citations

                    Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta
                

Authors:

David S Booth , Nicole King

In:

Source: eLife
Publication Date: (2020)
Issue: 9: 1-24

Research Area:

Gene Expression

Platform:

4D-Nucleofector® 96-well Systems

Experiment

Priming
To prime S. rosetta cells for nucleofection, we treated them with a cocktail that removes the extracellular matrix as follows. Aliquots of washed cells were pelleted at 800 g and 22°C for 5 min. Thesupernatant was gently removed with gel-loading tips and each pellet was resuspended in 100 ml ofpriming buffer (40 mM HEPES-KOH, pH 7.5; 34 mM lithium citrate; 50 mM l-cysteine; 15% [wt/vol] PEG 8000; and 1 mM papain [Millipore Sigma, St. Louis, MO; Cat. No. P3125-100MG]). After incubating cells for 30–40 min, 10 ml of 50 mg/ml bovine serum albumin was added to each aliquot ofprimed cells to quench proteolysis from the priming buffer. Finally, the cells were centrifuged at1250 g and 22°C for 5 min, the supernatant was removed, and the pellet was resuspended in 25 mlof SF Buffer (Lonza, Basel, Switzerland; Cat. No. V4SC-2960). The resuspended cells were stored onice while preparing nucleofection reagents.
Nucleofection
Each nucleofection reaction was prepared by adding 16 ml of ice-cold SF Buffer to 4 ml of the SpCas9RNP that was assembled as described above. (For reactions that used two different gRNAs, each gRNA was assembled with SpCas9 separately and 4 ml of each RNP solution was added to SF buffer at this step). 2 ml of the repair oligonucleotide template was added to the SpCas9 RNP diluted in SF buffer. Finally, 2 ml of primed cells were added to the solution with SpCas9 RNP and the repair template. The whole solution, which has a total volume of 24 ml (30 ml for two different SpCas9 RNPs and repair templates), was placed in one well of a 96-well nucleofection plate. The well was pulsed in a Lonza shuttle nucleofector (Lonza, Cat. No. AAF-1002B and AAM-1001S) with the CM156 pulse.
Recovery
Immediately after transfection, 100 ml of ice-cold recovery buffer (10 mM HEPES-KOH, pH 7.5; 0.9 M sorbitol; 8% [wt/vol] PEG 8000) was added to each transfection and gently mixed by firmly tapping the side of the plate or cuvette. After the cells rested in recovery buffer at room-temperature for 5 min, the whole volume of a nucleofection well was transferred to 2 ml of low nutrient media in one well of a six well plate. After 30 min, 10 ml of 10 mg/ml E. pacifica (prepared by resuspending a frozen 10 mg pellet of E. pacifica in ASW) was added to each well and the six well plate was incubated at 22°C and 60% relative humidity for downstream experiments.

Abstract

In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.

Open in PubMed