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Analysis of off target generated by CRISPR/Cas9 in DM1 mutant haploid embryonic stem cells

ABSTRACT: Analysis of off target generated by CRISPR/Cas9 in DM1 mutant haploid embryonic stem cells

PROVIDER: PRJNA490829 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7829270_1.fastq.gz	Fastqsanger.gz
	SRR7829270_2.fastq.gz	Fastqsanger.gz
	SRR7829271_1.fastq.gz	Fastqsanger.gz
	SRR7829271_2.fastq.gz	Fastqsanger.gz
	SRR7829269_1.fastq.gz	Fastqsanger.gz

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Analysis of off target generated by CRISPR/Cas9 in DM1 mutant haploid embryonic stem cells

Project description:The androgenetic embryonic stem cells we generated carrying several genes mutations could be injected into the oocyte and produce the semi-cloned mice. Those semi-cloned mice could mimic several DM1-associated phenotypes. We try to identify if any off-target exist in those cells inducing by CRISPR/Cas9.

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Alternative splicing and gene expression defects in motorneurons derived from DM1 hiPSCs and MBNL knockouts hiPSCs generated by CRISPR/Cas9.

Project description:Myotonic dystrophy type I (DM1) is the most frequent neuromuscular disease (NMD) in adults and only symptomatic treatments have been proposed to patients. DM1 has long been defined by muscular defects but several studies suggested the involvement of the neuromuscular junction (NMJ). In this aim, our purpose raised the question about the pathological contribution of the motoneurons (MNs) in DM1 physiopathology. By using a micropatterned 96-well plate as a co-culture platform, we generated a functional humanized system in 11 days which associates hiPS-derived MNs and human primary skeletal muscle cells. To focus only on DM1 motoneurons-dependent phenotypes, we generated hybrid systems using wild-type skeletal muscle cells combined with mutated hiPS-derived MNs. In this context, we have identified several alterations affecting synaptic vesicular trafficking, AChR clustering and communication between pre- and post-synaptic compartments. More interestingly, hiPS-derived MNs depleted for MBNL1 and MBNL2 reproduced alterations at the pre- and post-synaptic levels. These experiments suggested that the functional defects associated to MNs can be directly attributed to the MBNL family proteins. These findings may be clinically relevant to open new therapeutic approaches taking into account the alterations affecting DM1 motoneurons.

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Project description:To search for factors regulating paternally imprinted genes (PEGs), we performed a genome-wide loss-of-function CRISPR/Cas9 screen in haploid parthenogenetic ESCs. This by staining a pooled CRISPR library with a PEG10 antibody and next FACS-sorted for cells that presented de-novo PEG10 expression.

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Project description:Alternative splicing and gene expression defects in motorneurons derived from DM1 hiPSCs and MBNL knockouts hiPSCs generated by CRISPR/Cas9.

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Project description:APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine to uracil deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3s can trigger the cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through the homology-directed repair (HDR) of Cas9-generated double-strand breaks . In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks can be further processed to yield mutations mainly involving insertions or deletions (indels). Mechanistically, both APOBEC3-mediated deamination and DNA repair proteins play important roles in the generation of these indels. Correspondingly, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can substantially repress these unwanted mutations in genomic DNA.

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Project description:Shuffling the yeast genome using CRISPR/Cas9-generated DSBs that target the transposable Ty1 elements

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