Indel profiling of primary hepatocytes by deep sequencing
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE209543 | GEO | 2022/07/29
REPOSITORIES: GEO
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Project description:Prime editor (PE) is a precise genome-editing tool capable of all possible base conversions, as well as insertions and deletions without DSBs or donor DNA. The efficient delivery of PE in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated a series of efficient split site when delivering PE by dual-AAV. Additionally, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. To test the editing efficiency in vivo, a novel dual-AAV split-ePE3 was packaged in AAV9 and delivered via tail vein injection in mice, achieving 24.4% precise genome editing 3 weeks post-injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality.
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Project description:Prime editor (PE) is a precise genome-editing tool capable of all possible base conversions, as well as insertions and deletions without DSBs or donor DNA. The efficient delivery of PE in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated a series of efficient split site when delivering PE by dual-AAV. Additionally, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. To test the editing efficiency in vivo, a novel dual-AAV split-ePE3 was packaged in AAV9 and delivered via tail vein injection in mice, achieving 24.4% precise genome editing 3 weeks post-injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality.
2024-12-01 | GSE270813 | GEO
Project description:We analyzed the expression of RNAs and microRNAs in hepatocytes isolated from ApcDeltaHep livers, either ROSA26 or deleted for the b-catenin binding site upstream of the DLK1/DIO3 locus by Crispr/Cas9 (DLK1DeltaWRE). We thus explored if editing of this regulatory site impaired the expression of the entire locus and which signaling pathways were modulated.
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Project description:The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ~35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
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Project description:Skeletal muscle satellite cells (SCs) are muscle stem cells responsible for muscle development and injury induced muscle regeneration. The pace of SC related study, however, is constrained partially by the technological limitations in generating genetically modified mice. Although the ease of use of CRISPR-Cas9 in genome manipulation has been documented in many cell lines and various species, its application in endogenous SCs remains elusive. In this study, we generated muscle-specific Cas9-expressing mice and achieved robust in vivo genome editing in juvenile SCs at the postnatal stage through AAV9 mediated short guide RNAs (sgRNAs) delivery. We also found adult quiescent SCs are reluctant to CRISPR/Cas9 editing despite efficient AAV9 transduction. To edit juvenile SCs in vivo, as a proof-of-concept, we delivered sgRNAs targeting MyoD, a key gene critical for muscle physiology and showed an efficient editing at MyoD locus, resulting in accumulation of SCs and defects in SCs differentiation which resembled the phenotypes reported in MyoD knockout mice. Further application of this system on potential key transcription factors (TFs) involved in SC fate transition, Myc, Bcl6 and Pknox2, unveiled their distinct functions in the early stage of SC activation and injury induced muscle regeneration. In addition, we revealed that Myc orchestrated SCs activation through impinging on 3D chromatin architecture. Altogether we established a robust muscle restricted CRISPR/Cas9-based gene editing platform in endogenous SCs and elucidated the functionality of key factors governing SC activities.
2021-10-27 | GSE134528 | GEO
Project description:Skeletal muscle satellite cells (SCs) are muscle stem cells responsible for muscle development and injury induced muscle regeneration. The pace of SC related study, however, is constrained partially by the technological limitations in generating genetically modified mice. Although the ease of use of CRISPR-Cas9 in genome manipulation has been documented in many cell lines and various species, its application in endogenous SCs remains elusive. In this study, we generated muscle-specific Cas9-expressing mice and achieved robust in vivo genome editing in juvenile SCs at the postnatal stage through AAV9 mediated short guide RNAs (sgRNAs) delivery. We also found adult quiescent SCs are reluctant to CRISPR/Cas9 editing despite efficient AAV9 transduction. To edit juvenile SCs in vivo, as a proof-of-concept, we delivered sgRNAs targeting MyoD, a key gene critical for muscle physiology and showed an efficient editing at MyoD locus, resulting in accumulation of SCs and defects in SCs differentiation which resembled the phenotypes reported in MyoD knockout mice. Further application of this system on potential key transcription factors (TFs) involved in SC fate transition, Myc, Bcl6 and Pknox2, unveiled their distinct functions in the early stage of SC activation and injury induced muscle regeneration. In addition, we revealed that Myc orchestrated SCs activation through impinging on 3D chromatin architecture. Altogether we established a robust muscle restricted CRISPR/Cas9-based gene editing platform in endogenous SCs and elucidated the functionality of key factors governing SC activities.
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