Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene

Abstract

Chlamydomonas reinhardtii is the most powerful photosynthetic eukaryotic unicellular model organism. However, its potential is not fully exploitable since as in most green plants specific targeting of nuclear genes is not routinely possible. Recently, we have shown by repair of an introduced truncated model gene that transformation of Chlamydomonas with single stranded DNA greatly suppresses random integration of the DNA in the genome whereas homologous recombination (HR) is left unchanged. However, endogenous genes still could not be targeted. Here we present optimized transformation conditions that further improved HR and suppressed non-homologous DNA integration (NHI). The improved transformation strategy allowed us now to specifically inactivate in two different Chlamydomonas strains the nuclear PHOT gene, which encodes for the blue light photoreceptor phototropin (PHOT). The option to target moderately expressed Chlamydomonas nuclear genes with high efficiency now further improves the utility of this this alga for basic science and biotechnology.