Compozr zfn

The generation of zebrafish ak2 knockout mutants using CompoZr ZFNs (Sigma-Aldrich) and the genotyping strategy have been previously described (Sood et al., 2013 (link)). We selected two mutations predicted to cause frameshifts with premature terminations, ak2^hg14 (c.45delAA, p.K16RfsX62) and ak2^hg15 (c.41insACGG, p.K16TfsX64), denoted here as ak2^del2 and ak2^ins4, respectively. We also recovered a mutant line (ak2^hg16, denoted as ak2^L124P) carrying a c.T371C/p.L124P missense mutation within ak2 exon 4 from a zebrafish DNA library of ENU-induced mutations. DNA extraction and amplification from fixed samples were performed using Extract-N-Amp Tissue PCR kit (Sigma-Aldrich) under standard conditions and REDExtract-N-Amp PCR Ready Mix (Sigma-Aldrich). Genomic sequences flanking the point mutation of ak2^L124P mutants were PCR amplified using the following primers: AK2-F3seq (5′-TGTAAAACGACGGCCAGTTCTCATTTGTAGCTGGATGAC-3′) and AK2-R4seq (5′-CAGGAAACAGCTATGACCCACTTACAGGACCCTCCATGC-3′). PCR products were sequenced with M13R primer and big-dye v3.1 sequencing mix (Life Technologies) after removal of unused primers and nucleotides with Exo-SAP-IT (Affymetrix). Sequence analysis was performed using software package Sequencher, version 5.0 (Gene Codes).

ZFNs were of the CompoZr ZFN format generated by Sigma-Aldrich, except for the RSK4 ZFNs that were engineered by Sangamo Biosciences, Inc. The ZFNs contain the eHiFi (ELD/KKR) FokI domains and function as obligate heterodimers with improved catalytic activity (23 (link)). The zinc finger moieties of the dimers were designed to target 30–33-bp recognition sites in total. The sequences of the major ZFNs used here (RSK2, RSK4 and PRMT1) are given in Supplementary Table S1. Plasmid and ssODN donors (Supplementary Table S2) were synthesized by Gene Oracle Inc. and Sigma-Aldrich (Genosys), respectively.