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CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotyp...

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Autores principales: Keatinge, Marcus, Tsarouchas, Themistoklis M., Munir, Tahimina, Porter, Nicola J., Larraz, Juan, Gianni, Davide, Tsai, Hui-Hsin, Becker, Catherina G., Lyons, David A., Becker, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8084196/
https://www.ncbi.nlm.nih.gov/pubmed/33914736
http://dx.doi.org/10.1371/journal.pgen.1009515
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author Keatinge, Marcus
Tsarouchas, Themistoklis M.
Munir, Tahimina
Porter, Nicola J.
Larraz, Juan
Gianni, Davide
Tsai, Hui-Hsin
Becker, Catherina G.
Lyons, David A.
Becker, Thomas
author_facet Keatinge, Marcus
Tsarouchas, Themistoklis M.
Munir, Tahimina
Porter, Nicola J.
Larraz, Juan
Gianni, Davide
Tsai, Hui-Hsin
Becker, Catherina G.
Lyons, David A.
Becker, Thomas
author_sort Keatinge, Marcus
collection PubMed
description Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.
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spelling pubmed-80841962021-05-06 CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury Keatinge, Marcus Tsarouchas, Themistoklis M. Munir, Tahimina Porter, Nicola J. Larraz, Juan Gianni, Davide Tsai, Hui-Hsin Becker, Catherina G. Lyons, David A. Becker, Thomas PLoS Genet Research Article Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest. Public Library of Science 2021-04-29 /pmc/articles/PMC8084196/ /pubmed/33914736 http://dx.doi.org/10.1371/journal.pgen.1009515 Text en © 2021 Keatinge et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Keatinge, Marcus
Tsarouchas, Themistoklis M.
Munir, Tahimina
Porter, Nicola J.
Larraz, Juan
Gianni, Davide
Tsai, Hui-Hsin
Becker, Catherina G.
Lyons, David A.
Becker, Thomas
CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
title CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
title_full CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
title_fullStr CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
title_full_unstemmed CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
title_short CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
title_sort crispr grna phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8084196/
https://www.ncbi.nlm.nih.gov/pubmed/33914736
http://dx.doi.org/10.1371/journal.pgen.1009515
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