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

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

  • Marcus Keatinge - , University of Edinburgh (Autor:in)
  • Themistoklis M. Tsarouchas - , University of Edinburgh (Autor:in)
  • Tahimina Munir - , University of Edinburgh (Autor:in)
  • Nicola J. Porter - , University of Edinburgh (Autor:in)
  • Juan Larraz - , University of Edinburgh (Autor:in)
  • Davide Gianni - , Biogen (Autor:in)
  • Hui Hsin Tsai - , Biogen (Autor:in)
  • Catherina G. Becker - , University of Edinburgh (Autor:in)
  • David A. Lyons - , University of Edinburgh (Autor:in)
  • Thomas Becker - , University of Edinburgh (Autor:in)

Abstract

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.

Details

OriginalspracheEnglisch
Aufsatznummere1009515
FachzeitschriftPLOS genetics
Jahrgang17
Ausgabenummer4
PublikationsstatusVeröffentlicht - 29 Apr. 2021
Peer-Review-StatusJa
Extern publiziertJa

Externe IDs

PubMed 33914736