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Spermatophyta

ABSTRACT: Cas9 sequencing for the transgene detection

PROVIDER: PRJDB20204 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	DRR640483_1.fastq.gz	Fastqsanger.gz
	DRR640484_1.fastq.gz	Fastqsanger.gz
	DRR640485_1.fastq.gz	Fastqsanger.gz
	DRR640486_1.fastq.gz	Fastqsanger.gz
	DRR640487_1.fastq.gz	Fastqsanger.gz

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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [sRNA-Seq]

Project description:Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of transcript cleavage by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein / RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation, rather than RNAi. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and editing rates. In addition, transgenes with the engineered protein / RNA-binding module had improved long-term durability of transgene expression, as after four generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.

2025-06-02 | GSE287530 | GEO

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

Project description:RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ~10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide 1.sgRNA1-6 binding sites were identified with ChipSeq by using HA antibody (there are 2 replicates for sgRNA1-3, one sample for sgRNA4-6,one control without sgRNA) 2.PCR products which amplifies " off-target genomic sites" were deep sequenced in the presence of WT Cas9+sgRNA or WT Cas9 alone( unique adaptor was used for each sgRNA and mixed for multiplex run)

2014-05-18 | E-GEOD-55887 | biostudies-arrayexpress

Project description:detection of transgene events by NGS

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Spermatophyta

Project description:Paulownia Leaf Transcription Group

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Project description:Rapid survey of de novo mutations in naturally growing tree species following March 2011 disasters in Fukushima: the effect of low dose rate radiation

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Exome-seq of Jak2VF erythroid progenitors cells transduced with Dnmt3a-Cas9-GFP lentivirus vs. emp-Cas9-GFP controls 17 weeks post transplantation

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2017-12-25 | E-MTAB-6310 | biostudies-arrayexpress

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