Efficient methods for multiple types of precise gene‐editing in Chlamydomonas

Abstract

Precise gene-editing using CRISPR/Cas9 technology remains a long-standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi-type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low-expression genes (CrTET1 and CrKU80), the introduction of a FLAG-HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live-cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3 0 -UTR of MAA7 and VIPP1 resulted in a stable knock-down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.

The synthetic future of algal genomes

“…Deletion or downregulation of components of the NHEJ pathway for DNA repair such as Ku80 increased the frequency of HR-mediated integration by up to 70% in Kluyveromcyes marxianus and other yeasts. 104 RNP-mediated gene editing to make knockouts and targeted insertions of small transgene cassettes has been demonstrated in Chlamydomonas , 98 , 105 , 106 and the frequency of homology-directed repair (HDR) in gene editing was improved by mutagenesis of Ku80 . 107 However, targeted integration of large, complex, multi-gene expression constructs for synthetic biology has not yet been demonstrated.…”

Bilin‐regulated LHCA1 accumulation is independent of photoreceptors PHOT, CRYs, and UVR8 in Chlamydomonas reinhardtii

“…The uvr8-1 hmox1 mutant was created by the recently established CRISPR-Cas9 method in C. reinhardtii (Chen et al, 2023;Greiner et al, 2017). Briefly, gRNA (5′-ACCGAGCAGCGCTATTGAAT-3′) targeting to the first exon of UVR8 gene was designed by Cas-Designer tool (http:// www.…”

“…The gRNA was transcribed in vitro and purified using HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2050S) and Monarch RNA Cleanup Kit (NEB, T2050L) following the recommended protocol. Purified gRNA was quantified and the Cas9-gRNA cleavage activity was validated in vitro as described in (Chen et al, 2023). gRNA (70-80 μg) was further assembled with SpCas9 protein (100-120 μg) in the cleavage buffer (2 mM HEPES pH 7.5, 15 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) at 37°C for 15 min to form Cas9-gRNA complex.…”