Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome

Abstract

SUMMARYThe success of the green alga Chlamydomonas reinhardtii as a model organism is to a large extent due to the wide range of molecular techniques that are available for its characterization. Here, we review some of the techniques currently used to modify and interrogate the C. reinhardtii nuclear genome and explore several technologies under development. Nuclear mutants can be generated with ultraviolet (UV) light and chemical mutagens, or by insertional mutagenesis. Nuclear transformation methods include biolistic delivery, agitation with glass beads, and electroporation. Transforming DNA integrates into the genome at random sites, and multiple strategies exist for mapping insertion sites. A limited number of studies have demonstrated targeted modification of the nuclear genome by approaches such as zinc-finger nucleases and homologous recombination. RNA interference is widely used to knock down expression levels of nuclear genes. A wide assortment of transgenes has been successfully expressed in the Chlamydomonas nuclear genome, including transformation markers, fluorescent proteins, reporter genes, epitope tagged proteins, and even therapeutic proteins. Optimized expression constructs and strains help transgene expression. Emerging technologies such as the CRISPR/Cas9 system, high-throughput mutant identification, and a whole-genome knockout library are being developed for this organism. We discuss how these advances will propel future investigations.

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

“…Homologous recombination is a prerequisite for generating mutant strains by insertion or deletion of distinct genes. In C. reinhardtii, the frequency of homologous recombination is exceptionally low, and most often, transforming DNA integrates randomly by the nonhomologous end-joining repair pathway (42). Until now, a rapid method to generate targeted mutations with a moderate success rate was still missing.…”

“…Usually, the screening of mutants relies on a distinct mutant phenotype that allows uncovering of specific gene functions based on complementation analysis. Commonly, insertional mutagenesis by random integration of a selection marker was used to generate mutants with a desired phenotype (42,43). More recently, an alternative approach used whole-genome sequencing to identify gene mutations, but it was hampered by the mutational profile of the strains analyzed (44).…”

Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii

“…Naturally occurring alleles, including nonsense polymorphisms, TE insertion polymorphisms (Supplemental Data Set 1), and gene content polymorphism, provide a potentially rich source of variation for functional studies (Alonso-Blanco and Koornneef, 2000) that should complement artificially induced mutant libraries (Zhang et al, 2014;Jinkerson and Jonikas, 2015). Research into such natural variation has greatly assisted in discovery of valuable traits for crop improvement (Xu et al, 2006) and may also yield insight into the genetic basis of trait variation in Chlamydomonas (Alonso-Blanco and Koornneef, 2000).…”

“…In addition, practical characteristics such as the ease with which it can be cultured, the ability to conduct conventional genetic crosses, and the relative simplicity of genetic manipulation make Chlamydomonas a preferred alga for experimental studies (Graham, 1997;Harris, 2008). The completion of the ;112-Mb genome sequence in 2007 (Merchant et al, 2007; Chlamydomonas JGI genome sequence version 4) has enabled new advances in algal genomics (Umen and Olson, 2012), and high-throughput genomic applications now complement traditional genetic approaches in studies of Chlamydomonas (Zhang et al, 2014;Jinkerson and Jonikas, 2015).…”

Glucose-Induced Trophic Shift in an Endosymbiont Dinoflagellate with Physiological and Molecular Consequences

“…It should also allow the isolation of algal mutants defective in photosynthetic functions. The establishment of heterotrophic growth in the green alga Chlamydomonas was a major factor in facilitating the use of this organism as a leading model for photosynthesis research (Levine and Goodenough, 1970;Rochaix, 2001;Jinkerson and Jonikas, 2015), and this feature of some Symbiodinium strains should be similarly powerful in elucidating the roles of photosynthetic activities in the cnidarian-alga association.…”