Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5’NGG3’ protospacer adjacent motifs (PAM). Examination of dmCas9 binding sites using two Trp53 targeting sgRNAs in Arf -/- MEF cell line (mouse).
Project description:This study demonstrates the applicability of an in vivo CRISPR/Cas9 genome-wide screen in the BALB/c-Trp53+/– mouse model of breast cancer to identify novel tumor suppressor genes involved in mammary tumorigenesis. Preneoplastic mammary organoids were established from using sorted basal cells isolated from BALB/c-Trp53+/– mice. CRISPR/Cas9 screening was used to target Trp53, Axin1 and Prkar1a. To explore the potential molecular basis of the morphological differences, RNA-seq analysis was used to compare the Axin1/Trp53 and Prkar1a/Trp53-edited organoids and to organoids targeted by Trp53-only guides.
