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Found: 26 179
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Direct preparation of Cas9 ribonucleoproteins with an extended 'gRNA-shRNA' construct in Escherichia coli for precise genome manipulation.
Gene perturbation approaches have emerged as powerful tools for elucidating gene function and treating hereditary disorders. Previously, we developed a method for streamlined production of ready-to-use Cas9 ribonucleoproteins (RNPs) in Escherichia coli BL21(DE3). In this study, we present an improved approach by assembling Cas9 RNPs with an extended 'gRNA-shRNA' construct in the RNase III deficient strain HT115(DE3). Transfection of these engineered Cas9 RNPs into mammalian cells enables multidimensional genome manipulation, including simultaneous knockdown and knockout of target genes. Furthermore, the design of shRNA specifically targeting human DNA ligase IV (LIG4) significantly enhances efficiency in homology-directed repair genome editing. Collectively, our findings establish a user-friendly CRISPR/Cas9 RNP tool with immense potential for precise genome editing, gene function analysis, and gene therapy.
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Gene editing as a therapeutic strategy for spinocerebellar ataxia type-3.
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a neurodegenerative disease caused by expanded polyglutamine repeats in exon 10 of the ataxin-3 gene, ATXN3. The accumulation of mutant ATXN3 protein leads to severe clinical manifestations and premature death. Clinically, SCA3 pathology is characterized by progressive ataxia leading to motor incoordination that may affect balance, gait and speech, and neuropathologically by a progressive degeneration of the spinal cord and cerebellum, as well as the cerebral cortex and basal ganglia. Although SCA3 is a rare disease, it is the most common autosomal dominant spinocerebellar ataxia worldwide. Its geographical distribution varies worldwide, with peak prevalence in certain regions of Brazil, Portugal and China. In 1994, the identification of the polyglutamine expansion in the ATXN3 gene made it possible not only to diagnose this pathology but also to dissect the mechanisms leading to cellular degeneration. As a monogenic disease for which only symptomatic treatment is available, the ATXN3 gene represents an attractive therapeutic target for gene editing strategies.
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Editorial: Genome editing applications of CRISPR/Cas9 in metabolic diseases, hormonal system and cancer research.
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Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing.
Transcription factors (TFs) are potent proteins that control gene expression and can thereby drive cell fate decisions. Fluorescent reporters have been broadly knocked into endogenous TF loci to investigate the biological roles of these factors; however, the sensitivity of such analyses in human pluripotent stem cells (hPSCs) is often compromised by low TF expression levels and/or reporter silencing. Complementarily, we report an inducible and quantitative reporter platform based on the Cre-LoxP recombination system that enables robust, quantifiable, and continuous monitoring of live hPSCs and their progeny to investigate the roles of TFs during human development and disease. Stem Cells 2019;37:1556-1566.
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Gene Editing: Practical Application in the Lab.
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Adaptation of high-efficiency CRISPR/Cas9-based multiplex genome editing system in white lupin by using endogenous promoters.
White lupin (Lupinus albus L.) is an important crop with high phosphorus (P) use efficiency; however, technologies for its functional genomic and molecular analyses are limited. Cluster regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been applied to gene editing and function genomics in many crops, but its application in white lupin has not been well documented. Here, we adapted the CRISPR/Cas9-based multiplex genome editing system by using the native U3/U6 and ubiquitin (UBQ) promoters to drive sgRNAs and Cas9. Two target sites (T1 and T2) within the Lalb_Chr05g0223881 gene, encoding a putative trehalase, were selected to validate its efficacy in white lupin based on the Agrobacterium rhizogenes-mediated transformation. We found that the T0 hairy roots were efficiently mutated at T1 and T2 with a frequency of 6.25%-35% and 50%-92.31%, respectively. The mutation types include nucleotide insertion, deletion, substitution, and complicated variant. Simultaneous mutations of the two targets were also observed with a range of 6.25%-35%. The combination of LaU6.6 promoter for sgRNA and LaUBQ12 promoter for Cas9 generated the highest frequency of homozygous/biallelic mutations at 38.46%. In addition, the target-sgRNA sequence also contributes to the editing efficiency of the CRISPR/Cas9 system in white lupin. In conclusion, our results expand the toolbox of the CRISPR/Cas9 system and benefit the basic research in white lupin.
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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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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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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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CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa.
In recent years, the clustered regularly interspaced palindromic repeats-Cas (CRISPR-Cas) technology has become the method of choice for precision genome editing in many organisms due to its simplicity and efficacy. Multiplex genome editing, point mutations, and large genomic modifications are attractive features of the CRISPR-Cas9 system. These applications facilitate both the ease and velocity of genetic manipulations and the discovery of novel functions. In this protocol chapter, we describe the use of a CRISPR-Cas9 system for multiplex integration and deletion modifications, and deletions of large genomic regions by the use of a single guide RNA (sgRNA), and, finally, targeted point mutation modifications in Paenibacillus polymyxa.
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