Additional file 1 of A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening
Creators: name: Carrington, Blake
name: Ramanagoudr-Bhojappa, Ramanagouda
name: Bresciani, Erica
name: Han, Tae-Un
name: Sood, Raman
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Sequences of all primers and sgRNA's used in this study. Figure S1. tcnba CRISPR selection and CRISPR-STAT analysis. A Screenshot of the tcnba target region from UCSC genome browser using the CRISPRscan track showing possible sgRNAs near the stop codon. B Evaluation of individual embryos by CRISPR-STAT to determine activity levels. In all plots, the X-axis shows the size of the peaks and the Y-axis shows the peak height. sgRNA-T2 showed higher activity and was selected for knock-in experiments. Figure S2. Design of tcnba ssODN and somatic and germline screening. A Detailed design of the ssODN aligned with the ref sequence (chr5:30,618,013-30,618,151) and sgRNA used for generating the DSB. B Sequence chromatogram of a clone from an injected embryo positive for the knock-in peak confirming the insertion of the desired sequence as well clean and precise integration at each end of the ssODN. C Representative plot (X-axis showing the size of the peaks and the Y-axis showing the peak height) from an F1 embryo heterozygous for the knock-in allele (denoted by red arrowhead) compared to a WT embryo. Figure S3. Sequence confirmation of the tcnbaFLAG/FLAG RT-PCR product. Figure S4. gata2b CRISPR selection and CRISPR-STAT analysis. A Screenshot of UCSC genome browser using the CRISPRscan and ZebrafishGenomics tracks showing possible sgRNAs near the stop codon. B Evaluation of individual embryos by CRISPR-STAT to determine activity levels. In all plots, the X-axis shows the size of the peaks and the Y-axis shows the peak height. sgRNA-T1 showed higher activity and was selected for knock-in experiments. Figure S5. Design of gata2b ssODN and somatic and germline screening. A Detailed design of the ssODN aligned with the ref sequence (chr6:40,803,222-40,803,359) and sgRNA used for generating the DSB. B Sequence of a TOPO clone from an injected embryo positive for the knock-in peak confirming the insertion of the desired sequence as well clean and precise integration at each end of the ssODN. C Representative plot (X-axis showing the size of the peaks and the Y-axis showing the peak height) from an F1 embryo heterozygous for the knock-in allele (denoted by red arrowhead) compared to a WT embryo. Figure S6. Sequence confirmation of gata2bHA/HA RT-PCR product. Figure S7. gba CRISPR selection and CRISPR-STAT analysis. A Screenshot of UCSC genome browser using the CRISPRscan track showing possible sgRNAs near the desired point mutation. B Evaluation of individual embryos by CRISPR-STAT to determine activity levels. In all plots, the X-axis shows the size of the peaks and the Y-axis shows the peak height. sgRNA-T1 showed higher activity and was selected for knock-in experiments. Figure S8. Design of gba ssODN and sequence confirmation of knock-in in injected embryos. A Detailed design of the ssODN aligned with the ref sequence (chr16:14,227,951-14,228,090), sequence of our WT cohort of fish, the sgRNA used for generating the DSB and the SalI restriction site that is generated by the ssODN. B Sequence of a TOPO clone from an injected embryos positive for the knock-in peak confirming the presence of the desired nucleotide changes as well clean and precise integration at each end of the ssODN.