Author for correspondence rkurodaiscchubuacjp MA 0000 0003 4016 9583 RK 0000

Author for correspondence rkurodaiscchubuacjp ma 0000

This preview shows page 1 - 3 out of 7 pages.

Author for correspondence ([email protected]) M.A., 0000-0003-4016-9583; R.K., 0000-0002-0268-7729 1 © 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev175976. doi:10.1242/dev.175976 DEVELOPMENT
Image of page 1

Subscribe to view the full document.

techniques (Jinek et al., 2012) to create gene-knockout snails (Fig. 1A,B). Out of four independent injection experiments, we obtained 10 CRISPR/Cas9-injected adult snails (numbered 1-10) and two control adult snails (11-12) (Fig. S1B,C). By using DNA extracted from the clipped foot while keeping the snails alive, heteroduplex mobility assays (HMA) (Zhu et al., 2014) were carried out on the snails (Fig. 1B), which showed mosaic mutational patterns at the F0 stage (Fig. 1C). Germline transmission to F1 embryos occurred in snails 2, 3, 5, 8 and 10, of which snails 2, 3 and 10 produced only dextral offspring, whereas snail 8 had only sinistral offspring (Fig. 1D; Fig. S2; Table S1). Remarkably, snail 5 produced both dextral and sinistral F1 (denoted as 5-F1-dex and 5-F1-sin) within an egg mass (Fig. S2B; Table S1). This must be a consequence of mosaicity of frameshift and non-frameshift mutations introduced into the tissues of the gonad at F0, which are originated from the 4d mesentoblast (Morrill, 1998). When and where in the gonad the Lsdia1 mRNA is transcribed and provided to the oocyte is an interesting unresolved issue. Fig. 1. Inactivation of Lsdia1 by CRISPR/Cas9 reverses snail shell coiling. (A) Structures of LsDia1 and LsDia2. GBD, Rho GTPase-binding domain; DID, diaphanous inhibitory domain; DD, dimerization domain; FH1 and FH2, formin homology domains 1 and 2; DAD, diaphanous autoregulatory domain. Proto- spacer adjacent motif (PAM) and target sequences were designed in the FH1 region of Lsdia1 (red arrowhead) to discriminate Lsdia1 from Lsdia2 (Fig. S1A). (B) Flow of experiments: Cas9 mRNA and guide RNA were injected into the dextral snail eggs at the one-cell stage, and these embryos were carefully raised to adult F0 snails. HMA was performed on DNA from the clipped foot of ten CRISPR/Cas9-injected (1-10) and two control (11-12) adult snails (Fig. S1B). Adult snails were self-fertilized to produce the F1 generation. Five to ten eggs from each egg mass were directly subjected to PCR amplification and HMA (Fig. S2A), and the rest of the embryos were reared to F1 juveniles to check their chirality. (C) HMA of the F0 generation by 2% agarose gel (top) and 10% polyacrylamide gel (bottom) electrophoresis. (D) Bar chart of shell coiling of the F1 generation. No offspring were produced from snails 6, 7 and 12. The total number of embryos studied ( n ) is shown on the top of the bar graph. (E) Flow of experiments: F1 were individually bred and their genotype was determined from clipped foot DNA. (F) Comparison of the target sequence for the germline transmitted F1 (snails 2, 3, 5, 8 and 10). (G) Bar chart of offspring (F2) phenotype examined at the juvenile stage for each F1 genotype (bottom of the bar chart), including wild-type Dex (+/+), Sin ( - / - ), control (snail 11) and non-edited F1 (snails 1, 4, 9). The total number of embryos studied ( n ) is shown on the top of the bar graph. Homozygous knockout mutants (blue line, P <0.0001). D in a red circle and S in a blue circle indicate dextral and sinistral snails, respectively. Eggs produced by self-fertilization of F1 were used to check the presence of LsDia1 protein
Image of page 2
Image of page 3
  • Fall '17
  • Brian Buchwitz

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern

Ask Expert Tutors You can ask 0 bonus questions You can ask 0 questions (0 expire soon) You can ask 0 questions (will expire )
Answers in as fast as 15 minutes