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1.
Sci Rep ; 12(1): 12193, 2022 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842430

RESUMO

CRISPR-Cas12a systems are becoming an attractive genome editing tool for cell engineering due to their broader editing capabilities compared to CRISPR-Cas9 counterparts. As opposed to Cas9, the Cas12a endonucleases are characterized by a lack of trans-activating crRNA (tracrRNA), which reduces the complexity of the editing system and simultaneously makes CRISPR RNA (crRNA) engineering a promising approach toward further improving and modulating editing activity of the CRISPR-Cas12a systems. Here, we design and validate sixteen types of structurally engineered Cas12a crRNAs targeting various immunologically relevant loci in-vitro and in-cellulo. We show that all our structural modifications in the loop region, ranging from engineered breaks (STAR-crRNAs) to large gaps (Gap-crRNAs), as well as nucleotide substitutions, enable gene-cutting in the presence of various Cas12a nucleases. Moreover, we observe similar insertion rates of short HDR templates using the engineered crRNAs compared to the wild-type crRNAs, further demonstrating that the introduced modifications in the loop region led to comparable genome editing efficiencies. In conclusion, we show that Cas12a nucleases can broadly utilize structurally engineered crRNAs with breaks or gaps in the otherwise highly-conserved loop region, which could further facilitate a wide range of genome editing applications.


Assuntos
Sistemas CRISPR-Cas , RNA Guia , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes , RNA Guia/genética , RNA Guia/metabolismo
2.
Sci Rep ; 12(1): 10036, 2022 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-35710827

RESUMO

Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.


Assuntos
Síndromes de Usher , Animais , Sistemas CRISPR-Cas , Endonucleases/genética , Edição de Genes , Humanos , Macaca mulatta/genética , Macaca mulatta/metabolismo , RNA Guia/metabolismo , RNA Mensageiro , Síndromes de Usher/genética
3.
CRISPR J ; 5(3): 422-434, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35686982

RESUMO

Knockout mice for human disease-causing genes provide valuable models in which new therapeutic approaches can be tested. Electroporation of genome editing tools into zygotes, in vitro or within oviducts, allows for the generation of targeted mutations in a shorter time. We have generated mouse models deficient in genes involved in metabolic rare diseases (Primary Hyperoxaluria Type 1 Pyruvate Kinase Deficiency) or in a tumor suppressor gene (Rasa1). Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein. In vitro or in vivo ribonucleoprotein (RNP) electroporation rendered more than 90% and 30% edited newborn animals, respectively. Mice lines with edited alleles were established and disease hallmarks have been verified in the three models that showed a high consistency of results and validating RNP electroporation into zygotes as an efficient technique for disease modeling without the need to outsource to external facilities.


Assuntos
Edição de Genes , Zigoto , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Camundongos , Camundongos Knockout , RNA Guia/genética , RNA Guia/metabolismo , Ribonucleoproteínas/genética , Zigoto/metabolismo
4.
Hum Reprod ; 37(8): 1760-1773, 2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35700449

RESUMO

STUDY QUESTION: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse? SUMMARY ANSWER: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner. WHAT IS KNOWN ALREADY: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet. STUDY DESIGN, SIZE, DURATION: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29). PARTICIPANTS/MATERIALS, SETTING, METHODS: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis. MAIN RESULTS AND THE ROLE OF CHANCE: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , RNA Guia , Blastocisto/metabolismo , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/fisiologia , Feminino , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Humanos , Masculino , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Gravidez , RNA Guia/metabolismo , Sêmen/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Anal Chim Acta ; 1217: 340009, 2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35690427

RESUMO

The clinical methods to detect RNA viruses and disease-related RNAs suffer from time-consuming processes, high false-positive rates, or limited sensitivity. Here, we propose a strategy for rapid RNA detection through intra-enzyme chain replacement-mediated Cas13a cascade cyclic reaction without target amplification. A hairpin RNA mediator (a cleavage substrate for target-activated Cas13a) and a guiding RNA recognized by the cleavage product through intra-enzyme chain replacement were designed and optimized. Upon the recognition and binding of the target RNA to the Cas13a/CrRNA complex, Cas13a is initially activated to cleave the mediator, and the cleavage products recognize the corresponding Cas13a/CrRNA complex by intra-enzyme chain replacement and initiate the circular cascade of Cas13a cleavage and activation. The accumulated active Cas13a cleaves fluorescent reporter probe for achieving target RNA detection. This "mix & read" RNA detection at room temperature was performed in total 30 min. Using miRNA-21 as the target, the changes in fluorescence intensity were linearly correlated to the concentrations from 10 fM to 50 pM with the detection limit of 75 aM, while no significant changes in fluorescence intensity were detected for non-targets. This method applied to the clinical sputum respiratory syncytial virus-positive samples gave results consistent with those from the clinical fluorescence immunoassay. Thus, intra-enzyme chain replacement-promoted Cas13a cascade cyclic reaction for detection of RNA viruses in the "mix & read" mode at room temperature is rapid, simple, convenient, and efficient for RNA detection and can be adapted to point-of-care testing for high throughput screening of RNA virus infections.


Assuntos
Sistemas CRISPR-Cas , RNA Guia , RNA Guia/metabolismo , RNA Viral/genética
6.
Methods Mol Biol ; 2518: 203-215, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35666447

RESUMO

CRISPR-Cas regulators have provided an excellent toolbox to control gene expression due to the versatility of its components and the easy programming of the single guide RNA (sgRNA) to target DNA sequences. Included in this are CRISPR activation (CRISPRa) systems. These systems allow users to activate transcription of a target gene through the localization of transcription activation domains (ADs) near promoter elements, which in turn recruit RNA polymerase (RNAP) to turn on transcription. A variety of different CRISPRa systems have been described that vary in AD type, recruitment strategies, and CRISPR-Cas systems. Recently, a highly modular CRISPRa system was described that allows for facile exchange of ADs and CRISPR-Cas components. This allows for the creation of CRISPRa systems with unique properties, for example, ability to activate from specific positions upstream of a gene of interest. Here, we describe a protocol for designing, characterizing, and applying the modular CRISPRa system for gene activation in E. coli. We first focus on how to identify activating sites upstream of promoters and the cloning of the targeting sgRNA. We then describe how to perform a fluorescence experiment to evaluate activation of a single target site. Finally, we explain how to adapt the system to expand the target range and how to characterize the activation pattern obtained from different CRISPRa designs.


Assuntos
Escherichia coli , RNA Guia , Bactérias/genética , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Ativação Transcricional
7.
Methods Mol Biol ; 2518: 237-251, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35666449

RESUMO

Epigenome editing has become more precise and effective by coupling epigenetic effectors to the dCas9 protein and targeting regulatory regions such as promoters and enhancers. Here, we describe a basic methodology for performing an epigenome editing experiment, starting from gRNA design and cloning to transiently transfecting the gRNA plasmid and the CRISPR/dCas9-based epigenetic effector and finalizing with chromatin immunoprecipitation (ChIP) to validate changes in epigenetic state at a targeted genomic region.


Assuntos
Sistemas CRISPR-Cas , RNA Guia , Sistemas CRISPR-Cas/genética , Epigenoma , Epigenômica , Edição de Genes/métodos , RNA Guia/genética , RNA Guia/metabolismo
8.
Org Biomol Chem ; 20(26): 5245-5248, 2022 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-35726625

RESUMO

CRISPR-Cas9-mediated DNA editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage by the Cas endonuclease. Because natural gRNA is susceptible to intracellular degradation, it is desirable to chemically protect it for efficient editing. Using 4'-thioribonucleoside 5'-triphosphates and T7 transcription, we have prepared 4'-thio-modified gRNAs that guide Cas9-mediated DNA cleavage. This approach is a simple way to obtain chemically modified RNA suitable for CRISPR-Cas9 DNA editing.


Assuntos
Sistemas CRISPR-Cas , Clivagem do DNA , RNA , RNA Guia/genética , RNA Guia/metabolismo
9.
J Biol Chem ; 298(7): 102085, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35636511

RESUMO

Inhibition of gene expression in Caenorhabditis elegans, a versatile model organism for studying the genetics of development and aging, is achievable by feeding nematodes with bacteria expressing specific dsRNAs. Overexpression of hypoxia-inducible factor 1 (hif-1) or heat-shock factor 1 (hsf-1) by conventional transgenesis has previously been shown to promote nematodal longevity. However, it is unclear whether other methods of gene overexpression are feasible, particularly with the advent of CRISPR-based techniques. Here, we show that feeding C. elegans engineered to stably express a Cas9-derived synthetic transcription factor with bacteria expressing promoter-specific single guide RNAs (sgRNAs) also allows activation of gene expression. We demonstrate that CRISPR activation via ingested sgRNAs specific for the respective promoter regions of hif-1 or hsf-1 increases gene expression and extends lifespan of C. elegans. Furthermore, and as an in silico resource for future studies aiming to use CRISPR activation in C. elegans, we provide predicted promoter-specific sgRNA target sequences for >13,000 C. elegans genes with experimentally defined transcription start sites. We anticipate that the approach and components described herein will help to facilitate genome-wide gene overexpression studies, for example, to identify modulators of aging or other phenotypes of interest, by enabling induction of transcription by feeding of sgRNA-expressing bacteria to nematodes.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ingestão de Alimentos , Longevidade/genética , RNA Guia/metabolismo
10.
J Agric Food Chem ; 70(23): 7240-7247, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35578739

RESUMO

Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum (F. graminearum) infection, reduces crop yield and contaminates grain with mycotoxins. We report a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based nucleic acid assay for an early and rapid diagnosis of wheat FHB. Guide RNA (gRNA) was screened for highly specific recognition of polymerase chain reaction (PCR) amplicon of the internal transcribed spacer (ITS) region and the transcription elongation factor 1α (EF1α) of F. graminearum. The trans-activation of Cas12a protein cleaves the single-stranded DNA probes with the terminal fluorophore and quencher groups, thus allowing us to report the presence of ITS and EF1α of F. graminearum. Owing to the dual recognition process through PCR primers and gRNA hybridization, the approach realized specific discrimination of F. graminearum from other pathogenic fungi. It also allowed us to detect as low as 1 fg/µL total DNA from F. graminearum, which is sufficient to diagnose a 4 day F. graminearum infection. CRISPR-Cas12a-based nucleic acid assay promises the molecular diagnosis of crop diseases and broadens the application of CRISPR tools.


Assuntos
Fusarium , Micoses , Sistemas CRISPR-Cas , Fusarium/metabolismo , Micoses/genética , Doenças das Plantas/microbiologia , RNA Guia/metabolismo , Triticum/genética , Triticum/microbiologia
11.
Chem Commun (Camb) ; 58(42): 6215-6218, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35507371

RESUMO

To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5' cap and 3' poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.


Assuntos
MicroRNAs , Animais , Expressão Gênica , Regulação da Expressão Gênica , Mamíferos/genética , Mamíferos/metabolismo , MicroRNAs/genética , RNA Guia/metabolismo , RNA Mensageiro
12.
Methods Mol Biol ; 2429: 281-306, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507169

RESUMO

Targeted genome editing in hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 can potentially provide a permanent cure for hematologic diseases. However, the utility of CRISPR/Cas9 systems for therapeutic genome editing can be compromised by their off-target effects. In this chapter, we outline the procedures for CRISPR/Cas9 off-target identification and validation in HSPCs. This method is broadly applicable to diverse CRISPR/Cas9 systems and cell types. Using this protocol, researchers can perform computational prediction and experimental identification of potential off-target sites followed by off-target activity quantification by next-generation sequencing.


Assuntos
Sistemas CRISPR-Cas , RNA Guia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , RNA Guia/genética , RNA Guia/metabolismo
13.
Prog Biophys Mol Biol ; 172: 60-76, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35577099

RESUMO

CRISPR/Cas system, a newly but extensively investigated genome-editing method, harbors practical solutions for various genetic problems. It relies on short guide RNAs (gRNAs) to recruit the Cas9 protein, a DNA cleaving enzyme, to its genomic target DNAs. The Cas9 enzyme exhibits some unique properties, like the ability to differentiate self vs. non-self - DNA strands using the base-pairing potential of crRNA, i.e., only CRISPR DNA is entirely complementary to the CRISPR repeat sequences at the crRNA whereas the presence of mismatches in the upstream region of the spacer permit CRISPR interference which is inhibited in case of CRISPR-DNA, allosteric regulation in its domains, and domain reorientation on sgRNA binding. Several groups have contributed their efforts in understanding the functioning of the CRISPR/Cas system, but even then, there is a lot more to explore in this area. The structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape is still ambiguous. A better understanding of the underlying energetics of the CRISPR/Cas9 system should reveal critical parameters to design better CRISPR/Cas9s.


Assuntos
Proteína 9 Associada à CRISPR , RNA Guia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , DNA/química , DNA/genética , Edição de Genes , RNA Guia/química , RNA Guia/genética , RNA Guia/metabolismo
14.
Nat Commun ; 13(1): 2351, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35534455

RESUMO

Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.


Assuntos
Edição de Genes , RNA Guia , Animais , Sistemas CRISPR-Cas/genética , Reparo do DNA por Junção de Extremidades , Edição de Genes/métodos , Mamíferos/genética , RNA Guia/genética , RNA Guia/metabolismo , Reparo de DNA por Recombinação
15.
Nat Commun ; 13(1): 2833, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35595757

RESUMO

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Células HEK293 , Humanos , RNA/metabolismo , RNA Guia/genética , RNA Guia/metabolismo
16.
ACS Synth Biol ; 11(5): 1958-1970, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35500195

RESUMO

Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce ß-carotene. After seven rounds of iterative evolution over two months, a ß-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in ß-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.


Assuntos
Sistemas CRISPR-Cas , Saccharomyces cerevisiae , Sistemas CRISPR-Cas/genética , Edição de Genes , Engenharia Genética , Mutagênese , RNA Guia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta Caroteno/metabolismo
17.
Methods Mol Biol ; 2489: 173-200, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524051

RESUMO

The CRISPR/Cas system, which has been widely applied to organisms ranging from microbes to animals, is currently being adapted for use in Streptomyces bacteria. In this case, it is notably applied to rationally modify the biosynthetic pathways giving rise to the polyketide natural products, which are heavily exploited in the medical and agricultural arenas. Our aim here is to provide the potential user with a practical guide to exploit this approach for manipulating polyketide biosynthesis, by treating key experimental aspects including vector choice, design of the basic engineering components, and trouble-shooting.


Assuntos
Policetídeos , Streptomyces , Animais , Vias Biossintéticas/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Policetídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Streptomyces/genética , Streptomyces/metabolismo
18.
Nat Protoc ; 17(5): 1332-1358, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35388178

RESUMO

The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Sistemas CRISPR-Cas/genética , Cromossomos , Edição de Genes/métodos , Mutação , RNA Guia/genética , RNA Guia/metabolismo
19.
Bioconjug Chem ; 33(5): 858-868, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35436106

RESUMO

Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Alcinos , Azidas/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia/genética , RNA Guia/metabolismo
20.
Commun Biol ; 5(1): 325, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35388146

RESUMO

CRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.


Assuntos
Sistemas CRISPR-Cas , Clivagem do DNA , DNA/genética , Conformação de Ácido Nucleico , RNA Guia/genética , RNA Guia/metabolismo
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