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Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9-based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.

Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR-Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.

Cancer is a genetic disease stemming from cumulative genetic/epigenetic aberrations. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome editing technology has been extensively applied in various cell types and organisms, both in vitro and in vivo, for efficient gene disruption and gene modification. CRISPR-Cas9 has shown great promise for the treatment of cancer. However, despite its advantages and tremendous potential, numerous challenges, such as fitness of edited cells, editing efficiency, delivery methods and potential off-target effects, remain to be solved for completely clinical application. Here, we present the potential applications and recent advances of CRISPR-Cas9 in cancer therapy, and discuss the challenges that might be encountered in clinical applications.

In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited disorders affecting different organ systems, such as blood and muscles. Both hematological and neuromuscular genetic disorders benefit from genome editing approaches but face different challenges in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In the last decade, many clinical trials were initiated and are now delivering encouraging results. The recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and transfusion-dependent β-thalassemia, represents a significant milestone in the field and highlights the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed up preclinical development of therapeutic solutions. Several corrective interventions have been proposed to genetically restore dystrophin production using the CRISPR toolbox and have demonstrated promising results in different DMD animal models. Although these advances represent a significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector doses, together with safety and immunological concerns. Collectively, the results obtained in the hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for patients affected by these debilitating conditions. As each field suffers from different and specific challenges, the clinical translation of CRISPR therapies may progress differentially depending on the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions. This review provides insights into the diverse applications of CRISPR-based technologies in both preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare advances in both fields while highlighting current trends, difficulties, and challenges to overcome.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) adaptive immune systems constitute a bacterial defence against invading nucleic acids derived from bacteriophages or plasmids. This prokaryotic system was adapted in molecular biology and became one of the most powerful and versatile platforms for genome engineering. CRISPR/Cas9 is a simple and rapid tool which enables the efficient modification of endogenous genes in various species and cell types. Moreover, a modified version of the CRISPR/Cas9 system with transcriptional repressors or activators allows robust transcription repression or activation of target genes. The simplicity of CRISPR/Cas9 has resulted in the widespread use of this technology in many fields, including basic research, biotechnology and biomedicine.

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.

The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

Genetic engineering of model organisms and cultured cells has for decades provided important insights into the mechanisms underlying cardiovascular development and disease. In the past few years the development of several nuclease systems has broadened the range of model/cell systems that can be engineered. Of these, the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has become the favorite for its ease of application. Here we will review this RNA-guided nuclease system for gene editing with respect to its usefulness for cardiovascular studies and with an eye toward potential therapy. Studies on its off-target activity, along with approaches to minimize this activity will be given. The advantages of gene editing versus gene targeting in embryonic stem cells, including the breadth of species and cell types to which it is applicable, will be discussed. We will also cover its use in iPSC for research and possible therapeutic purposes; and we will review its use in muscular dystrophy studies where considerable progress has been made toward dystrophin correction in mice. The CRISPR/Ca9s system is also being used for high-throughput screening of genes, gene regulatory regions, and long noncoding RNAs. In addition, the CRISPR system is being used for nongene-editing purposes such as activation and inhibition of gene expression, as well as for fluorescence tagging of chromosomal regions and individual mRNAs to track their cellular location. Finally, an approach to circumvent the inability of post-mitotic cells to support homologous recombination-based gene editing will be presented. In conclusion, applications of the CRISPR/Cas system are expanding at a breath-taking pace and are revolutionizing approaches to gain a better understanding of human diseases.

Summary Tomato (Solanum lycopersicum), which is used for both processing and fresh markets, is a major crop species that is the top ranked vegetable produced over the world. Tomato is also a model species for research in genetics, fruit development and disease resistance. Genetic resources available in public repositories comprise the 12 wild related species and thousands of landraces, modern cultivars and mutants. In addition, high quality genome sequences are available for cultivated tomato and for several wild relatives, hundreds of accessions have been sequenced, and databases gathering sequence data together with genetic and phenotypic data are accessible to the tomato community. Major breeding goals are productivity, resistance to biotic and abiotic stresses, and fruit sensorial and nutritional quality. New traits, including resistance to various biotic and abiotic stresses and root architecture, are increasingly being studied. Several major mutations and quantitative trait loci (QTLs) underlying traits of interest in tomato have been uncovered to date and, thanks to new populations and advances in sequencing technologies, the pace of trait discovery has considerably accelerated. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing (GE) already proved its remarkable efficiency in tomato for engineering favorable alleles and for creating new genetic diversity by gene disruption, gene replacement, and precise base editing. Here, we provide insight into the major tomato traits and underlying causal genetic variations discovered so far and review the existing genetic resources and most recent strategies for trait discovery in tomato. Furthermore, we explore the opportunities offered by CRISPR/Cas9 and their exploitation for trait editing in tomato.

Summary Grain size and weight are important components of a suite of yield-related traits in crops. Here, we showed that the CRISPR-Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1-recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double-copy mutant showing larger effect than the respective single copy mutants. The TaGW7-centered gene co-expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co-localization of TaGW7 with α- and β-tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7-associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR-Cas9 system with cross-species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.

Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones (Solanum spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the S-locus which is composed of tightly linked genes, S-RNase (S-locus RNase) and multiple SLFs (S-locus F-box proteins), which are expressed in the style and pollen, respectively. Using S-RNase genes known to function in the Solanaceae gametophytic SI mechanism, we identified S-RNase alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the S-locus in potato, we genetically mapped the S-RNase gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the S-RNase gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine S-RNase KO T<SUB>0</SUB> lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T<SUB>1</SUB> progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through S-RNase KO for use in diploid potato breeding approaches.

Cultivation of superior varieties is the key to maintaining the sustainable development of the Litopenaeus vannamei (L.vannamei) industry. CRISPR/Cas9 technology represents a generation of genetic breeding technology based on gene editing. However, the conventional delivery strategy of CRISPR/Cas9 components could not be used due to the particular physiological traits and practical difficulties of L. vannamei embryos. We designed and established polyethylenimine (PEI)-coated nanoparticles with carboxylated SNWTs core to safely deliver CRISPR/Cas9 plasmids into early embryos for target gene editing. The results showed that the transfection efficiency of this strategy was 36%, which was approximately 4-fold higher than the efficiency of the classical lipid transfection method. The transcription factor Pax6, which has notable effects on early embryonic eye development, provides clear phenotypic proof for this strategy. Unnatural base alterations were found in up to 20% of transfected embryos. This study establishes a foundation for the application of CRISPR technology in L. vannamei and provides an innovative approach for large-scale gene function studies in aquaculture.

Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repair (HDR) efficiency causing low knock-in rates. We present a detailed pipeline using HDR donor plasmids containing antibiotic resistance cassettes for rapid selection of gene-edited cells. Our protocol streamlines N- or C-terminal tagging in human cells, enabling HDR donor plasmid preparation in a single cloning step.

The CRISPR/Cas9 system acts as the prokaryotic immune system and has important applications in gene editing. The protein Cas9 is one of its crucial components. The role of Cas9 is to search for specific target sequences on the DNA and cleave them. In this Letter, we introduce a model of facilitated diffusion for Cas9 and fit its parameters to single-molecule experiments. Our model confirms that Cas9 search for targets by sliding, but shows that its sliding length is rather short. We then investigate how Cas9 explores a long stretch of DNA containing randomly placed targets. We solve this problem by mapping it into the theory of Anderson localization in condensed matter physics. Our theoretical approach rationalizes experimental evidences on the distribution of Cas9 molecules along the DNA.

The Gene Ontology (GO) project is the largest resource for cataloguing gene function. The combination of solid conceptual underpinnings and a practical set of features have made the GO a widely adopted resource in the research community and an essential resource for data analysis. In this chapter, we provide a concise primer for all users of the GO. We briefly introduce the structure of the ontology and explain how to interpret annotations associated with the GO.

The Gene Ontology (GO) is a formidable resource but there are several considerations about it that are essential to understand the data and interpret it correctly. The GO is sufficiently simple that it can be used without deep understanding of its structure or how it is developed, which is both a strength and a weakness. In this chapter, we discuss some common misinterpretations of the ontology and the annotations. A better understanding of the pitfalls and the biases in the GO should help users make the most of this very rich resource. We also review some of the misconceptions and misleading assumptions commonly made about GO, including the effect of data incompleteness, the importance of annotation qualifiers, and the transitivity or lack thereof associated with different ontology relations. We also discuss several biases that can confound aggregate analyses such as gene enrichment analyses. For each of these pitfalls and biases, we suggest remedies and best practices.

Transfusion-dependent -thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses -globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients -one with TDT and the other with SCD -received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.) 2] Mutations in HBB that cause TDT 4 result in reduced ( + ) or absent ( 0 ) -globin synthesis and an imbalance between the -like and -like globin (e.g., , , and ) chains of hemoglobin, which causes ineffective erythropoiesis. Sickle hemoglobin is the result of a point mutation in HBB that replaces glutamic acid with valine at amino acid position 6. Polymerization of deoxygenated sickle hemoglobin causes erythrocyte deformation, hemolysis, anemia, painful vaso-occlusive episodes, irreversible end-organ damage, and a reduced life expectancy. reatment options primarily consist of transfusion and iron chelation in patients with TDT 7 and pain management, transfusion, and hydroxyurea in those with SCD. 8 Recently approved therapies, including luspatercept 9 and crizanlizumab, 10 have reduced transfusion requirements in patients with TDT and the incidence of vaso-occlusive episodes in those with SCD, respectively, but neither treatment addresses the underlying cause of the disease nor fully ameliorates disease manifestations. Allogeneic bone marrow transplantation can cure both TDT and

Gene editing stands for the methods to precisely make changes to a specific nucleic acid sequence. With the recent development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, gene editing has become efficient, convenient and programmable, leading to promising translational studies and clinical trials for both genetic and non-genetic diseases. A major concern in the applications of the CRISPR/Cas9 system is about its off-target effects, namely the deposition of unexpected, unwanted, or even adverse alterations to the genome. To date, many methods have been developed to nominate or detect the off-target sites of CRISPR/Cas9, which laid the basis for the successful upgrades of CRISPR/Cas9 derivatives with enhanced precision. In this review, we summarize these technological advancements and discuss about the current challenges in the management of off-target effects for future gene therapy.

Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.