Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta
- Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, United States
Figures
Figure 1
Introduction to Salpingoeca rosetta as a simple model for multicellularity and the ancestry of animal cell biology.
(A) Choanoflagellates (blue) are the closest living relatives of animals (red) and last shared a common ancestor (purple) ~800 million years ago (Parfrey et al., 2011). (B) The collar complex, an …
Figure 2 with 2 supplements
Engineered cycloheximide resistance in S. rosetta provides a proof-of-principle for Cas9-mediated genome editing.
(A) Schematic of Cas9-mediated genome editing to engineer cycloheximide resistance in S. rosetta. Nucleofection was used to deliver SpCas9 (gray) bound to gRNA (cyan), which together form the SpCas9 …
Figure 2—figure supplement 1
An approach for selecting cycloheximide resistance in S. rosetta.
(A) Cycloheximide inhibits S. rosetta growth. We seeded each well of a 24-well plate with 0.5 ml of cells at 2 × 104 cells/ml in a 3-fold serial dilution of cycloheximide, including a condition …
Figure 2—figure supplement 2
Engineered cycloheximide resistance establishes genome editing conditions.
(A) The design of a cycloheximide resistant allele, rpl36aP56Q, in S. rosetta. The protospacer adjacent motif (PAM, orange) next to the 56th codon of rpl36a (Target, cyan), which is located on the …
Figure 3 with 2 supplements
Genome editing of rosetteless enables targeted disruption of multicellular development in S. rosetta.
(A) An engineered mutation in rosetteless introduces a premature termination sequence (PTS) to knockout the expression of rosetteless. The rosetteless gene (exons shown as numbered black boxes, …
Figure 3—figure supplement 1
Phenotypes of rosetteless mutants correspond to their genotypes.
(A) The consensus genotype at the site of rosetteless editing in cell populations selected for cycloheximide resistance indicates the presence of the rtlsPTS1 allele. In a wild-type strain (top) and …
Figure 3—figure supplement 2
Wild-type and mutant strains proliferate similarly.
Growth curves for wild-type (A), rpl36aP56Q (B), rtlsPTS1 rpl36aP56Q (C), and rtlstl1 (D) show similar rates of proliferation. The growth for each strain was characterized by seeding cells at a …
Figure 4 with 1 supplement
S. rosetta preferentially introduces genome-edited mutations from DNA templates.
(A) Schematic of a gRNA targeting SpCas9 to a genomic locus of interest. A gRNA (cyan, knobs indicate 5’ ends) that encodes a 20 nt targeting sequence from the sense strand of a genomic locus …
Figure 4—figure supplement 1
Characterization of editing outcomes at the rosetteless locus with different types of repair templates.
(A) Double-stranded DNA repair templates (black indicates homology arms from the sense strand, gray indicates homology arms from the antisense strand and green is the PTS as in Figure 4) were …
Additional files
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Source code 1
Quantification of DNA repair outcomes.
BASH script for quantifying the frequency of repair outcomes from deep sequencing data that were preprocessed and aligned in a Galaxy server (Afgan et al., 2018).
- https://cdn.elifesciences.org/articles/56193/elife-56193-code1-v3.sc
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Supplementary file 1
Tables of critical resources.
Table A: Media recipes for making artificial seawater (Hallegraeff et al., 2004; Skelton et al., 2009), high nutrient media (modified from King et al., 2009; Levin and King, 2013; Booth et al., 2018), and low nutrient media. Table B: Oligonucleotide sequences for gRNAs, repair oligonucleotides, and primers that were used to construct and to validate genome edited strains. Table C: S. rosetta strains Genotypes and sources of S. rosetta strains used in this study. Table D: Deep sequencing library primers Sequences for primers (adapted from Lin et al., 2014 used to generate libraries for deep sequencing (Figure 4 and S5)
- https://cdn.elifesciences.org/articles/56193/elife-56193-supp1-v3.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56193/elife-56193-transrepform-v3.pdf
