Gene Editing in Green Alga Chlamydomonas reinhardtii via CRISPR-Cas9 Ribonucleoproteins

Kelterborn, Simon; Boehning, Francisca; Сизова, И. А.; Baidukova, Olga; Evers, Heide; Hegemann, Peter

doi:10.1007/978-1-0716-1791-5_3

Cited by 25 publications

(26 citation statements)

References 11 publications

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Section: Generation Of Mutants By Crispr-cas9mentioning

confidence: 99%

“…SpCas9-His was assembled with Alt-R ® CRISPR-Cas9 trans-activating crRNA (tracrRNA) and crRNA to form the Cas9 ribonucleotide complex (RNP) as described in Kelterborn et al (2022). The Cas9 RNP and PCR-amplified AphVII cassette were co-introduced to Chlamydomonas by electroporation (Kelterborn et al, 2022) with modifications. Cells at the logarithmic growth phase (OD730 = 0.3-0.5) in TAP medium were harvested at 600×g (5 min at room temperature) and suspended in transformation reagent (Max Efficiency, Cat No.…”

Section: Generation Of Mutants By Crispr-cas9mentioning

confidence: 99%

“…The cell suspension (1.0×10 8 cells mL −1 ) was incubated at 40°C for 30 min with gentle shaking (350 rpm) in a thermo shaker (Allsheng Instruments, Hangzhou, China). Although cells were cooled for 1 h at room temperature in the original protocol (Kelterborn et al, 2022), we modified this step to rapid cooling for 3 min on ice.…”

Section: Generation Of Mutants By Crispr-cas9mentioning

confidence: 99%

“…We used a protocol described in Kelterborn et al (2022) for genome editing with modifications (See details in the Materials and Methods). Because CRISPR-Cas9 in…”

Section: Generation Of Tar1 Mutants By Crispr-cas9 From Different Wil...mentioning

confidence: 99%

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A YAK1-type protein kinase, triacylglycerol accumulation regulator 1, in the green alga Chlamydomonas reinhardtii is a potential regulator of cell division and differentiation into gametes during photoautotrophic nitrogen deficiency

Tsuji

Kinoshita

Tsukahara

et al. 2023

J. Gen. Appl. Microbiol.

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Section: Generation Of Mutants By Crispr-cas9mentioning

confidence: 99%

Section: Generation Of Mutants By Crispr-cas9mentioning

confidence: 99%

Section: Generation Of Mutants By Crispr-cas9mentioning

confidence: 99%

“…We used a protocol described in Kelterborn et al (2022) for genome editing with modifications (See details in the Materials and Methods). Because CRISPR-Cas9 in…”

Section: Generation Of Tar1 Mutants By Crispr-cas9 From Different Wil...mentioning

confidence: 99%

See 2 more Smart Citations

A YAK1-type protein kinase, triacylglycerol accumulation regulator 1, in the green alga Chlamydomonas reinhardtii is a potential regulator of cell division and differentiation into gametes during photoautotrophic nitrogen deficiency

Tsuji

Kinoshita

Tsukahara

et al. 2023

J. Gen. Appl. Microbiol.

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show abstract

“…For CRISPR-Cas9-based editing of the Chlamydomonas genome, we used technique in which recombinant Streptococcus pyogenes Cas9 is pre-assembled with synthetic guide RNA (gRNA) and transformed directly into the cells as ribonucleoprotein (RNP) complexes 24 , 25 . The Cas9 RNP complex induces double-stranded DNA breaks that are mostly repaired by error-prone non-homologous end-joining or the more rarely occurring homology-directed repair (HDR) mechanisms that use homologous DNA as repair templates.…”

Section: Resultsmentioning

confidence: 99%

Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation

et al. 2022

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The green unicellular alga Chlamydomonas reinhardtii with two photoreceptors called channelrhodopsins is a model organism that gave birth to a new scientific field of biomedical studies, optogenetics. Although channelrhodopsins are helping to decipher the activity of the human brain, their functionality has never been extensively studied in the organism of origin, mainly due to the difficulties connected to reverse genetic interventions. In this study, we present a CRISPR-Cas9-based technique that enables a precise in vivo exchange of single amino acids in a selected gene. To shed light on the function of channelrhodopsins ChR1 (C1) and ChR2 (C2) in vivo, we deleted both channelrhodopsins independently in the wild-type strain and introduced point mutations in the remaining channel, causing modified photocycle kinetics and ion selectivity. The mutated strains, ΔC1C2-E123T, ΔC1C2-E90R and ΔC1C2-E90Q, showed about 100-fold decrease in photosensitivity, a reduced photophobic response and faster light adaptation rates due to accelerated photocycle kinetics and reduced Ca2+ conductance. Moreover, the ΔC1C2-E90Q with an additionally reduced H+ permeability produced an electrical response only in the presence of Na+ ions, highlighting a contribution and importance of H+ conductance to photocurrents in the wild-type algae. Finally, in the ΔC1C2-E90R strain with the channelrhodopsin selectivity converted to anions, no photo-responses were detected. We conclude that the precise photocycle kinetics and the particular ion selectivity of channelrhodopsins are the key parameters for efficient phototaxis in low light conditions.

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Recent progress and challenges in CRISPR-Cas9 engineered algae and cyanobacteria

et al. 2023

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Gene Editing in Green Alga Chlamydomonas reinhardtii via CRISPR-Cas9 Ribonucleoproteins

Cited by 25 publications

References 11 publications

A YAK1-type protein kinase, triacylglycerol accumulation regulator 1, in the green alga <i>Chlamydomonas</i> <i>reinhardtii</i> is a potential regulator of cell division and differentiation into gametes during photoautotrophic nitrogen deficiency

A YAK1-type protein kinase, triacylglycerol accumulation regulator 1, in the green alga <i>Chlamydomonas</i> <i>reinhardtii</i> is a potential regulator of cell division and differentiation into gametes during photoautotrophic nitrogen deficiency

Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation

Recent progress and challenges in CRISPR-Cas9 engineered algae and cyanobacteria

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