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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.

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.

Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D. officinale. These efficient research tools will not only help create novel D. officinale varieties, but will also facilitate the molecular genetic investigation of orchid biology.

As the most important genetic factor for melanophore differentiation and melanogenesis, mitf has been studied for over 30 years. The phenotypes of mitf mutants have also received continuous attention for nearly a century. Due to the third round of genome duplication, teleosts have two mitf genes. Mutation analysis demonstrates that mitfa plays an important role in zebrafish pigmentation. However, there have been no functional studies on body color regulation by mitfb or studies of mitfa;mitfb double mutants. In the present study, we mutated both mitf genes in tilapia using CRISPR/Cas9. Disruption of mitfa resulted in light yellow body color with weak gray vertical bars due to significantly reduced numbers of melanophores and increased sizes of xanthophores, while disruption of mitfb led to slight hypo-pigmentation. Double mutation of mitfa and mitfb resulted in dramatic hypo-pigmentation in trunk and fins due to loss of additional melanophores and increase in the number of iridophores compared to the single mutants, but had no influence on retinal pigment epithelium (RPE) pigmentation. The mitfa<SUP loc="post">−/−</SUP>;mitfb<SUP loc="post">−/−</SUP> mutants were yellow with black spots at early juvenile stage (60 dpf), yellow-reddish at late juvenile stage (120 dpf) and red and yellow (including iris) at adult stage (180 dpf), due to increased erythrophores and enlarged xanthophores. Our results demonstrated that both mitf genes are important for body color formation in tilapia, but mitfa plays a more important role than mitfb. Additionally, by comparing the phenotypes of the mitfa<SUP loc="post">−/−</SUP>;mitfb<SUP loc="post">−/−</SUP>, pmela<SUP loc="post">−/−</SUP>;pmelb<SUP loc="post">−/−</SUP> and hps4<SUP loc="post">−/−</SUP> mutants, we found that disruption of genes with different functions in different aspects of melanogenesis could lead to different body colors in tilapia. Mitf is the most important gene for melanophore differentiation and is probably also critical for differentiation of erythrophores, xanthophores and iridophores. To our knowledge, this is the first report showing that both mitf genes are involved in body color formation by loss of function study, and the first report showing that erythrophore numbers and xanthophore sizes were affected by mitf. Our research not only serves as a model for studying mitf function in tilapia and the closely related cichlids, but also provides new strategies for breeding red and yellow tilapia for 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.

Summ a r yTransfusion-dependent β-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 γ-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.govnumbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.)T ransfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) are the most common monogenic diseases worldwide, with an annual diagnosis in approximately 60,000 patients with TDT and 300,000 patients with SCD.[1][2][3] Both diseases are caused by mutations in the hemoglobin β subunit gene (HBB).Mutations in HBB that cause TDT 4 result in reduced (β + ) or absent (β 0 ) β-globin synthesis and an imbalance between the α-like and β-like globin (e.g., β, γ, and δ) chains of hemoglobin, which causes ineffective erythropoiesis.5,6 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.5 Treatment 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 CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia

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.