Fig. 5
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- ZDB-FIG-220906-5
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- Colijn et al., 2022 - High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products
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Fig. 5. Genotyping of embryos from the F1 generation reveals germline transmission of the 6-bp indel, which is confirmed via sequencing. (A) Image of the fragment analyzer gel output data. Lanes 1–12 are F1 embryos collected from a cross between F0 fish #5 and a wildtype AB zebrafish breeder. Single bands (Lanes 2, 3, 5, 7, 8, 10, 11, and 12) represent wildtype amplicons. The doublets (Lanes 1, 4, 6, and 9) represent heterozygous carriers of the identified 6-bp indel. (The less intense bands visible at 300 bp are PCR artifacts and are not present with the use of an alternate primer pair.) (B) Representation of the Danio rerio cluap1 locus with the location of the 6-bp indel. The CRISPR target site is in the TSS of cluap1 in exon 2. (C) Representation of exon 2 with the sequencing results for the WT and mutant alleles. The bold letter indicates a single bp change, the dashes indicate bp deletions, and the magenta indicates the TSS. The 6-bp indel allele, now referred to as cluap1stl839, disrupts the TSS, leading to a predicted loss-of-function cluap1 allele. TSS: Translation Start Site (ATG triplet). |
Reprinted from Developmental Biology, 484, Colijn, S., Yin, Y., Stratman, A.N., High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products, 22-29, Copyright (2022) with permission from Elsevier. Full text @ Dev. Biol.