CRISPR-induced Deletion in Zebrafish abcc9 Gene

A target site within the abcc9 open-reading frame was chosen using CHOPCHOP (http://chopchop.cbu.uib.no/). sgRNA template was purchased at Integrated DNA Technologies (IDT) as standard desalted same-day oligos and synthesis was carried out using the Ambion MEGAscript T7 or SP6 kits (Ambion). Purification of the in vitro synthesized mRNA was achieved with the RNeasy Mini Kit (Qiagen)^{64 (link)}. gRNA and Cas9-encoding mRNA were co-injected into one-cell stage zebrafish embryos. Each embryo was injected with 2 nl of solution containing ~12.5 ng μl⁻¹ of sgRNA and ~300 ng μl⁻¹ of Cas9 mRNA. Injected embryos were grown to adulthood to generate “founder” fish (F0), and screened for germline transmission of CRISPR-induced indel mutations using an adapted method for detecting simple sequence-length polymorphisms (SSLPs). Each putative founder adult fish was crossed with a wild-type adult fish (F1). Sequencing analysis confirmed successful introduction of 13n deletion and segregation in a Mendelian fashion. Homozygous fish (F2) were generated by inbreeding heterozygous mutant carriers.

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Smeland M.F., McClenaghan C., Roessler H.I., Savelberg S., Hansen G.Å., Hjellnes H., Arntzen K.A., Müller K.I., Dybesland A.R., Harter T., Sala-Rabanal M., Emfinger C.H., Huang Y., Singareddy S.S., Gunn J., Wozniak D.F., Kovacs A., Massink M., Tessadori F., Kamel S.M., Bakkers J., Remedi M.S., Van Ghelue M., Nichols C.G, & van Haaften G. (2019). ABCC9-related Intellectual disability Myopathy Syndrome is a KATP channelopathy with loss-of-function mutations in ABCC9. Nature Communications, 10, 4457.

Publication 2019

Ambion MEGAscript T7 RNeasy Mini Kit

Adult Cell Crispr Deletion Embryos Fish Germline Heterozygous Homozygous Indel mutations Mrna Oligos Polymorphisms Sequencing analysis Synthesis Transmission Zebrafish

Corresponding Organization : University Hospital of North Norway

Other organizations : Washington University in St. Louis, University Medical Center Utrecht, Hubrecht Institute for Developmental Biology and Stem Cell Research, UiT The Arctic University of Norway

Top 5 similar protocols

Variable analysis

independent variables

Injection of sgRNA and Cas9-encoding mRNA into one-cell stage zebrafish embryos

dependent variables

Introduction of 13n deletion in the abcc9 open-reading frame
Segregation of CRISPR-induced indel mutations in a Mendelian fashion
Generation of homozygous mutant fish (F2) by inbreeding heterozygous mutant carriers

control variables

Injection volume (2 nl)
Concentration of sgRNA (~12.5 ng μl^-1)
Concentration of Cas9 mRNA (~300 ng μl^-1)
Crossing putative founder adult fish (F0) with wild-type adult fish to generate F1 offspring

positive controls

None specified

negative controls

None specified

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