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. 2020 Nov 5;11(11):1311. doi: 10.3390/genes11111311

Figure 1.

Figure 1

Detection of CRISPR/Cas9-induced mutations at the mCherry locus in mylz2-mCherry transgenic amphioxus. (A) T7EI cleavage assay showing no detectable mutation in uninjected embryos (control), or embryos injected with mCherry-sgRNA and Cas9 mRNA transcribed from pXT7-Cas9 (pXT7) or pCS2-nls-zCas9-nls (pCS2) plasmids. —means no T7 endonuclease I was added and + means T7 endonuclease I was added. (B) RT-qPCR analysis of Cas9 mRNA and mCherry-sgRNA expression in embryos injected with Cas9 protein and mCherry-sgRNA or Cas9 mRNA and mCherry-sgRNA. Samples were collected and examined at unfertilized egg (egg), two-cell (2-cell) and early gastrula (EG) stages. (C) T7EI cleavage assay showing no detectable mutation in uninjected embryos (control) and different levels of mutations in embryos injected, respectively, with mCherry-sgRNA and each of the three commercial Cas9 protein (TaKaRa, Thermo and NEB). —means no T7 endonuclease I was added and + means T7 endonuclease I was added. (D) Mutations were detected using DNA sequencing in the injected embryos. The wild-type (WT) reference sequence is shown on the top. The underlined sequence is the target site and GGG (green) is the PAM sequence. Deletion is shown by the dashed line and insertion is highlighted by inserted letters. Indels (+, insertion; –, deletion) are listed on the right of each allele. (E) Mutation efficiencies in embryos injected with different molar ratios of TaKaRa Cas9 to mCherry-sgRNA in which the amount of Cas9 protein was kept constant while that of mCherry-sgRNA was different. (F) Mutation efficiencies in embryos injected with different molar ratios of TaKaRa Cas9 to mCherry-sgRNA in which amount of mCherry-sgRNA was kept constant while that of Cas9 protein was different. Red arrowheads in panels C, E, F, H and I mark bands released by T7 endonuclease I digestion. (G) Observation of red fluorescent signal in two-day transgenetic larvae. In contrast to uninjected (control) larvae which showed a specific fluorescent signal in the notochord and somites, the injected embryos showed no detectable fluorescent signals. The blue fluorescent signal shows the amphioxus cell nuclei stained with DAPI. Scale bars in all images are 100 μm. (H,I) Mutation efficiencies in different stages of embryos injected with TaKaRa Cas9 and mCherry-sgRNA. Un-eggs, unfertilized eggs; LB, late blastula; MG, mid-gastrula; RS, rotation stage; LM, late neurula; MO, mouth-opening stage.