Project description:This project aims to compare gRNA design algorithms and single- versus dual-targeting using a benchmark CRISPR-knockout library composed of gRNAs targeting essential and non-essential genes.
Project description:This project aims to compare gRNA design algorithms using a benchmark CRISPR-knockout library composed of gRNAs targeting essential and non-essential genes.
Project description:This project also aims to compare gRNA design algorithms using a benchmark CRISPR-knockout library composed of gRNAs targeting essential and non-essential genes.
Project description: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.
Project description:We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.
Project description:Immortalised HaCaT keratinocytes were transduced with Cas9 and the CRISPR-KO v1.1 genome-wide gRNA library. The gRNA library was prepared from genomic DNA isolated 14 days post library transduction. gRNA representation will be compared to the original CRISPR-KO v1.1 library to reveal genes essential for HaCaT survival and growth.