Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages
Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages
Citació
- Arnan C, Ullrich S, Pulido-Quetglas C, Nurtdinov R, Esteban A, Blanco-Fernandez J et al. Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages. BMC Genomics. 2022 May 26;23(1):402. DOI:10.1186/s12864-022-08612-7
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Resum
CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.