables targeted manipulation of genomes, thereby offering the opportunity to treat such diseases. While ethical and regulatory guidelines need to be developed and considered, the prospect of genome editing for curative treatment is certainly exciting. Here, we review the current state of therapeutics based on genome editing techniques. We highlight recent breakthroughs, describe clinical trials employing genome editing-based medicine, discuss the benefits and pitfalls, and take a look into the future of genome editing.

New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery.

The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions.

PIWI-Interacting RNA (piRNA) and Epigenetic Editing in Environmental Health Sciences.

PURPOSEOF REVIEW: The epigenome modulates gene expression in response to environmental stimuli. Modifications to the epigenome are potentially reversible, making them a promising therapeutic approach to mitigate environmental exposure effects on human health. This review details currently available genome and epigenome editing technologies and highlights ncRNA, including piRNA, as potential tools for targeted epigenome editing. RECENT FINDINGS: Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease (CRISPR/Cas) research has significantly advanced genome editing technology, with broad promise in genetic research and targeted therapies. Initial epigenome-directed therapies relied on global modification and suffered from limited specificity. Adapted from current genome editing tools, zinc finger protein (ZFP), TALE, and CRISPR/nuclease-deactivated Cas (dCas) systems now confer locus-specific epigenome editing, with promising applicability in the field of environmental health sciences. However, high incidence of off-target effects and time taken for screening limit their use. FUTURE DEVELOPMENT: ncRNA serve as a versatile biomarker with well-characterized regulatory mechanisms that can easily be adapted to edit the epigenome. For instance, the transposon silencing mechanism of germline PIWI-interacting RNAs (piRNA) could be engineered to specifically methylate a given gene, overcoming pitfalls of current global modifiers. Future developments in epigenome editing technologies will inform risk assessment through mechanistic investigation and serve as potential modes of intervention to mitigate environmentally induced adverse health outcomes later in life.

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases.

Advancements in genome editing make possible to exploit the functions of enzymes for efficient DNA modifications with tremendous potential to treat human genetic diseases. Several nuclease genome editing strategies including Meganucleases (MNs), Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) have been developed for the correction of genetic mutations. CRISPR-Cas has further been engineered to create nickase genome editing tools including Base editors and Prime editors with much precision and efficacy. In this review, we summarized recent improvements in nuclease and nickase genome editing approaches for the treatment of genetic diseases. We also highlighted some limitations for the translation of these approaches into clinical applications.

Anti-Arthritogenic Property of Interleukin 10-Expressing Human Amniotic MSCs Generated by Gene Editing in Collagen-Induced Arthritis

Although stem cells are promising tools for the treatment of arthritis, their therapeutic effects remain controversial. In this study, we investigated the therapeutic properties of interleukin (IL)-10-overexpressing human amniotic mesenchymal stem cells (AMMs) generated via gene editing in a collagen-induced mouse model. IL-10 was inserted into the genomic loci of AMMs via transcription activator-like effector nucleases. In vitro immunomodulatory effects of IL-10-overexpressing AMMs (AMM/I) were evaluated and their anti-arthritogenic properties were determined in collagen-induced arthritis (CIA) mice. Transplantation of AMM/I attenuates CIA progression. In addition, the regulatory T cell population was increased, while T helper-17 cell activation was suppressed by AMM/I administration in CIA mice. Consistently, AMM/I injection increased proteoglycan expression, while reducing inflammation and the expression levels of the pro-inflammatory factors, IL-1 β, IL-6, monocyte chemoattractant protein-1, and tumor necrosis factor- α, in joint tissues. In conclusion, use of IL-10-edited human AMM/I may be a novel therapeutic strategy for the treatment of arthritis.

Genome centric engineering using ZFNs, TALENs and CRISPR-Cas9 systems for trait improvement and disease control in Animals.

Livestock is an essential life commodity in modern agriculture involving breeding and maintenance. The farming practices have evolved mainly over the last century for commercial outputs, animal welfare, environment friendliness, and public health. Modifying genetic makeup of livestock has been proposed as an effective tool to create farmed animals with characteristics meeting modern farming system goals. The first technique used to produce transgenic farmed animals resulted in random transgene insertion and a low gene transfection rate. Therefore, genome manipulation technologies have been developed to enable efficient gene targeting with a higher accuracy and gene stability. Genome editing (GE) with engineered nucleases-Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) regulates the targeted genetic alterations to facilitate multiple genomic modifications through protein-DNA binding. The application of genome editors indicates usefulness in reproduction, animal models, transgenic animals, and cell lines. Recently, CRISPR/Cas system, an RNA-dependent genome editing tool (GET), is considered one of the most advanced and precise GE techniques for on-target modifications in the mammalian genome by mediating knock-in (KI) and knock-out (KO) of several genes. Lately, CRISPR/Cas9 tool has become the method of choice for genome alterations in livestock species due to its efficiency and specificity. The aim of this review is to discuss the evolution of engineered nucleases and GETs as a powerful tool for genome manipulation with special emphasis on its applications in improving economic traits and conferring resistance to infectious diseases of animals used for food production, by highlighting the recent trends for maintaining sustainable livestock production.

CRISPR/Cas9 a simple, inexpensive and effective technique for gene editing.

In recent years, the number of tools and techniques that enable genetic material to be added, removed or altered at specific locations in the genome has increased significantly. The objective is to know the structure of genomes, the function of genes and improve gene therapy.In this work we intend to explain the functioning of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) and the advantages that this technique may have compared to previously developed techniques, such as RNA interference (RNAi), Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in gene and genome editing.We will start with the story of the discovery, then its biological function in the adaptive immune system of bacteria against bacteriophage attack, and ending with a description of the mechanism of action and its use in gene editing. We will also discuss other Cas enzymes with great potential for use in genome editing as an alternative to Cas9.CRISPR/Cas9 is a simple, inexpensive, and effective technique for gene editing with multiple applications from the development of functional genomics and epigenetics. This technique will, in the near future, have great applications in the development of cell models for use in medical and pharmaceutical processes, in targeted therapy, and improvement of agricultural and environmental species.

Genetic Manipulation Strategies for β-Thalassemia: A Review.

Thalassemias are monogenic hematologic diseases that are classified as α- or β-thalassemia according to its quantitative abnormalities of adult α- or β-globin chains. β-thalassemia has widely spread throughout the world especially in Mediterranean countries, the Middle East, Central Asia, India, Southern China, and the Far East as well as countries along the north coast of Africa and in South America. The one and the only cure for β-thalassemia is allogenic hematopoietic stem cell transplantations (HSCT). Nevertheless, the difficulty to find matched donors has hindered the availability of this therapeutic option. Therefore, this present review explored the alternatives for β-thalassemia treatment such as RNA manipulation therapy, splice-switching, genome editing and generation of corrected induced pluripotent stem cells (iPSCs). Manipulation of β-globin RNA is mediated by antisense oligonucleotides (ASOs) or splice-switching oligonucleotides (SSOs), which redirect pre-mRNA splicing to significantly restore correct β-globin pre-mRNA splicing and gene product in cultured erythropoietic cells. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) are designer proteins that can alter the genome precisely by creating specific DNA double-strand breaks. The treatment of β-thalassemia patient-derived iPSCs with TALENs have been found to correct the β-globin gene mutations, implying that TALENs could be used as a therapy option for β-thalassemia. Additionally, CRISPR technologies using Cas9 have been used to fix mutations in the β-globin gene in cultured cells as well as induction of hereditary persistence of fetal hemoglobin (HPFH), and α-globin gene deletions have proposed a possible therapeutic option for β-thalassemia. Overall, the accumulated research evidence demonstrated the potential of ASOs-mediated aberrant splicing correction of β-thalassemia mutations and the advancements of genome therapy approaches using ZFNs, TALENs, and CRISPR/Cas9 that provided insights in finding the permanent cure of β-thalassemia.

CRISPR/Cas9 Technique for Temperature, Drought, and Salinity Stress Responses.

Global warming and climate change have severely affected plant growth and food production. Therefore, minimizing these effects is required for sustainable crop yields. Understanding the molecular mechanisms in response to abiotic stresses and improving agricultural traits to make crops tolerant to abiotic stresses have been going on unceasingly. To generate desirable varieties of crops, traditional and molecular breeding techniques have been tried, but both approaches are time-consuming. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALENs) are genome-editing technologies that have recently attracted the attention of plant breeders for genetic modification. These technologies are powerful tools in the basic and applied sciences for understanding gene function, as well as in the field of crop breeding. In this review, we focus on the application of genome-editing systems in plants to understand gene function in response to abiotic stresses and to improve tolerance to abiotic stresses, such as temperature, drought, and salinity stresses.

Application of CRISPR-Mediated Gene Editing for Crop Improvement.

Plant gene editing has become an important molecular tool to revolutionize modern breeding of crops. Over the past years, remarkable advancement has been made in developing robust and efficient editing methods for plants. Despite a variety of available genome editing methods, the discovery of most recent system of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) has been one of the biggest advancement in this path, with being the most efficient approach for genome manipulation. Until recently, genetic manipulations were confined to methods, like Agrobacterium-mediated transformations, zinc-finger nucleases, and TAL effector nucleases. However this technology supersedes all other methods for genetic modification. This RNA-guided CRISPR-Cas system is being rapidly developed with enhanced functionalities for better use and greater possibilities in biological research. In this review, we discuss and sum up the application of this simple yet powerful tool of CRISPR-Cas system for crop improvement with recent advancement in this technology.

Gene Editing-Based Technologies for Beta-hemoglobinopathies Treatment.

Simple Summaryβ-thalassemia syndromes are clinically and genetically heterogeneous blood disorders presented by β-chain deficiency in hemoglobin production. Despite improvements in transfusion practices and chelation treatment, many lingering challenges have encouraged researchers to develop newer therapeutic strategies such as gene editing. One of the most powerful arms of genetic manipulation is gene editing tools, which have been recently applied to improve β-thalassemia symptoms. Nevertheless, several obstacles, such as off-target effects, protospacer-adjacent motif requirement, efficient gene transfer and expression methods, DNA-damage toxicity, and immunotoxicity issues still need to be addressed in order to improve the safety and efficacy of the gene editing approaches. Hence, additional efforts are needed to address these problems, evaluate the safety of genome editing tools at the clinical level and follow the outcomes of gene editing tools-mediated therapeutic approaches in related patients.Beta (β)-thalassemia is a group of human inherited abnormalities caused by various molecular defects, which involves a decrease or cessation in the balanced synthesis of the β-globin chains in hemoglobin structure. Traditional treatment for β-thalassemia major is allogeneic bone marrow transplantation (BMT) from a completely matched donor. The limited number of human leukocyte antigen (HLA)-matched donors, long-term use of immunosuppressive regimen and higher risk of immunological complications have limited the application of this therapeutic approach. Furthermore, despite improvements in transfusion practices and chelation treatment, many lingering challenges have encouraged researchers to develop newer therapeutic strategies such as nanomedicine and gene editing. One of the most powerful arms of genetic manipulation is gene editing tools, including transcription activator-like effector nucleases, zinc-finger nucleases, and clustered regularly interspaced short palindromic repeat–Cas-associated nucleases. These tools have concentrated on γ- or β-globin addition, regulating the transcription factors involved in expression of endogenous γ-globin such as KLF1, silencing of γ-globin inhibitors including BCL11A, SOX6, and LRF/ZBTB7A, and gene repair strategies. In this review article, we present a systematic overview of the appliances of gene editing tools for β-thalassemia treatment and paving the way for patients’ therapy.

SDF-1-edited human amniotic mesenchymal stem cells stimulate angiogenesis in treating hindlimb ischaemia.

Although stem cells have extensively been studied as a novel vehicle for tissue repair, their sustained efficacy remains controversial. In this study, we aimed to investigate the angiogenic potency over time of stromal cell‐derived factor‐1 (SDF‐1) gene‐edited amniotic mesenchymal stem cells (AMM/S) in a hindlimb ischaemia model. An SDF‐1 transgene was inserted into the AMM cell genome via transcription activator‐like effector nuclease (TALEN) mediated knock‐in, and cell migration, Matrigel tube formation, and in vivo Matrigel plug assays were performed. AMM/S were also transplanted into hindlimb ischaemia model mice. Blood perfusion, therapeutic potential, histology, capillary density and in vivo angiogenic assays were performed. AMM/S exhibited high expression of the SDF‐1 gene, and robustly promoted migration, proliferation and microvascular formation. AMM/S transplantation significantly increased blood perfusion and limb loss prevention compared with AMM. AMM/S also significantly inhibited increased capillary density and expression of angiogenic factors in the ischaemic hindlimb. Our study demonstrated that AMM/S provides a significant therapeutic effect in ischaemic hindlimbs by enhancing angiogenesis.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

Advance of Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 System and Its Application in Crop Improvement.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is the third generation of novel targeted genome editing technology after zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). It is also one of the most promising techniques for mutating and modifying genes. The CRISPR-Cas9 system has the advantages of simplicity, high efficiency, high specificity, and low production cost, thus greatly promoting the study of gene function. Meanwhile, it has attracted the attention of biologists. After the development and improvement in recent years, CRISPR-Cas9 system has become increasingly mature and has been widely used in crop improvement. Firstly, this review systematically summarizes the generation and advantages of CRISPR-Cas9 system. Secondly, three derivative technologies of the CRISPR-Cas9 system are introduced. Thirdly, this review focuses on the application of CRISPR-Cas9 system in gene knockout, gene knock-in, and gene regulation, as well as the improvement of yield, quality, and biological resistance of important crops such as rice, wheat, soybean, corn, and potato. Finally, this review proposes the potential challenges of CRISPR-Cas9 system, and discusses the future development of CRISPR-Cas9 system.