2.2. Construction of Donor Plasmid and Guide RNA Vectors for Knock-In
This protocol is extracted from research article:
RedEfish: Generation of the Polycistronic mScarlet: GSG-T2A: Ttpa Zebrafish Line
Antioxidants (Basel), Jun 16, 2021; DOI: 10.3390/antiox10060965

The donor plasmid containing homology arms to the ttpa genomic sequence (GenBank:BX248497, RefSeq:NM_199731), mScarlet sequence, Gly-Ser-Gly linker (GSG) sequence and 2A self-cleaving peptide (T2A) was constructed and supplied by In Vivo Biosystems, Inc. (Eugene, OR, USA). Constructs were assembled by Gibson ligation and confirmed by digest at restriction sites Pvul and Pvull, followed by sequencing. A single guide RNA (sgRNA) sequence was designed with the custom designed short CRISPR RNA (crRNA) sequence fused to the scaffold trans-activating CRISPR RNA (tracRNA) sequence (Figure 1).

Knock-in strategy and designed protein sequence. LHA refers to the left homology arm and RHA refers to the right homology arm of the donor plasmid containing the mScarlet coding sequence. Amino acid sequences are denoted by color to indicate protein: mScarlet (red), GSG-T2A linker site (green) and exon 1 of ttpa (blue).

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