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Characterizing and controlling CRISPR repair outcomes in nondividing human cells

ABSTRACT: Characterizing and controlling CRISPR repair outcomes in nondividing human cells

PROVIDER: PRJNA1299347 | ENA |

REPOSITORIES: ENA

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Characterizing and controlling CRISPR repair outcomes in nondividing human cells

Project description:Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We showed that CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells. Neurons also took far longer to fully resolve this damage, and upregulated non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allowed us to direct DNA repair toward desired editing outcomes: in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

2025-08-31 | GSE304183 | GEO

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

Project description:Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

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Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

Project description:Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

2024-07-30 | GSE272812 | GEO

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

Project description:Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ~1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6,872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: 1) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, 2) deletions are predominantly associated with microhomology, and 3) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model (Lindel) that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.

2019-06-07 | GSE131421 | GEO

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

Project description:Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

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Outcomes of intrahepatic cholangiocarcinoma specific CRISPR screening in vivo

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Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.

2018-06-04 | E-MTAB-6808 | biostudies-arrayexpress

CHARACTERIZING TOXICITY PATHWAYS OF FLUOXETINE TO PREDICT ADVERSE OUTCOMES IN ADULT PIMEPHALES PROMELAS

Project description:CHARACTERIZING TOXICITY PATHWAYS OF FLUOXETINE TO PREDICT ADVERSE OUTCOMES IN ADULT PIMEPHALES PROMELAS

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Outcomes of intrahepatic cholangiocarcinoma specific CRISPR screening in vivo [mouse exome]

Project description:Outcomes of intrahepatic cholangiocarcinoma specific CRISPR screening in vivo [mouse exome]

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Outcomes of intrahepatic cholangiocarcinoma specific CRISPR screening in vivo [RNA-seq]

Project description:Outcomes of intrahepatic cholangiocarcinoma specific CRISPR screening in vivo [RNA-seq]

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