Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

Abstract

The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent non-homologous end-joining and polymerase theta (POLQ)-mediated end-joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect homology-directed repair of the inactivated paromomycin resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate homology-directed repair/gene inactivation. Our data suggests that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced homology-directed repair and random integration of transgenes, while KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in Chlamydomonas reinhardtii and can be used for future development of algal biotechnology.

Gene targeting in polymerase theta‐deficient Arabidopsis thaliana

“…We found the frequency of GT in the Polθ‐deficient genetic background to be reduced as compared with Col‐0, which may argue for a yet unidentified role for Polθ in HR itself, as was recently suggested for the model alga species Chlamydomonas reinhardtii (Sizova et al., 2021 ). Such a role, however, contradicts numerous studies performed in other eukaryotic model systems (e.g., Mara et al., 2019 ; McVey et al., 2016 ; Zelensky et al., 2017 ).…”

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

“…Five colonies harboring an integrated DNA donor at the CrKU80 ‐gRNA1 target site were identified from the #3 Pm R pool (Figure S2f–h). Spot growth experiments containing low dose of the DNA‐damaging agent zeocin showed CrKU80 mutant cells were more sensitive to zeocin than WT cells (Figure S2i), consistent with previous reports (Ferenczi et al., 2021; Rohr et al., 2004; Sizova et al., 2021). These data indicated that the null mutation of CrKU80 was obtained.…”

“…That is, the long DNA donor may mediate HR‐dependent illegitimate recombination, while the integration of short donor DNA might participate in the MMEJ pathway, in which exogenous DNA fragments can be integrated into the genome without base deletion at the junction site (Sakuma et al., 2016; Yao et al., 2017). The MMEJ pathway reportedly plays a predominant role in the repair of CRISPR/Cas9‐induced DSBs in Chlamydomonas (Sizova et al., 2021).…”

“…MMEJ‐based editing using 25‐bp micro‐homologous arms combined with the CRISPR/Cas9 system has enabled precise knock‐in at target sites in worms, frogs, human cells, zebrafish, yeast and Chlamydomonas (Hayashi & Tanaks, 2019; Hisano et al., 2015; Nakade et al., 2014; Picariello et al., 2020). More recently, NHEJ, HR and MMEJ were shown to contribute to the repair of CRISPR/Cas9‐induced DSBs in Chlamydomonas , with MMEJ playing a predominant role (Akella et al., 2021; Ferenczi et al., 2021; Sizova et al., 2021). This knowledge had not yet been applied to engineering Chlamydomonas strains, but it did suggest that providing a donor with microhomology may facilitate precise gene editing via DNA integration in Chlamydomonas .…”

Algal model species for advancing biological sciences

“…The availability of the first algal genomes laid the foundation for developing novel molecular tools with CRISPR/Cas-based genome editing likely being the most impactful tool (e.g., Jiang et al 2014, Hopes et al 2016, Nymark et al 2016, Badis et al 2021. Further developments of these CRISPR/Cas tools in recent years have made some algal species as genetically tractable as well-established models in biology (e.g., Moosburner et al 2020, Sizova et al 2021, Belshaw et al 2022. Although significant progress has also been made concerning the development of biological resources and data banks for algae (e.g., Maheswari et al 2008, Vandepoele et al 2013, Grigoriev et al 2021 are not yet matching those of wellestablished model organisms in terms of their diversity and comprehension (e.g., Rhee et al 2003, Chen et al 2005, Keseler et al 2011.…”