Predictable and precise template-free CRISPR editing of pathogenic variants

Shen, Max W.; Arbab, Mandana; Hsu, Jonathan Y.; Worstell, Daniel; Culbertson, Sannie J.; Krabbe, Olga; Cassa, Christopher A.; Liu, David R.; Gifford, David K.; Sherwood, Richard I.

doi:10.1038/s41586-018-0686-x

Cited by 462 publications

(587 citation statements)

References 30 publications

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“…1E presents examples of the mutated alleles. This mutational landscape resembled profiles reported for chromosomal repair of CRISPR/Cas9 double stranded breaks by NHEJ in other mammalian cells (25). GenBank at NCBI assigned These sequence reads from amplicon-NGS libraries from the mutated PGRN locus of the DhuPGRN-H69 lines have been assigned the SRA accessions SRR9735031-34, BioProject ID PRJNA556235, and BioSample accession SAMN12344265.…”

Section: Progranulin Mutation In Cholangiocytes By Crispr Knockoutsupporting

confidence: 57%

Liver fluke granulin promotes exosome-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

Arunsan

Chaidee

et al. 2019

Preprint

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Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with fish-borne liver flukes is a major risk factor for cholangiocarcinoma (CCA). The parasite secretes a growth factor termed liver fluke granulin (Ov-GRN-1), a homologue of the human progranulin (huPGRN), which contributes significantly to biliary tract fibrosis and morbidity during infection. Here, exosome-mediated transfer of mRNAs from the human cholangiocyte cell line (H69) was investigated following exposure to Ov-GRN-1, to naïve recipient H69 cells. To minimize the influence of endogenous huPGRN, the gene encoding huPGRN was inactivated using CRISPR/Cas9-based gene knock-out. Several huPGRN-depleted cell lines, termed DhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of huPGRN transcripts and protein, both in gene-edited cells and within exosomes released by these cells. Profiles of exosomal RNAs (exRNA) from DhuPGRN-H69 for CCAassociated characteristics revealed a paucity of transcripts for estrogen-and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific mRNAs including MAPK/AKT pathway members were induced by exposure to Ov-GRN-1. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 exposed to Ov-GRN-1. Of these, CCA-associated exRNAs modified the CCA microenvironment in naïve recipient cells co-cultured with exosomes from DhuPGRN-H69 exposed to Ov-GRN-1, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells comparing with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCAconducive disposition.

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Section: Progranulin Mutation In Cholangiocytes By Crispr Knockoutsupporting

confidence: 57%

Liver fluke granulin promotes exosome-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

Arunsan

Chaidee

et al. 2019

Preprint

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“…1a. Recently, several laboratories showed that CRISPR/Cas9 indel patterns and in-frame mutational probabilities are predictable from sgRNA-targeted DNA sequences 9,10 . Taking advantage of these findings, we first examined if the model is applicable to Munoz data.…”

Section: Essential Protein Domains Are Hyper-sensitive To Crispr/cas9mentioning

confidence: 99%

De novo Identification of Essential Protein Domains from CRISPR/Cas9 Tiling-sgRNA Knockout Screens

Zhang

Villarreal

et al. 2019

Preprint

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High-throughput CRISPR/Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. To facilitate de novo identification of essential protein domains from such screens, we developed ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refers to the protein regions that are associated with strong sgRNA dropout effect in the screens. We used ProTiler to analyze a published CRISPR tiling screen dataset, and identified 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlapped with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also revealed unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which we validated experimentally. Surprisingly, the CKHS regions were negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR/Cas9 mediated amino acids loss.

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“…HPS pathogenesis and therapeutic options continue to be investigated, including through the use of organoids (Korogi et al, 2019; Strikoudis et al, 2019) and explorations of gene therapy (Ikawa et al, 2015; Iyer et al, 2019; Shen et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Hermansky–Pudlak syndrome: Mutation update

et al. 2020

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Hermansky-Pudlak syndrome (HPS) is a group of 10 autosomal recessive multisystem disorders, each defined by the deficiency of a specific gene. HPS-associated genes encode components of four ubiquitously expressed protein complexes: Adaptor protein-3 (AP-3) and biogenesis of lysosome-related organelles complex-1 (BLOC-1) through -3. All individuals with HPS exhibit albinism and a bleeding diathesis; additional features occur depending on the defective protein complex. Pulmonary fibrosis is associated with AP-3 and BLOC-3 deficiency, immunodeficiency with AP-3 defects, and gastrointestinal symptoms are more prevalent and severe in BLOC-3 deficiency. Therefore, identification of the HPS subtype is valuable for prognosis, clinical management, and treatment options. The prevalence of HPS is estimated at 1-9 per 1,000,000. Here we summarize 264 reported and novel variants in 10 HPS genes and estimate that~333 Puerto Rican HPS subjects and~385 with other ethnicities are reported to date. We provide pathogenicity predictions for missense and splice site variants and list variants with high minor allele frequencies. Current cellular and clinical aspects of HPS are also summarized. This review can serve as a manifest for molecular diagnostics and genetic counseling aspects of HPS.

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Predictable and precise template-free CRISPR editing of pathogenic variants

Cited by 462 publications

References 30 publications

Liver fluke granulin promotes exosome-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

Liver fluke granulin promotes exosome-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

De novo Identification of Essential Protein Domains from CRISPR/Cas9 Tiling-sgRNA Knockout Screens

Hermansky–Pudlak syndrome: Mutation update

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