Clustered Regularly Interspaced Short Palindromic repeats/CRISPR-associated Nuclease 9 Research Articles

Application progress of CRISPR/Cas9 genome-editing technology in edible fungi.

Edible fungi are not only delicious but are also rich in nutritional and medicinal value, which is highly sought after by consumers. As the edible fungi industry continues to rapidly advance worldwide, particularly in China, the cultivation of superior and innovative edible fungi strains has become increasingly pivotal. Nevertheless, conventional breeding techniques for edible fungi can be arduous and time-consuming. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) is a powerful tool for molecular breeding due to its ability to mediate high-efficiency and high-precision genome modification, which has been successfully applied to many kinds of edible fungi. In this review, we briefly summarized the working mechanism of the CRISPR/Cas9 system and highlighted the application progress of CRISPR/Cas9-mediated genome-editing technology in edible fungi, including Agaricus bisporus, Ganoderma lucidum, Flammulina filiformis, Ustilago maydis, Pleurotus eryngii, Pleurotus ostreatus, Coprinopsis cinerea, Schizophyllum commune, Cordyceps militaris, and Shiraia bambusicola. Additionally, we discussed the limitations and challenges encountered using CRISPR/Cas9 technology in edible fungi and provided potential solutions. Finally, the applications of CRISPR/Cas9 system for molecular breeding of edible fungi in the future are explored.

Comprehensive analysis of U-box E3 ubiquitin ligases gene family revealed BnPUB18 and BnPUB19 negatively regulated drought tolerance in Brassica napus

Plant U-box (PUB) proteins play crucial role in response to abiotic stresses, especially in drought stress. Rapeseed (Brassica napus L.) is an important oil crop sensitive to drought stress, and drought has become one of the main factors restricting the yield of rapeseed. However, there are few reports on BnPUB genes responding to drought stress in rapeseed. Here, a total of 190 BnPUB genes were surveyed from the semi-winter cultivar Zhongshuang11 (ZS11) genome and clustered into 7 subclades according to phylogenetic analysis. Further analysis of the gene structures, protein domains, conserved motifs, and promoter cis-acting elements revealed the diversity of BnPUB genes in rapeseed. Furthermore, the analysis of duplication and synteny events elucidates the adaptive evolution of BnPUBs. Public transcriptome data and quantitative real time polymerase chain reaction (qRT-PCR) results showed that BnPUB genes were differentially expressed in diverse tissues, and most notably, BnPUB18 and BnPUB19 genes were more susceptible to drought and dehydration. Finally, gene-edited lines that knocked out all copies of BnPUB18 and BnPUB19 genes were generated using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology, and yielded significant improvements to drought tolerance. These findings not only provide extensive systematic information on BnPUB genes in rapeseed, but also offers valuable germplasm resources for breeding drought tolerant rapeseed.

Application of DNA-free CRISPR/Cas-mediated Genome Editing in Crops: A Review

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system was instigated first into eukaryotes in the last ten years are becoming productive and worldwide application for genome modification. Genome engineering through insertion of foreign DNA insert have numerous disadvantages which could be overcome by use of DNA-free genome editing. Various ways of DNA-free genome editing mediated by CRISPR/Cas systems are CRISPR/Cas delivery as ribonucleoprotein, delivery of CRISPR/Cas as virus-like particles and agrobacterium-based delivery of CRISPR/Cas. Crop improvement through DNA-free genome editing via CRISPR/Cas have been applied in rice, wheat, maize, tomato, soybean and rare species like Nicotiana benthamiana etc. It is method of choice for precise genome editing without genome shuffle in an organism.

Effect of CRISPR/Cas9-Edited PD-1/PD-L1 on Tumor Immunity and Immunotherapy.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease9 (CRISPR/Cas9) gene editing technology implements precise programming of the human genome through RNA guidance. At present, it has been widely used in the construction of animal tumor models, the study of drug resistance regulation mechanisms, epigenetic control and innovation in cancer treatment. Tumor immunotherapy restores the normal antitumor immune response by restarting and maintaining the tumor-immune cycle. CRISPR/Cas9 technology has occupied a central position in further optimizing anti-programmed cell death 1(PD-1) tumor immunotherapy. In this review, we summarize the recent progress in exploring the regulatory mechanism of tumor immune PD-1 and programmed death ligand 1(PD-L1) based on CRISPR/Cas9 technology and its clinical application in different cancer types. In addition, CRISPR genome-wide screening identifies new drug targets and biomarkers to identify potentially sensitive populations for anti-PD-1/PD-L1 therapy and maximize antitumor effects. Finally, the strong potential and challenges of CRISPR/Cas9 for future clinical applications are discussed.

CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

CRISPR/Cas9 in cancer: An attempt to the present trends and future prospects.

Cancer is the second leading cause of death globally. Series of sequential, repeated genetic changes and epigenetic modifications are leading to the formation of tumors. These tumors subsequently causing the infected cells to invade and transform their surrounding cells by metastasis are some hallmarks in cancer. Although tremendous efforts have been extended for structurally characterizing the numerous genomic mutations undergoing in cancer cells, there is a lack of information regarding the functions of many mutated genes. Clustered Regularly Interspaced Short Palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) has become a robust method for building changes in genome of many organisms. Recent reports have suggested that modification of CRISPR/Cas9 can provide plot form to probe the mechanisms in tumorigenesis and in cancer therapies. This review focuses on the historical perspectives of CRISPR/Cas9. The study highlights the applications and also role in cancer cell genome editing, which is helpful to understand the dynamics. Intense research in progress on mechanism of action of CRISPR/Cas9 has been reviewed and critically discussed. Further, relevant literature on animal models focusing on various approaches has been highlighted to emphasize the therapeutics of CRISPR/Cas9 with current trends and future challenges.

CRISPR/Cas9-edited triple-fusion reporter gene imaging of dynamics and function of transplanted human urinary-induced pluripotent stem cell-derived cardiomyocytes.

To investigate the post-transplantation behaviour and therapeutic efficacy of human urinary-induced pluripotent stem cell-derived cardiomyocytes (hUiCMs) in infarcted heart. We used clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology to integrate a triple-fusion (TF) reporter gene into the AAVS1 locus in human urine-derived hiPSCs (hUiPSCs) to generate TF-hUiPSCs that stably expressed monomeric red fluorescent protein for fluorescence imaging, firefly luciferase for bioluminescence imaging (BLI) and herpes simplex virus thymidine kinase for positron emission tomography (PET) imaging. Transplanted cardiomyocytes derived from TF-hUiPSCs (TF-hUiCMs) engrafted and proliferated in the infarcted heart as monitored by both BLI and PET imaging and significantly improved cardiac function. Under ischaemic conditions, TF-hUiCMs enhanced cardiomyocyte (CM) glucose metabolism and promoted angiogenic activity. This study established a CRISPR/Cas9-mediated multimodality reporter gene imaging system that can determine the dynamics and function of TF-hUiCMs in myocardial infarction, which is helpful for investigating the application of stem cell therapy.

A review of application of base editing for the treatment of inner ear disorders

Abstract The development of applications for the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system has increased greatly in recent years, especially in the area of gene therapy by efficient in vivo genome editing. Although great success has been achieved in repairing and rewriting genomes through homology-directed repair coupled with Cas9 nuclease cleavage, its in vivo efficiency is insufficient for gene therapy. Base editing is a next-generation genome-editing tool that does not involve double-stranded DNA breaks and uses components of the CRISPR system together with other enzymes to make point mutations directly in cellular DNA or RNA. Base editors, composed of an engineered deaminase and a catalytically impaired CRISPR/Cas9 variant, are powerful tools for targeted base editing in cells and organisms. In non-dividing cells, base editors can directly transform one base or base pair into another, efficiently installing a point mutation. Undesired by-products of editing are seldom generated during this procedure. Herein we review the different base-editing platforms, including their deaminase recruitment strategies and editing outcomes, and the in vivo delivery of base editors. Additionally, we summarize therapeutic applications of base editing in disorders of the inner ear.

Inhibition of Hepatitis B Virus with the Help of CRISPR/Cas9 Technology.

Hepatitis B infection caused by hepatitis B virus (HBV) is a serious health issue worldwide. Existing therapeutic strategies hardly eradicate HBV infections, and they fail to attain complete cure. Advanced treatment strategies are urgently needed to successfully terminate further spread of HBV infection and eliminate hidden reservoirs of virus. Recently, a novel RNA-guided gene editing tool, known as the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system, has been established. It facilitates site-specific mutagenesis and reveals a new way to develop applicable techniques for disease treatment, such as extermination of infectious agents like HBV This study highlights the current developments in CRISPR/Cas9 technology and its importance for target-specific inhibition of HBV genome. Benefits, challenges, feasible solutions, and proposed guidelines for forthcoming study in CRISPR/Cas9 are described to highlight the possible cures of and treatments for chronic HBV infection.

Generation of CCR5-ablated Human Induced Pluripotent Stem Cells as a Therapeutic Approach for Immune-mediated Diseases.

C-C chemokine receptor type 5 (CCR5) is a receptor for some pro-inflammatory chemokines which plays important roles in immunological disorder and host responses to infectious agents. Additionally, the prognosis of some immune-mediated diseases in the people who are naturally carrying the CCR5 32bp deletions is optimistic. However, the clinical application of CCR5 32bp mutant cells is very limited due to the rare availability of donors who are homozygous for CCR5 D32. The transfection efficiency of nucleofected placental mesenchymal stem cells derived - human induced pluripotent stem cells (PMSC-hiPSCs) was examined through the evaluation of green fluorescent protein (GFP) expression using flow cytometry. The nucleofected clonal populations were selected using colony picking. The CCR5 gene disrupted clonal populations were evaluated and confirmed by PCR and Sanger sequencing methods. Also, off-target sites were evaluated by the "Loss of a primer binding site" technique. The results of the flow cytometry revealed that among the six applied nucleofection programs for PMSC-iPSCs, the program of A-033 has achieved the best transfection efficiency (27.7%). PCR and then sequencing results confirmed the CCR5 gene was disrupted in two clonal populations of 16 (D6) and 62 (D20) by the Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) system. The "Loss of a primer binding site" technique showed that no exonic off-target mutations were induced in both CCR5 gene disrupted clonal populations. We establish a CRISPR/Cas9 mediated CCR5 ablated PMSC-hiPSCs without detectable off-target damage. This approach can provide a stable supply of autologous/allogeneic CCR5-disrupted PMSC-hiPSCs that might be a feasible approach for the treatment of immune-mediated diseases.

CRISPR/Cas9 technology as a potent molecular tool for gene therapy.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) is an RNA-guided gene editing tool which offers several advantageous characteristics in comparison with the conventional methods (e.g., zinc finger nucleases and transcription activator-like effector nucleases) such as cost-effectiveness, flexibility, and being easy-to-use. Despite some limitations such as efficient delivery and safety, CRISPR-Cas9 is still the most convenient tool for gene editing purposes. Due to the potential capability of the CRISPR-Cas9 system in genome editing and correction of casual mutations, it can be considered as a possible therapeutic system in the treatment of disorders associated with the genome mutations and in particular cancer treatment. In this review, we will discuss CRISPR-Cas-based gene editing along with its classifications and mechanism of action. Furthermore, the therapeutic application of the CRISPR-Cas9 system in mutational disorders, delivery systems, as well as its advantages and limitations with a special emphasis on cancer treatment will be discussed.

Therapeutic applications of CRISPR/Cas9 system in gene therapy.

Gene therapy is based on the principle of the genetic manipulation of DNA or RNA for treating and preventing human diseases. The clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) system, derived from the acquired immune system in bacteria and archaea, has provided a new tool for accurate manipulation of genomic sequence to attain a therapeutic result. The advantage of CRISPR which made it an easy and flexible tool for diverse genome editing purposes is that a single protein (Cas9) complex with 2 short RNA sequences, function as a site-specific endonuclease. Recently, application of CRISPR/Cas9 system has become popular for therapeutic aims such as gene therapy. In this article, we review the fundamental mechanisms of CRISPR-Cas9 function and summarize preclinical CRISPR-mediated gene therapy reports on a wide variety of disorders.

Generation of knockout mouse models of cyclin-dependent kinase inhibitors by engineered nuclease-mediated genome editing

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors p16Ink4a (Cdkn2a, cyclin-dependent kinase inhibitor 2a), p19Arf (an alternative reading frame product of Cdkn2a,), and p27Kip1 (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The p16Ink4a and p19Arf knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27Kip1 knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

Disruption of the Tff1 gene in mice using CRISPR/Cas9 promotes body weight reduction and gastric tumorigenesis

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. Tff1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated Tff1-knockout (KO) mice, without a neomycin resistant (NeoR) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our Tff1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established Tff1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that Tff1 expression influences gender differences.

527. Improvement of Pre-Clinical Non-Human Primate Model for Pluripotent Stem Cell Based Therapies by Introducing Marker Genes in Safe Harbor Locus

Induced pluripotent stem cells (iPSCs) are being developed as sources for clinical cellular regenerative therapies, as well as valuable in vitro human disease models. Derivation of iPSCs from non-human primates (NHP) affords the opportunity to test the safety, feasibility and efficacy of proposed iPSC-derived cellular delivery in species with physiology, immunology and scale similar to humans. However, there is a need for stable and safe labeling methods for iPSCs and their differentiated progeny allowing analysis of survival, proliferation, tissue integration and biodistribution, in vitro and in vivo. Typically, marker genes have been inserted into target cells by transduction with randomly-integrating viral vectors. However, these methods raise concerns regarding genotoxicity and transgene silencing, particularly in pluripotent stem cells, limiting their utility for tracking and eventual clinical applications. Targeted integration into genomic “safe harbors” offer a promising alternative approach to mark target cells, potentially circumventing these issues. The adeno-associated virus integration site 1 (AAVS1) has been proposed as a suitable safe harbor for human cells, and we now investigate its utility in our rhesus macaque NHP iPSC model. We have efficiently knocked-in both a truncated CD19 (hΔCD19) marker gene a non-immunogenic and clinical relevant marker, or green fluorescent protein (GFP) at the homologous AAVS1 site in rhesus iPSCs (RhiPSCs) using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) system. PCR and Southern blot analyses demonstrated highly efficient knock-in into the AAVS1 locus, with over one third of clones screened containing only targeted but not random integrations. (Table 1Table 1). Edited RhiPSC-GFP/hΔCD19 clones retained a normal karyotype and pluripotency - as shown by teratoma formation. Directed differentiation of these clones to neutrophils, hepatocytes or cardiomyocytes was not hindered by the knock-in of marker genes into the AAVS1 sites. Notably, transgene expression was stable in undifferentiated RhiPSCs and differentiated cell types derived from the RhiPSC (Figure 1Figure 1), in contrast to prior experience with viral vector delivery. We have established a computational platform to assess off-target effects of guide RNAs in the rhesus genome. Genetically marked RhiPSCs afford a unique opportunity to develop clinically relevant models for iPSC-based cell therapies.Table 1Summary of CRISPR-mediated gene editing in rhesus iPSCsOriginal iPSC cloneReporter geneClones with TI/Clones screened1Clones without RI/Clones with TI2ZG15-M11-10hΔCD194/42/8GFP14/145/9ZG32-3-4hΔCD19ND1/4GFPND2/4ZH26-HS41hΔ CD19ND1/4Total18/18(100%)11/29 (37.9%) View Table in HTML RI: random integration, TI; targeted integration, ND: not determined1based on by PCR analysis2based on Southern blot analysisView Large Image | Download PowerPoint Slide

CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV.

Hepatitis B virus (HBV) infection remains a major global health problem because current therapies rarely eliminate HBV infections to achieve a complete cure. A different treatment paradigm to effectively clear HBV infection and eradicate latent viral reservoirs is urgently required. In recent years, the development of a new RNA-guided gene-editing tool, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system, has greatly facilitated site-specific mutagenesis and represents a very promising potential therapeutic tool for diseases, including for eradication of invasive pathogens such as HBV. Here, we review recent advances in the use of CRISPR/Cas9, which is designed to target HBV specific DNA sequences to inhibit HBV replication and to induce viral genome mutation, in cell lines or animal models. Advantages, limitations and possible solutions, and proposed directions for future research are discussed to highlight the opportunities and challenges of CRISPR/Cas9 as a new, potentially curative therapy for chronic hepatitis B infection.

CRISPR-Cas9 systems: versatile cancer modelling platforms and promising therapeutic strategies.

The RNA-guided nuclease CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9) and its variants such as nickase Cas9, dead Cas9, guide RNA scaffolds and RNA-targeting Cas9 are convenient and versatile platforms for site-specific genome editing and epigenome modulation. They are easy-to-use, simple-to-design and capable of targeting multiple loci simultaneously. Given that cancer develops from cumulative genetic and epigenetic alterations, CRISPR-Cas9 and its variants (hereafter referred to as CRISPR-Cas9 systems) hold extensive application potentials in cancer modeling and therapy. To date, they have already been applied to model oncogenic mutations in cell lines (e.g., Choi and Meyerson, Nat Commun 2014;5:3728) and in adult animals (e.g., Xue et al., Nature 2014;514:380-4), as well as to combat cancer by disabling oncogenic viruses (e.g., Hu et al., Biomed Res Int 2014;2014:612823) or by manipulating cancer genome (e.g., Liu et al., Nat Commun 2014;5:5393). Given the importance of epigenome and transcriptome in tumourigenesis, manipulation of cancer epigenome and transcriptome for cancer modeling and therapy is a promising area in the future. Whereas (epi)genetic modifications of cancer microenvironment with CRISPR-Cas9 systems for therapeutic purposes represent another promising area in cancer research. Herein, we introduce the functions and mechanisms of CRISPR-Cas9 systems in genome editing and epigenome modulation, retrospect their applications in cancer modelling and therapy, discuss limitations and possible solutions and propose future directions, in hope of providing concise and enlightening information for readers interested in this area.