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Title: CRISPR /Cas9 mutagenesis in Volvox carteri
Summary

Volvox carteriand other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis inV. carteriusingCRISPR/Cas9 components expressed from transgenes. We usedV. carterinitrate reductase gene (nitA) regulatory sequences to conditionally expressStreptococcus pyogenesCas9, andV. carteriU6RNAgene regulatory sequences to constitutively express single‐guideRNA(sgRNA) transcripts.Volvox carteriwas bombarded with both Cas9 vector and one of several sgRNAvectors programmed to target different test genes (glsA,regAandinvA), and transformants were selected for expression of a hygromycin‐resistance marker present on the sgRNAvector. Hygromycin‐resistant transformants grown with nitrate as sole nitrogen source (inducing fornitA) were tested for Cas9 and sgRNAexpression, and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sgRNAplasmid withglsAprotospacer sequence yielded progeny (at a rate of ~0.01%) with a gonidialess (Gls) phenotype similar to that observed for previously describedglsAmutants, and sequencing of theglsAgene in independent mutants revealed short deletions within the targeted region ofglsA, indicative of Cas9‐directed non‐homologous end joining. Similarly, bombardment of a morphologically wild‐type strain with the Cas9 plasmid and sgRNAplasmids targetingregAorinvAyieldedregAandinvAmutant transformants/progeny, respectively (at rates of 0.1–100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development inV. carteri, and of developmental novelty in the volvocine algae.

 
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NSF-PAR ID:
10130360
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
The Plant Journal
Volume:
97
Issue:
4
ISSN:
0960-7412
Page Range / eLocation ID:
p. 661-672
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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