Dataset Information

Identification of genes involved in class switch recombination by genome-wide CRISPR/Cas9 knock-out screen

ABSTRACT: This screen aims to identify genes involved in class switch recombination (CSR), a process that allows B cells to change the isotype they express from IgM/IgD to IgG, IgA or IgE. For that purpose, the murine B cell line CH12 stably expressing Cas9 was used. This cell line can be induced to undergo CSR from IgM to IgA very efficiently in vitro when cultured with TGF-b, IL-4 and an anti-CD40 antibody. CH12Cas9 cells were transduced with the second-generation mouse Brie (mBrie) retroviral gRNA library (pMX-mBrie) and selected with puromycin for two weeks to allow for the Cas9-mediated generation of knockouts. Cells were then stimulated to undergo CSR for 72h. Those which failed to undergo CSR (which remained IgM+) or that succeed (which became IgA+) were sorted using magnetic beads. Genes, whose loss-of-function affect the efficiency of CSR, were identified by looking at gRNA enrichment in the IgM+ versus IgA+ populations through deep sequencing.

ORGANISM(S): Mus musculus

PROVIDER: GSE184145 | GEO | 2021/09/18

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of their target site recognition. Most previous studies have examined predicted off-target binding sites that differ from the perfect target site by one to four mismatches, which represent only a subset of genomic regions. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites. For two guide RNAs targeting the murine Snurf gene promoter, we observed very high binding specificity at the intended target site while off-target binding was observed at 2- to 6-fold lower intensities. We also identified significant gRNA-independent off-target binding. Interestingly, we found that these regions are highly enriched in the PAM site, a sequence required for target site recognition by CRISPR. To determine the relationship between Cas9 binding and endonuclease activity, we used targeted sequence capture as a high-throughput approach to survey a large number of the potential off-target sites identified by ChIP-seq or computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results demonstrate that even a simple configuration of a Cas9:gRNA nuclease can support very specific DNA cleavage activity and that most interactions between the CRISPR nuclease complex and genomic PAM sites do not lead to DNA cleavage. ChIP-seq using dCas9 to determine genome-wide binding of CRISPR/Cas9 noED: Cas9 doublemutant protein without an effector domain KRAB: Cas9 doublemutant protein fused to the KRAB repressor domain S1 gRNA: guide RNA targeting GCTCCCTACGCATGCGTCCC(AGG) in the mouse genome S2 gRNA: guide RNA targeting AATGGCTCAGGTTTGTCGCG(CGG) in the mouse genome VEGFA TS3 gRNA: guide RNA targeting GGTGAGTGAGTGTGTGCGTG(TGG) in the human genome

Dataset Information

Identification of genes involved in class switch recombination by genome-wide CRISPR/Cas9 knock-out screen

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure