Dataset Information

Whole genome CRISPR screens in SC-islet grafts under allogeneic rejection (Hu. Mice) [CRISPR]

ABSTRACT: A whole genome screen using a CRISPR lentivirus library (Doench et al., 2016) was performed. The Brunello CRISPR library consists of a pool of 76,441 human targeting guide RNAs (gRNA) and 1000 control gRNAs [non-targeting (NT) or intergenic], in a lentiviral vector that expresses Cas9. The pooled library targets 19,114 human genes, most of them by four gRNA per gene. To avoid multiple different gRNA in cells and a nonspecific effect on the screen results (Doench, 2018), a low infection lentivirus titer (multiplicity of infection that is <1) was used. Library transduced cells (LT SC-islets) were allowed at least 10 days for CRISPR editing, before transplantation to the NSG-MHCnull mouse model, where PBMCs were injected to half of the cohort (hPi mice: n=6, control mice: n=6) (Figures 3A). hPi mice retained levels of circulating T cells throughout the experiment (Figures S3A and S3B). Graft function and subsequent failure due to human PBMC injection was assessed (Figures 3B and S3C). When hPi graft failure was confirmed, 10 weeks after PBMC injection (Figure 3B), both control and hPi grafts were recovered from kidney sites, genomic DNA (gDNA) was extracted, and gRNA regions were amplified by PCR for Illumina sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE200085 | GEO | 2022/09/01

REPOSITORIES: GEO

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Whole genome CRISPR screens in SC-islet grafts under allogeneic rejection (Hu. Mice) [CRISPR]

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