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1.
Cell Rep ; 42(1): 111997, 2023 01 31.
Article in English | MEDLINE | ID: mdl-36656716

ABSTRACT

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer.


Subject(s)
Alcohol Dehydrogenase , Focal Adhesion Kinase 1 , Neoplasms , Humans , Aldehyde Oxidoreductases/metabolism , Focal Adhesion Kinase 1/genetics , Neoplasms/genetics , Nitric Oxide/metabolism , Phosphorylation , Protein Processing, Post-Translational , Tumor Microenvironment , Alcohol Dehydrogenase/metabolism
2.
Mol Ther Nucleic Acids ; 24: 403-415, 2021 Jun 04.
Article in English | MEDLINE | ID: mdl-33868784

ABSTRACT

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human MYOD transcription factor. Restoration of DMD expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.

3.
Nucleic Acids Res ; 48(5): e25, 2020 03 18.
Article in English | MEDLINE | ID: mdl-31943080

ABSTRACT

Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.


Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA, Circular/genetics , RNA, Guide, Kinetoplastida/genetics , Alleles , Base Sequence , CRISPR-Associated Protein 9/metabolism , Cell Line, Tumor , DNA, Circular/metabolism , Gene Editing/methods , HEK293 Cells , Haplotypes , Hep G2 Cells , Humans , Plasmids/chemistry , Plasmids/metabolism , RNA, Guide, Kinetoplastida/metabolism
4.
Cell Mol Life Sci ; 76(13): 2633-2645, 2019 Jul.
Article in English | MEDLINE | ID: mdl-30887099

ABSTRACT

The RNA-guided CRISPR-Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.


Subject(s)
Breast/metabolism , CRISPR-Cas Systems , Gene Editing/methods , Genetic Vectors/administration & dosage , Ovarian Neoplasms/genetics , RNA, Guide, Kinetoplastida , Uterine Cervical Neoplasms/genetics , Cells, Cultured , Female , Gene Targeting , Genetic Vectors/genetics , HEK293 Cells , Humans
5.
Nucleic Acids Res ; 46(22): e131, 2018 12 14.
Article in English | MEDLINE | ID: mdl-30551175

ABSTRACT

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.


Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA, Circular/genetics , Gene Editing/methods , Base Sequence , Biosensing Techniques , CRISPR-Associated Protein 9/metabolism , Cell Line , Chromosomes, Human, Pair 18/chemistry , Chromosomes, Human, Pair 18/metabolism , DNA End-Joining Repair , DNA, Circular/metabolism , Fibroblasts , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Genes, Reporter , Genetic Loci , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Genome, Human , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism
6.
Gigascience ; 7(3): 1-19, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29635374

ABSTRACT

Background: Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings: We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Conclusion: Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.


Subject(s)
DNA Methylation/genetics , Genome/genetics , RNA, Guide, Kinetoplastida/genetics , CRISPR-Cas Systems/genetics , CpG Islands/genetics , HEK293 Cells , Humans , Methyltransferases/genetics
7.
FEBS Lett ; 591(13): 1892-1901, 2017 07.
Article in English | MEDLINE | ID: mdl-28580607

ABSTRACT

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) systems have emerged as the method of choice for genome editing, but large variations in on-target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9-mediated gene editing efficiency for 20 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa cells and in human fibroblasts. Furthermore, we investigate the gRNA sequence determinants of CRISPR-Cas9 activity using a surrogate reporter system and find that the efficiency of Cas9-mediated gene editing is dependent on guide sequence secondary structure formation. This knowledge can aid in the further improvement of tools for gRNA design.


Subject(s)
CRISPR-Cas Systems/genetics , Chromatin/genetics , Gene Editing/methods , DNA Cleavage , Genetic Loci/genetics , Genomics , HEK293 Cells , HeLa Cells , Humans , Point Mutation
8.
J Biotechnol ; 247: 42-49, 2017 Apr 10.
Article in English | MEDLINE | ID: mdl-28259533

ABSTRACT

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells.


Subject(s)
Bacterial Proteins/genetics , Endonucleases/genetics , Gene Knockout Techniques/methods , INDEL Mutation , Mammals/genetics , Rec A Recombinases/genetics , Animals , Bacterial Proteins/metabolism , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Repair , Endonucleases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Rec A Recombinases/metabolism , Recombinant Fusion Proteins/metabolism , Streptococcus pyogenes/genetics , Streptococcus pyogenes/metabolism
9.
Cell Mol Life Sci ; 73(13): 2543-63, 2016 07.
Article in English | MEDLINE | ID: mdl-26755436

ABSTRACT

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.


Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Animals , Cells, Cultured , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Fibroblasts/cytology , Fibroblasts/metabolism , Fluorescence , Gene Knockout Techniques/methods , Genes, Reporter , Genetic Vectors/genetics , HEK293 Cells , Homologous Recombination , Humans , MCF-7 Cells , Receptor, IGF Type 1 , Receptors, Somatomedin/genetics , Swine , Transcription Activator-Like Effector Nucleases/genetics , Transcription Activator-Like Effector Nucleases/metabolism
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