Genome editing in plants using the compact editor CasΦ
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA852208 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19824900.fastq.gz | Fastqsanger.gz | |||
| SRR19825000.fastq.gz | Fastqsanger.gz | |||
| SRR19825100.fastq.gz | Fastqsanger.gz | |||
| SRR19825200.fastq.gz | Fastqsanger.gz | |||
| SRR19825300.fastq.gz | Fastqsanger.gz |
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Project description:CRISPR-Cas systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wildtype CasΦ enzyme, and yielded more offspring plants with inherited edits. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, flexible PAM and wide range of working temperatures make CasΦ an excellent addition to existing plant genome editing systems.
2022-12-14 | GSE206798 | GEO
Project description:Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.
| S-EPMC9942878 | biostudies-literature
Project description:Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid onset dementia and death. However, elimination of endogenous PrP can halt prion disease progression. Here, we describe CHARM, a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable targeted DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates the endogenous DNA methyltransferases, thereby reducing transgene size and bystander effects. When delivered to the mouse brain by an adeno-associated viral (AAV) vector, PRNP-targeted CHARM ablates PrP expression across the brain. We temporally limit editor expression by implementing a kinetically-tuned self-silencing approach. CHARM represents a broadly applicable strategy to programmably prevent expression of pathogenic proteins, including those implicated in other neurodegenerative diseases.
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Project description:N6-methyladenonsine (m6A) is the most prevalent modification on mRNA and plays critical roles in mRNA processing and metabolism. However, how the m6A modification on individual gene of interest regulates gene function and the links to phenotypic outcome in plants are mostly unknown. Here, we described the construction and characterization of programmable m6A editing tools by fusing the m6A writer, core catalytical domain of MTA and MTB complex, and eraser, ALKBH5, to catalytically dead Cas13a (dCas13a), respectively, for targeting methylation and demethylation of specific mRNA. We demonstrated that our m6A editors could efficiently and specifically add and remove the m6A modification on specific RNA transcript in both Nicotiana benthamiana and Arabidopsis. Moreover, targeting SHORT-ROOT (SHR) transcript with methylation editor could significantly increase its m6A levels with limited off-target effects and enhance its expression, giving rise to induced plant growth with enlarged leaf size and root, increased plant height, plant biomass and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study m6A modification of specific genes in plants, and might also have great potential applications for crop improvement in future
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Project description:Purpose: The purpose of this experiment is to expand the repertoire of C. elegans edited transcripts and identify the roles of ADR-1 as indirect regulator of editing and ADR-2 as the only active deaminase in vivo. Methods: Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline. Results: Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3’ UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2; and mutations within its dsRNA binding domains abolished both binding and editing regulation. Conclusions: ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo and raises the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing by deaminase-independent mechanisms. Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline.
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Project description:Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.
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