Presentation Title

Targeted knockout of katanin genes in C. elegans by CRISPR and RNAi

Faculty Mentor

Renee Baran

Start Date

17-11-2018 8:30 AM

End Date

17-11-2018 10:30 AM

Location

CREVELING 71

Session

POSTER 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Microtubules are dynamic polymers that function in cell shape and motility, cell division, and intracellular transport. Microtubule severing by the katanin complex may contribute to how microtubules are regulated during axon growth in C. elegans. In C. elegans, mei-1codes for the catalytic subunit of katanin. Mutations in mei-1are lethal in embryos. However, F47G4.4 and F47G4.5, two putative regulatory subunits, are expressed by neurons but are not well-characterized. Worms with deletions in these genes have normal movement, suggesting the two genes have redundant function in neurons. Strains containing fluorescent markers targeted to different parts the GABAergic neurons were mated to strains with the RB1734 (F47G4.4) and RB2014 (F47G4.5) deletion alleles. Knocking out both genes in the same animal would provide the true elimination of the function of katanin in neurons. However, a double mutant cannot easily be made through genetic crosses because F47G4.4 and F47G4.5 are adjacent on the same chromosome. To generate a double mutant phenotype, we are using two strategies: RNAi and CRISPR/Cas9 gene editing. RNAi targets the mRNA gene product, resulting in null or hypomorphic phenotypes. CRISPR introduces double strand breaks and small deletions in a gene. RNAi against F47G4.5 in the F47G4.4 mutant caused misexpression of synaptic vesicles in neuron commissures in one trial, but this result needs additional verification. To knockout F47G4.4 in the F47G4.5 deletion mutant, I am creating reagents to disrupt Exon 3 of F47G4.4 using CRISPR-cas9 gene editing. This system requires a guide RNA to target the gene, a repair vector that inserts GFP into Exon 3, and the cas9 enzyme. Sequences flanking exon 3 were amplified by PCR, purified and combined with a repair vector using Gibson Assembly. The completed repair vector will be used in injections into the F47G4.5 deletion worms to make the double knockout.

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Nov 17th, 8:30 AM Nov 17th, 10:30 AM

Targeted knockout of katanin genes in C. elegans by CRISPR and RNAi

CREVELING 71

Microtubules are dynamic polymers that function in cell shape and motility, cell division, and intracellular transport. Microtubule severing by the katanin complex may contribute to how microtubules are regulated during axon growth in C. elegans. In C. elegans, mei-1codes for the catalytic subunit of katanin. Mutations in mei-1are lethal in embryos. However, F47G4.4 and F47G4.5, two putative regulatory subunits, are expressed by neurons but are not well-characterized. Worms with deletions in these genes have normal movement, suggesting the two genes have redundant function in neurons. Strains containing fluorescent markers targeted to different parts the GABAergic neurons were mated to strains with the RB1734 (F47G4.4) and RB2014 (F47G4.5) deletion alleles. Knocking out both genes in the same animal would provide the true elimination of the function of katanin in neurons. However, a double mutant cannot easily be made through genetic crosses because F47G4.4 and F47G4.5 are adjacent on the same chromosome. To generate a double mutant phenotype, we are using two strategies: RNAi and CRISPR/Cas9 gene editing. RNAi targets the mRNA gene product, resulting in null or hypomorphic phenotypes. CRISPR introduces double strand breaks and small deletions in a gene. RNAi against F47G4.5 in the F47G4.4 mutant caused misexpression of synaptic vesicles in neuron commissures in one trial, but this result needs additional verification. To knockout F47G4.4 in the F47G4.5 deletion mutant, I am creating reagents to disrupt Exon 3 of F47G4.4 using CRISPR-cas9 gene editing. This system requires a guide RNA to target the gene, a repair vector that inserts GFP into Exon 3, and the cas9 enzyme. Sequences flanking exon 3 were amplified by PCR, purified and combined with a repair vector using Gibson Assembly. The completed repair vector will be used in injections into the F47G4.5 deletion worms to make the double knockout.