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Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

Sebastian Ullrich, View ORCID ProfileCarme Arnan, Carlos Pulido-Quetglas, Ramil Nurtdinov, Alexandre Esteban, Joan Blanco-Fernandez, Estel Aparicio-Prat, Rory Johnson, Sílvia Pérez-Lluch, Roderic Guigó
doi: https://doi.org/10.1101/2021.04.26.441397
Sebastian Ullrich
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
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Carme Arnan
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
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  • ORCID record for Carme Arnan
Carlos Pulido-Quetglas
4Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Switzerland
5Department for BioMedical Research, University of Bern, Bern, Switzerland
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Ramil Nurtdinov
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
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Alexandre Esteban
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
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Joan Blanco-Fernandez
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
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Estel Aparicio-Prat
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
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Rory Johnson
4Department of Medical Oncology, Inselspital, Bern University Hospital, University of Bern, Switzerland
5Department for BioMedical Research, University of Bern, Bern, Switzerland
6School of Biology and Environmental Science, University College Dublin, Ireland
7Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Ireland
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Sílvia Pérez-Lluch
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
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Roderic Guigó
1Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Barcelona (BIST), Catalonia, Spain
2Universitat Pompeu Fabra (UPF), Barcelona, Catalonia, Spain
3Institut Hospital del Mar d’Investigacions Mèdiques (IMIM), Barcelona, Catalonia, Spain
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  • For correspondence: roderic.guigo@crg.cat
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ABSTRACT

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify both 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 the usage of a second gRNA targeting the same gene synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next take advantage of our paired-guide (pgRNA) system to design a library to simultaneously target 874 pc-genes and 166 lncRNAs which are 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 for deeper characterization. Two of these, FURIN and NFE2, code for proteins related to cell differentiation and macrophage function; the other two, LINC02432 and MIR3945HG, are lncRNAs associated with cancerous and infectious diseases, respectively. The CRISPR-Cas9 coupled to pgRNAs system is, therefore, a suitable tool to target simultaneously pc-genes and lncRNAs for genomic perturbation assays.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted April 27, 2021.
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Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages
Sebastian Ullrich, Carme Arnan, Carlos Pulido-Quetglas, Ramil Nurtdinov, Alexandre Esteban, Joan Blanco-Fernandez, Estel Aparicio-Prat, Rory Johnson, Sílvia Pérez-Lluch, Roderic Guigó
bioRxiv 2021.04.26.441397; doi: https://doi.org/10.1101/2021.04.26.441397
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Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages
Sebastian Ullrich, Carme Arnan, Carlos Pulido-Quetglas, Ramil Nurtdinov, Alexandre Esteban, Joan Blanco-Fernandez, Estel Aparicio-Prat, Rory Johnson, Sílvia Pérez-Lluch, Roderic Guigó
bioRxiv 2021.04.26.441397; doi: https://doi.org/10.1101/2021.04.26.441397

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