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Large-scale screening of genes responsible for silique length and seed size in Brassica Napus via pooled CRISPR library.
BACKGROUND: Enhancing rapeseed (Brassica napus, B. napus) yield is critical for ensuring global vegetable oil security. However, yield is heavily influenced by silique development and seed size, the enhancement of which is limited by scarce genetic resources. The CRISPR/Cas9 system has emerged as a powerful tool for constructing genome-wide mutant libraries, even in polyploid crops with complex genomes. RESULTS: The transcriptome-wide association study (TWAS) data, tissue-specific expression profiles data and reported genes were integrated to identify candidate genes regulating silique development and seed size. We constructed a sgRNA library targeting these genes and generated a CRISPR/Cas9 editing mutant library through genetic transformation. Specifically, 6124 sgRNAs were designed for 1739 candidate genes with ≦ 4 orthologues. 681 T0 plants were obtained through genetic transformation, which harbor 453 sgRNAs. Of 408 T0 plants analyzed, 151 (37.00%) exhibited successful gene editing events, targeting 84 candidate genes. Ten homozygous mutant plants were isolated and preliminary phenotypic analysis was performed in mutants targeting the BnaHRDs. The results suggest that mutations in BnaHRD.A03 and BnaHRD.C03 may modulate plant height (PH), main inflorescence length (MIL), silique length (SL), effective silique number per plant (ENS), seed number per silique (SNPS), and thousand-seed weight (TSW). CONCLUSIONS: This study harnessed the CRISPR/Cas9 technology to establish a preliminary library of gene-edited mutants in B. napus, thereby laying a robust foundation for the future screening of candidate genes pertaining to silique development and seed size. Furthermore, this study provides a methodological framework for rapid functional gene discovery in B. napus through CRISPR-based approaches.
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CRISPR Editing in Biological and Biomedical Investigation.
The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) system has sparked advancements in biological and biomedical research. The scientific breakthrough of the development of CRISPR-Cas9 technology has allowed us to recapitulate human diseases by generating animal models of interest ranging from zebrafish to non-human primates. The CRISPR-Cas9 system can also be used to delineate the mechanisms underlying the development of human disorders and to precisely correct disease-causing mutations. Repurposing this technology enables wider applications in transcriptome and epigenome manipulation and holds promise to reach the clinic. In this review, we highlight the latest advances of the CRISPR-Cas9 system in different platforms and discuss the hurdles and challenges this technology is facing. J. Cell. Biochem. 118: 4152-4162, 2017. © 2017 Wiley Periodicals, Inc.
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A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris.
Pichia pastoris has gained much attention as a popular microbial cell factory for the production of recombinant proteins and high-value chemicals from laboratory to industrial scale. However, the lack of convenient and efficient genome engineering tools has impeded further applications of Pichia pastoris towards metabolic engineering and synthetic biology. Here, we report a CRISPR-based toolbox for gene editing and transcriptional regulation in P. pastoris. Based on the previous attempts in P. pastoris, we constructed a CRISPR/Cas9 system for gene editing using the RNA Pol-III-driven expression of sgRNA. The system was used to rapidly recycle the selectable marker with an eliminable episomal plasmid and achieved up to 100% knockout efficiency. Via dCas9 fused with transcriptional repressor (Mix1/RD1152) or activator (VPR), a flexible toolbox for regulation of gene expression was developed. The reporter gene eGFP driven by yeast pGAP or pCYC1 promoter showed strong inhibition (above 70%) and up to ~ 3.5-fold activation. To implement the combinatorial genetic engineering strategy, the CRISPR system contained a single Cas9-VPR protein, and engineered gRNA was introduced in P. pastoris for simultaneous gene activation, repression, and editing (CRISPR-ARE). We demonstrated that CRISPR-ARE was highly efficient for eGFP activation, mCherry repression, and ADE2 disruption, individually or in a combinatorial manner with a stable expression of multiplex sgRNAs. The simple and multifunctional toolkit demonstrated in this study will accelerate the application of P. pastoris in metabolic engineering and synthetic biology. KEY POINTS: • An eliminable CRISPR/Cas9 system yielded a highly efficient knockout of genes. • Simplified CRISPR/dCas9-based tools enabled transcriptional regulation of targeted genes. • CRISPR-ARE system achieved simultaneous gene activation, repression, and editing in P. pastoris.
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The Trend of CRISPR-Based Technologies in COVID-19 Disease: Beyond Genome Editing.
Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art technologies in genomic and proteomic engineering. CRISPR-Cas system is one of the emerging techniques in the field of biotechnology. To date, the CRISPR-Cas system has been extensively applied in gene editing, targeting genomic sequences for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches. With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools such as the CRISPR-Cas system for use in diagnostic emergencies. This system can compete with other existing biotechnological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this article, we review the various platforms of the CRISPR-Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine development.
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Sanguine About Sangamo: An Interview with Edward Lanphier.
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Enhancing tiny millets through genome editing: current status and future prospects.
This study aims to address the critical need for genetic improvement of small millets, which are vital yet underutilized cereal crops cultivated in semi-arid regions of Africa and Asia. Given their high nutritional value and climate resilience, small millets hold significant potential for food security and sustainable agriculture in arid regions. However, traditional breeding methods have proven to be time-consuming and inefficient in enhancing desirable traits. This study highlights the transformative potential of genome editing technologies, particularly the CRISPR/Cas9 system, in accelerating the development of improved small millet varieties. The findings presented in this paper detail recent advancements in using CRISPR/Cas for enhancing resistance to biotic stresses, including bacterial, viral, and fungal pathogens. Additionally, we explore how genome editing can be applied to improve abiotic stress tolerance, addressing challenges such as drought, cold, heat, and herbicides in small millets. We discuss the existing challenges faced by breeders, including issues related to ploidy levels, off-target effects, and limitations in organelle genome modification. The review also suggests potential strategies for overcoming these bottlenecks, aiming to develop stress-resistant super cultivars. Overall, this paper provides an overview of the current state of genome editing research in small millets while identifying future opportunities to enhance key traits for nutrient enrichment and climate resilience, ultimately paving the way for advancements in these crucial crops.
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Innovative approach in malaria research: Harnessing CRISPR-Cas9 for antimalarial drug-resistance studies in Africa.
Malaria remains a major global health challenge, particularly in sub-Saharan Africa, where Plasmodium falciparum is increasingly resistant to frontline antimalarial therapies. CRISPR-Cas9, a groundbreaking genome editing tool, has become a transformative force in biomedical research, offering valuable insights into the genetic mechanisms underlying drug resistance. This review provides a comprehensive overview of the CRISPR-Cas9 system, its origin, evolution, and application in antimalarial drug-resistance research, with particular emphasis on African studies. We examine the role of CRISPR in elucidating resistance pathways, validating molecular markers, and enhancing diagnostic strategies. Additionally, a systematic scoping review highlights the geographic scope and focus of CRISPR-related malaria research conducted across Africa. Significantly low application and adoption were observed. Despite existing technological and regulatory barriers, CRISPR holds strong potential for accelerating antimalarial drug discovery and advancing precision medicine. Its strategic application in malaria-endemic regions could significantly bolster efforts toward disease control and eventual elimination.
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CRISPR-Cas12a Genome Editing at the Whole-Plant Level Using Two Compatible RNA Virus Vectors.
The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.
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CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities.
CRISPR/Cas technology holds promise for the development of therapies to treat inherited diseases. Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder with a variable multisystemic character for which no cure is yet available. Here, we review CRISPR/Cas-mediated approaches that target the unstable (CTG•CAG)n repeat in the DMPK/DM1-AS gene pair, the autosomal dominant mutation that causes DM1. Expansion of the repeat results in a complex constellation of toxicity at the DNA level, an altered transcriptome and a disturbed proteome. To restore cellular homeostasis and ameliorate DM1 disease symptoms, CRISPR/Cas approaches were directed at the causative mutation in the DNA and the RNA. Specifically, the triplet repeat has been excised from the genome by several laboratories via dual CRISPR/Cas9 cleavage, while one group prevented transcription of the (CTG)n repeat through homology-directed insertion of a polyadenylation signal in DMPK. Independently, catalytically deficient Cas9 (dCas9) was recruited to the (CTG)n repeat to block progression of RNA polymerase II and a dCas9-RNase fusion was shown to degrade expanded (CUG)n RNA. We compare these promising developments in DM1 with those in other microsatellite instability diseases. Finally, we look at hurdles that must be taken to make CRISPR/Cas-mediated editing a therapeutic reality in patients.
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An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA.
As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produced on-demand and in high purity, and a fixed Cas9-binding chemically-modified 79-mer, produced cost-effectively on large-scale, a strategy that provides access to site-specific modifications that enhance sgRNA activity and in vivo stability. Click ligation of the two components generates an artificial triazole linkage that is tolerated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well as gene-editing in cells with no unexpected off-target effects.
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