Homology-Directed Transgene-Free Gene Editing in Chlamydomonas reinhardtii
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Chlamydomonas reinhardtii is a microalgal model organism with a suite of molecular and genetic techniques, but routine editing of its nuclear genome is yet to be realized. DNA-based transformation techniques are prohibitively inefficient and lead to predominantly nonhomologous (i.e. off-target) integration. Standard CRISPR-based gene editing protocols have proved too ineffective to enable routine application. We have found that the use of CRISPR-Cpf1 in conjunction with single-stranded DNA (ssODN) repair templates achieves nuclear gene editing efficiencies as high as 20% as a proportion of total cells (Ferenczi et al. Proc Natl Acad Sci U S A 114:13567–13572, 2017). This produces edits with predictable outcomes in a transgene- and selection marker-free manner. The possibility to purchase all necessary reagents commercially with no preparation time (besides design) facilitates rapid and routine genetic engineering in this organism. Here we describe the use of this technique to knockout locus FKB12, which leads to rapamycin resistance and lends itself to an easy assay when adopting this gene-editing protocol.
Key wordsCRISPR Cpf1 Chlamydomonas reinhardtii ssODN Homology-directed repair (HDR)
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) PHYCONET Proof of Concept Fund, Grant PHYCPoC-31. A.F. was supported by the BBSRC East of Scotland BioScience (EASTBIO) National Productivity Investment Fund (NPIF) Industrial Cooperative Awards in Science & Technology (CASE) studentship BB/R505493/1.We thank Prof Andrew Hudson and Dr. Anja Hemschemeier for reviewing the manuscript.
- 4.Zhang R, Patena W, Armbruster U, Gang SS, Blum SR, Jonikas MC (2014) High-throughput genotyping of Green Algal mutants reveals random distribution of mutagenic insertion sites and endonucleolytic cleavage of transforming DNA. Plant Cell 26(4):1398–1409. https://doi.org/10.1105/tpc.114.124099CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Greiner A, Kelterborn S, Evers H, Kreimer G, Sizova I, Hegemann P (2017) Targeting of photoreceptor genes in Chlamydomonas reinhardtii via Zinc-finger nucleases and CRISPR/Cas9. Plant Cell. https://doi.org/10.1105/tpc.17.00659
- 10.Shin SE, Lim JM, Koh HG, Kim EK, Kang NK, Jeon S, Kwon S, Shin WS, Lee B, Hwangbo K, Kim J, Ye SH, Yun JY, Seo H, Oh HM, Kim KJ, Kim JS, Jeong WJ, Chang YK, Jeong BR (2016) CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Sci Rep 6:27810. https://doi.org/10.1038/srep27810CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163(3):759–771. https://doi.org/10.1016/j.cell.2015.09.038CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Ferenczi A, Pyott DE, Xipnitou A, Molnar A (2017) Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA. Proc Natl Acad Sci U S A 114(51):13567–13572. https://doi.org/10.1073/pnas.1710597114CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Hutner SH, Provasoli L, Schatz A, Haskins CP (1950) Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94(2):152–170Google Scholar
- 18.Harris EH, Stern DB, Witman GB (2009) The Chlamydomonas sourcebook, 2nd edn. Academic Press, San DiegoGoogle Scholar