Computational protocol: Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm

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Protocol publication

[…] Three sgRNA sequences targeting brca2 were designed using CRISPRdirect. From these sequences, synthetic dsDNA consisting of a 5′ random sequence, T7 promotor, target-specific sequence (minus PAM) and a constant region were designed according to Boel et al.. These dsDNA sequences were ordered as G-blocks at Integrated DNA Technologies (IDT). G-blocks were dissolved in 20 µl nuclease-free water. PCR conditions can be found in Supplementary data (Supplementary Table .PCR product was purified with the Qiaquick PCR purification kit (Qiagen) following general guidelines of the manufacturer. In vitro transcription was performed using the MEGAshortscript™ Kit (Thermo Fisher). The MEGAclear™ Kit (Thermo Fisher) was used to purify the transcription reaction. RNA quality was verified on the Experion (Bio-Rad). 25 pg sgRNA was co-injected with 250 pg Cas9 protein (Cas9 wild type nuclease protein with NLS, ToolGen) during the 1-cell stage. Phenol red 6% was used to visualise injection. At 24 hpf, 10 embryos were harvested for DNA extraction, using the KAPA Express Extract DNA Extraction kit. PCR was performed according to manufacturer’s protocol using primers flanking the crispr region. Samples were deep sequenced on the MiSeq and somatic indel frequency was analysed using BATCH-GE. If a high out-of-frame indel percentage was identified, mosaic fish (F0) were grown to adulthood. These were out-crossed and offspring (F1) was tested for the presence of the mutation. If present, offspring was grown to adulthood and genotyped for the mutation. [...] Data was analysed using SPSS statistics 24 (IBM). Unless mentioned otherwise, groups were compared using the student’s t-test (two-sided, α = 0.05). Levene’s test for equality of variances was performed. Graphs were made with Sigmaplot (Systat Software Inc.). […]

Pipeline specifications

Software tools CRISPRdirect, BATCH-GE, SPSS, SigmaPlot
Applications Genome edition, Miscellaneous
Organisms Danio rerio, Homo sapiens
Diseases Neoplasms, Sotos Syndrome