Tissue- and time-directed electroporation of CAS9 protein-gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration

Npj Regenerative Medicine
Ji-Feng FeiElly M Tanaka

Abstract

A rapid method for temporally and spatially controlled CRISPR-mediated gene knockout in vertebrates will be an important tool to screen for genes involved in complex biological phenomena like regeneration. Here we show that in vivo injection of CAS9 protein-guide RNA (gRNA) complexes into the spinal cord lumen of the axolotl and subsequent electroporation leads to comprehensive knockout of Sox2 gene expression in SOX2+ neural stem cells with corresponding functional phenotypes from the gene knockout. This is particularly surprising considering the known prevalence of RNase activity in cerebral spinal fluid, which apparently the CAS9 protein protects against. The penetrance/efficiency of gene knockout in the protein-based system is far higher than corresponding electroporation of plasmid-based CRISPR systems. We further show that simultaneous delivery of CAS9-gRNA complexes directed against Sox2 and GFP yields efficient knockout of both genes in GFP-reporter animals. Finally, we show that this method can also be applied to other tissues such as skin and limb mesenchyme. This efficient delivery method opens up the possibility for rapid in vivo genetic screens during axolotl regeneration and can in principle be applied to other ve...Continue Reading

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Citations

May 31, 2018·Integrative and Comparative Biology·Clare V H Baker, Melinda S Modrell
Nov 1, 2017·Proceedings of the National Academy of Sciences of the United States of America·Ji-Feng FeiElly M Tanaka
Oct 5, 2019·Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution·Varun B Dwaraka, S Randal Voss
Oct 12, 2018·Developmental Neurobiology·David L Stocum
Jan 3, 2019·Developmental Neurobiology·Katharina Lust, Elly M Tanaka
May 4, 2020·Advanced Drug Delivery Reviews·Xiangrong SongYuquan Wei
May 6, 2021·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Lydia TilleyPrayag Murawala
Feb 7, 2019·Developmental Neurobiology·Polina D FreitasJames R Monaghan

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Methods Mentioned

BETA
gene knockout
gene
amputation
transgenic
fluorescence imaging
transfection
PCR
genotyping

Software Mentioned

Prism
GraphPad

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