Abstract
F0 CRISPants were made using a sgRNA that targets both L and S homeologues of Xenopus laevis NeuroD2, in the DNA binding domain, resulting formation of a non-functional protein (Banerjee et al., 2024). Whole brains were dissected at Nieuwkoop and Faber (NF) stage 47 (Nieuwkoop & Faber, 1994) and snap frozen on dry ice in groups of four. Four such biological replicate samples were used for each condition. Total RNA was extracted using a Qiagen RNeasy mini kit with an on-column DNaseI treatment. Libraries were prepared using 1 mg of RNA with RIN >9.5 and sequenced using Illumina Nextseq P2 by the Otago Genomics Facility, University of Otago, New Zealand, generating ~30 M reads per sample. Single reads of 100 bp were mapped to XENLA_10.1 genome build (Xenbase, accessed November 2024, RRID:SCR_003280) using DRAGEN v 4.0.4. Galaxy EU (RRID:SCR_006281) was used to generate read counts using FeatureCounts (Liao et al., 2014) and to normalise read counts and identify differentially expressed genes with EdgeR (Robinson et al., 2010). Sequencing data and read counts are available at NCBI GEO (Edgar et al., 2002, RRID:SCR_005012) under accession GSE307156. Genes with fold change of > 2 and Benjamini-Hochberg corrected FDR <0.05 (PAdj) between control and CRISPant brains were considered differentially expressed. Enrichr (RRID:SCR_001575) was used with a custom background list of brain expressed genes to identify enriched gene ontologies (Chen et al., 2013; Kuleshov et al., 2016). Significantly enriched pathways (PAdj<0.05) are listed, with genes, in the accompanying file.