Article Title: Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing
Figure Lengend Snippet: Validation of independent methods used to abrogate MleKlf5a and MleKlf5b gene function. MleKlf5a ( A ) and MleKlf5b ( E ) exon-intron schematics show the location of splice-blocking morpholino oligonucleotide (sbMO) targets (blue boxes) and single-guide RNA (sgRNA) targets (black triangles) used in this study. The orange bars indicate the location of the DNA binding domain. ( B, F ) Electrophoretic gels of PCR products obtained using different sets of MleKlf5a and MleKlf5b sbMO RT-PCR primers on cDNA obtained from a single individual KLF-MO embryo exemplar (left) and control embryo exemplar (right). Schematics to the right of the gel images highlight examples of wildtype (wt) amplicon, exon-skipping (-E) and/or intron retention (+I) amplicons captured with primers for each gene ( Table 1 ). ( B ) MleKlf5a-MO gel: 2-log DNA ladder (L) used for band size reference, unlabeled lanes are not relevant to this study. Lane 1 shows both a 960 bp wt and a 860 bp third exon-skipped (-E3) MleKlf5a amplicon. Lane 2 shows a ∼3 kb third intron retention (+I3) MleKlf5a amplicon. Control gel: 2-log DNA ladder (L) used for band size reference. Lane 1 shows a single 960 bp wt MleKlf5a amplicon. (F) MleKlf5b-MO gel: 2-log DNA ladder (L) used for band size reference. Lane 1 shows both a 480 bp wt and a 430 bp sixth exon-skipped (-E6) MleKlf5b amplicon. Lane 2 shows a ∼1.2 kb seventh intron-retained (+I7) MleKlf5b amplicon. Control gel: 2-log DNA ladder (L) used for band size reference. Lane 1 shows a single 480 bp wt MleKlf5b amplicon. Wildtype (wt) and mis-spliced transcripts due to -E and/or +I were present in KLF-MO embryos ( n = 21 KLF-MO embryos). ( C-G) Discordance plots produced using ICE software show elevated sequence discordance downstream of predicted Cas9 cut sites relative to control genomic sequence. ( D, H ) Corresponding Sanger sequence traces from genomic DNA extracted from a single individual exemplar KLF-Cas9 embryo show signal degradation downstream of the Cas9 cut site as compared to a single individual exemplar wildtype embryo ( n = 17 KLF-Cas9 embryos). Sanger sequencing signal degradation is caused by the introduction of indels in KLF-Cas9 embryos. The sgRNA target sites are underlined, the position of the predicted Cas9 cut sites are represented by a vertical dashed line.
Article Snippet: Gene-specific primers were used on cDNA (OneTaq One-Step RT-PCR, New England Biolabs) to evaluate aberrant transcript splicing via gel electrophoresis.
Techniques: Blocking Assay, Binding Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Produced, Software, Sequencing