Targeting of Photoreceptor Genes inChlamydomonas reinhardtiivia Zinc-Finger Nucleases and CRISPR/Cas9

Abstract

The fast-growing biflagellated single-celled chlorophyte is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from and , and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes, (encoding channelrhodopsin 1 [ChR1]), (encoding ChR2), ,, ,, , and and created the and double mutants. Characterization of the ,, and mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae.

Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA

“…This necessitates the positive selection of mutants through cointegration of antibiotic resistance markers. The recent use of single-stranded oligodeoxynucleotides (ssODNs) has reduced nontarget integration, but has left gene targeting extremely inefficient (29)(30)(31)(32). Previous efforts to use targeting endonucleases, including zinc finger nucleases and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)associated protein 9 (Cas9), have not resolved these shortcomings in gene targeting efficiency (33)(34)(35)(36).…”

Microalgal Hydrogen Production

“…[197][198][199][200][201] Recently, scientists have developed many other genome editing tools, such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas9 or Cpf1 system, [202][203][204][205][206][207] TALENS (Transcriptional Activator-Like Effector Nucleases), [208] chemical mutagenesis-mediated TILLING (Targeting Induced Local Lesions In Genomes), [193,209] ZFN (Zinc Finger Nuclease), etc. [208,210] Carbon nanotube is likely to improve transformation Small Methods 2020, 4,1900514 efficiency in Chlamydomonas, [211] but related new technologies are still being tested. [212] Genomic sequencing of many microalgae has been completed: Thalassiosira pseudonana, [213] Cyanidioschyzon merolae, [214] Ostreococcus tauri, [215] Chlamydomonas, [49,216,217] Ostreococcus lucimarinus, [218] Phaeodactylum tricornutum, [219] Micromonas pusilla, etc.…”

Efficient CRISPR/Cas-mediated homologous recombination in the model diatomThalassiosira pseudonana

“…Gene targeting via HR has been demonstrated in a number of photosynthetic eukaryotes to date including Cyanidioschizon merolae (Minoda et al 2004), Ostreococcus tauri (Lozano et al 2014), Chlamydomonas reinhardtii (Greiner et al 2015), Nannochloropsis sp. (Kilian et al 2011), Phaeodactylumm tricornutum (Daboussi et al 2014;Weyman et al 2015), Physcomitrella patens (Kamisugi et al 2006;Kamisugi, Cuming, and Cove 2005;Kamisugi, Whitaker, and Cuming 2016), Arabidopsis (Schiml, Fauser, and Puchta 2014), Oryza (Begemann et al 2017) and…”