Ontology highlight
ABSTRACT:
PROVIDER: PRJNA748082 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15194300.fastq.gz | Fastqsanger.gz | |||
| SRR15194400.fastq.gz | Fastqsanger.gz | |||
| SRR15194301.fastq.gz | Fastqsanger.gz | |||
| SRR15194401.fastq.gz | Fastqsanger.gz | |||
| SRR15194302.fastq.gz | Fastqsanger.gz |
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Project description:Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing
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Project description:Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows for robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.
2021-09-10 | GSE180773 | GEO
Project description:The type V-I CRISPR-Cas system is becoming increasingly attractive for its potential utility in gene editing. However, natural nucleases often exhibit low efficiency, limiting their application. Here, we utilized structure-guided rational design and combinatorial protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. Accordingly, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene-editing activity in mammalian cells and plants. Cas-SF01 displays comparable or superior editing performance compared to SpCas9 or recently engineered Cas12 nucleases. Further analysis of PAM recognition showed that Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. Additionally, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we demonstrated that Cas-SF01 has robust gene-editing activity in both the monocot plant rice and dicot plant pepper. Our results suggest that Cas-SF01 can serve as a robust gene-editing platform with high efficiency and specificity for future genome editing applications across different organisms.
2023-11-05 | GSE236755 | GEO
Project description:Simple and efficient delivery of CRISPR genome editing systems in primary cells remains a major challenge. Here, we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells. PAGE couples a cell-penetrating Cas protein with a cell-penetrating endosomal escape peptide in a 30-minute incubation that yields up to ~98% editing efficiency in primary human and mouse T cells. PAGE provides a broadly generalizable platform for next generation genome engineering in primary cells. CITATION INFORMATION: Zhang Zhen, Baxter Amy E, Ren Diqiu, Qin Kunhua, Chen Zeyu, Collins Sierra M., Huang Hua, Komar Chad A., Bailer Peter F., Parker Jared B., Blobel Gerd A., Kohli Rahul M., Wherry E. John*, Berger Shelley,*, and Shi Junwei*. Peptide-assisted genome editing permits efficient CRISPR engineering of primary T cells.
2023-03-20 | GSE223805 | GEO
Project description:Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools. Here, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs (PAMs) based on bioinformatics analysis combined with plasmid interference assay in S. spinosa. By reprogramming this endogenous CRISPR-Cas system, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the Restriction-Modification (RM) systems was systematically disarmed. We showed that by disarming one type I RM system and two type II RM systems simultaneously, the transformation efficiency of foreign plasmids was significantly increased. Based on the engineered strain with simultaneous deletion of these three RM genes, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster as well as gene insertion at high efficiency. Collectively, we developed a reliable and high-efficiency genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
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Project description:As a potent and accurate genome-editing tool, CRISPR-Cas9 has been widely used in biomedical research and evaluated as gene therapy in treating human diseases. Although distinct engineered Cas9s, dCas9s and additional endonucleases have been identified, as these bacterial enzymes do not naturally express in mammalian cells, whether and how bacterial Cas9 proteins are regulated by mammalian hosts remains poorly understood. Here, we identified Keap1 as an endogenous E3 ligase that targets Cas9/dCas9/Fanzor1 for ubiquitination and degradation. Cas9-“ETGE” mutants evading Keap1 recognition displayed enhanced gene editing ability in cells. dCas9-“ETGE” mutants displayed extended protein half-life on chromatin, leading to significantly improved CRISPa and CRISPRi efficacy. Cas9 binding to Keap1 also inactivate Keap1 function via competing with Keap1 substrates or binding partners, while engineered Cas9 mutants showed less perturbation. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
2025-01-20 | PXD050935 | Pride
Project description:We employed CRISPR/Cas-mediated genome editing to generate S2 cell lines expressing GFP-tagged dCoREST, dL(3)mbt, dLSD1 and dG9a, respectively. This allowed us to determine the genome-wide binding profiles for these proteins by ChIP-seq using the same antibody (anti-GFP) in each case.
2019-11-04 | E-MTAB-8341 | biostudies-arrayexpress
Project description:The CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1), Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) are orthogonal systems capable of operating simultaneously in zebrafish. We implemented multichannel CRISPR recording using up to three CRISPR systems, and show that LbaCas12a may provide superior information density compared to previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. These results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.
2021-10-25 | GSE186338 | GEO
Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.
2022-03-03 | E-MTAB-11497 | biostudies-arrayexpress