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PLoS One. 2018 Jan 18;13(1):e0191503. doi: 10.1371/journal.pone.0191503. eCollection 2018.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.

Author information

1
Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
2
Epilepsy Genetics Program and F.M. Kirby Neurobiology Center, Department of Neurology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

Abstract

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.

PMID:
29346415
PMCID:
PMC5773193
DOI:
10.1371/journal.pone.0191503
[Indexed for MEDLINE]
Free PMC Article

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