Correction of RNA splicing defect in β654-thalassemia mice using CRISPR/Cas9 gene-editing technology

β654-thalassemia is a prominent Chinese subtype of β-thalassemia, representing 17% of total β-thalassemia cases in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73-nt by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant β-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β654 mice. Gene-edited mice were generated by microinjection of sgRNAs and Cas9 mRNAs into 1-cell embryos of β654 or control mice. 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited-β654 founders and their offspring, were significantly improved compared to the non-edited β654 mice, consistent with the restoration of wild-type β-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and live-born homozygous β654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide a groundwork for the exploration of β654-thalassemia therapy in the future.

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Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3′ Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF/SF2 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

Journal of Virology ◽

10.1128/jvi.77.3.2105-2115.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2105-2115 ◽

Cited By ~ 28

Author(s):

Xuefeng Liu ◽

Akila Mayeda ◽

Mingfang Tao ◽

Zhi-Ming Zheng

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Viral Rna ◽

Splicing Factor ◽

Rna Expression ◽

Bovine Papillomavirus ◽

Phosphatidylinositol 3 Kinase ◽

Selection Of ◽

Phosphatidylinositol 3

ABSTRACT Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5′ splice site alternatively splices to a proximal 3′ splice site (at nucleotide 3225) to express L2 or to a distal 3′ splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3′ splice sites and have been identified as regulating alternative 3′ splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3′ splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3′ splice site while increasing use of the distal 3′ splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3′ splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3′ splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.

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Alternative Splicing of a Novel Glycophorin Allele GPHe(GL) Generates Two Protein Isoforms in the Human Erythrocyte Membrane

Blood ◽

10.1182/blood.v90.1.391.391_391_397 ◽

1997 ◽

Vol 90 (1) ◽

pp. 391-397

Author(s):

Cheng-Han Huang ◽

Olga O. Blumenfeld ◽

Marion E. Reid ◽

Ying Chen ◽

Geoff L. Daniels ◽

...

Keyword(s):

Erythrocyte Membrane ◽

Splice Site ◽

Rna Splicing ◽

Phenotypic Diversity ◽

Full Length ◽

Protein Isoforms ◽

Membrane Domain ◽

Acceptor Splice Site ◽

Variable Expression ◽

Aberrant Rna

The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at −6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.

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Gene Replacement Therapies for Genodermatoses: A Status Quo

Frontiers in Genetics ◽

10.3389/fgene.2021.658295 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ulrich Koller ◽

Johann W. Bauer

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Gene Editing ◽

Gene Replacement ◽

Mechanical Trauma ◽

Wild Type ◽

Number Of Patients ◽

Genes Encoding ◽

Skin Fragility ◽

Recent Developments

Epidermolysis bullosa (EB) is a genodermatosis, characterized by the formation of extended blisters and lesions on the skin and mucous membranes upon minimal mechanical trauma. The disease is caused by mutations in genes encoding proteins that are essential for skin stability. Functional impairment, reduction, or absence of one of these proteins results in skin fragility due to reduced connectivity between dermis and epidermis. Currently, gene therapy represents the only treatment option with the potential to cure this severe blistering skin disease. Two promising forms of gene therapy are potentially feasible for EB: gene replacement and genome editing. While genome editing for genodermatoses remains at the preclinical stage, gene replacement approaches are clinically advanced and have been applied already to a small number of patients with junctional and dystrophic forms of EB. Here, the viral transduction of the “wild-type” transgene into skin stem cells, followed by autologous grafting of corrected epidermal sheets, led to the regeneration of stable skin. Recent developments regarding designer nuclease-based gene editing strategies enable the establishment of alternative options to restore the gene function in genodermatoses. This is particularly true in cases wherein genetic constellation hinders gene therapy-based gene replacement.

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Alternative Splicing of a Novel Glycophorin Allele GPHe(GL) Generates Two Protein Isoforms in the Human Erythrocyte Membrane

Blood ◽

10.1182/blood.v90.1.391 ◽

1997 ◽

Vol 90 (1) ◽

pp. 391-397 ◽

Cited By ~ 13

Author(s):

Cheng-Han Huang ◽

Olga O. Blumenfeld ◽

Marion E. Reid ◽

Ying Chen ◽

Geoff L. Daniels ◽

...

Keyword(s):

Erythrocyte Membrane ◽

Splice Site ◽

Rna Splicing ◽

Phenotypic Diversity ◽

Full Length ◽

Protein Isoforms ◽

Membrane Domain ◽

Acceptor Splice Site ◽

Variable Expression ◽

Aberrant Rna

Abstract The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at −6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.

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Highly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system

10.1101/2021.09.04.458978 ◽

2021 ◽

Author(s):

Hanseop Kim ◽

Wi-jae Lee ◽

Chan Hyoung Kim ◽

Yeounsun Oh ◽

Lee Wha Gwon ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Deoxyribonucleic Acid ◽

Specific Gene ◽

Target Sequence ◽

Sequence Specificity ◽

Wild Type ◽

Dna Cleaving ◽

Target Dna ◽

Near Future

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

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Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression

10.1101/488510 ◽

2018 ◽

Author(s):

Ching-Fu Tu ◽

Chin-kai Chuang ◽

Kai-Hsuan Hsiao ◽

Chien-Hong Chen ◽

Chie-Min Chen ◽

...

Keyword(s):

Gene Editing ◽

Clinical Symptoms ◽

Glucose Solution ◽

Clinical Manifestations ◽

Immunofluorescence Staining ◽

Wild Type ◽

Guide Rnas ◽

Porcine Epidemic Diarrhoea Virus ◽

Diarrhoea Virus ◽

Cas9 Mrna

Porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs; 4 live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I and III) and 3-day-old (in exp. II) KO and wild-type (WT) piglets were inoculated with TCID50 1x103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow’s colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals were euthanized for necropsy, and their intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.

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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

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The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance

Microorganisms ◽

10.3390/microorganisms9020335 ◽

2021 ◽

Vol 9 (2) ◽

pp. 335

Author(s):

Novaria Sari Dewi Panjaitan ◽

Yu-Tze Horng ◽

Chih-Ching Chien ◽

Hung-Chi Yang ◽

Ren-In You ◽

...

Keyword(s):

Survival Rate ◽

Klebsiella Pneumoniae ◽

Laser Scanning ◽

Bacterial Resistance ◽

Capsular Polysaccharide ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Intracellular Bacteria ◽

Wild Type ◽

Macrophage Phagocytosis

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

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First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-021-02103-5 ◽

2021 ◽

Author(s):

Daria Nitarska ◽

Robert Boehm ◽

Thomas Debener ◽

Rares Calin Lucaciu ◽

Heidi Halbwirth

Keyword(s):

Transgenic Plants ◽

Genome Editing ◽

Ornamental Plants ◽

Targeted Mutagenesis ◽

Cloning And Expression ◽

Euphorbia Pulcherrima ◽

Loss Of Function ◽

Wild Type ◽

Promising Tool ◽

Reddish Orange

AbstractThe CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

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