A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast

Novarina, Daniele; Koutsoumpa, Andriana; Milias-Argeitis, Andreas

doi:10.1016/j.xpro.2022.101358

Cited by 4 publications

(3 citation statements)

References 18 publications

(32 reference statements)

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“…Following this reasoning, we constructed a hxk -null strain, in which the three isozymes of hexokinases (Hxk1, Hxk2, and Glk1) were deleted using CRISPR-Cas9 19 , 20 . To estimate the glucose carryover per surface area ( ) hxk-null , we grew the hxk -null strain in a minimal medium supplemented with galactose until the mid-exponential phase.…”

Section: Resultsmentioning

confidence: 99%

“…The hexokinase-null ( hxk -null) mutant was constructed on the S288C-derived prototrophic YSBN6 strain ( MATa ho::HphMX4 ) 23 via CRISPR-Cas9 using a MoClo Yeast Toolkit 19 , 20 . Target sequences and repair fragments used for CRISPR-Cas9 deletions can be found in Table S2 .…”

Section: Methodsmentioning

confidence: 99%

“…Assuming equilibrium, the amount of intracellular glucose can then be estimated using the total cell volume (V tot ) and the extracellular glucose concentration (c glc, ex ). By subtracting the calculated intracellular amount of glucose from the total glucose determined in the pellet (n glc, pellet ), one can then estimate the amount of glucose carryover (n glc, co ) and normalize it to the total cell surface area ( S tot,hxk−null ), with the following equation: Following this reasoning, we constructed a hxk-null strain, in which the three isozymes of hexokinases (Hxk1, Hxk2, and Glk1) were deleted using CRISPR-Cas9 19,20 . To estimate the glucose carryover per surface area (…”

Section: Evaluation Of the Contribution Of The Sticking Effect To The...mentioning

confidence: 99%

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Quantifying intracellular glucose levels when yeast is grown in glucose media

Li,

Heinemann

2023

Sci Rep

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In Saccharomyces cerevisiae, intracellular glucose levels impact glucose transport and regulate carbon metabolism via various glucose sensors. To investigate mechanisms of glucose sensing, it is essential to know the intracellular glucose concentrations. Measuring intracellular glucose concentrations, however, is challenging when cells are grown on glucose, as glucose in the water phase around cells or stuck to the cell surface can be carried over during cell sampling and in the following attributed to intracellular glucose, resulting in an overestimation of intracellular glucose concentrations. Using lactose as a carryover marker in the growth medium, we found that glucose carryover originates from both the water phase and from sticking to the cell surface. Using a hexokinase null strain to estimate the glucose carryover from the cell surface, we found that glucose stuck on the cell surface only contributes a minor fraction of the carryover. To correct the glucose carryover, we revisited l-glucose as a carryover marker. Here, we found that l-glucose slowly enters cells. Thus, we added l-glucose to yeast cultures growing on uniformly 13C-labeled d-glucose only shortly before sampling. Using GC–MS to distinguish between the two differently labeled sugars and subtracting the carryover effect, we determined the intracellular glucose concentrations among two yeast strains with distinct kinetics of glucose transport to be at 0.89 mM in the wild-type strain and around 0.24 mM in a mutant with compromised glucose uptake. Together, our study provides insight into the origin of the glucose carryover effect and suggests that l-glucose added to the culture shortly before sampling is a possible method that yet has limitations with regard to measurement accuracy.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Evaluation Of the Contribution Of The Sticking Effect To The...mentioning

confidence: 99%

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Quantifying intracellular glucose levels when yeast is grown in glucose media

Li,

Heinemann

2023

Sci Rep

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Rapid and reversible regulation of cell cycle progression in budding yeast using optogenetics

Koutsoumpa,

Milias-Argeitis

2024

Preprint

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The regulatory complexity of the eukaryotic cell cycle poses technical challenges in experiment design and data interpretation, leaving gaps in our understanding of how cells coordinate cell cycle-related processes. Traditional methods, such as knockouts and deletions are often ineffective to compensatory interactions in the cell cycle control network, while chemical agents that cause cell cycle arrest can have undesired pleiotropic effects. Synthetic inducible systems targeting specific cell cycle regulators offer potential solutions but are limited by the need for external inducers, which make fast reversibility technically challenging. To address these issues, we developed an optogenetic tool (OPTO-Cln2) that enables light-controlled and reversible regulation of G1 progression in budding yeast. Through extensive validation and benchmarking via time-lapse microscopy, we verify that OPTO-Cln2-carrying strains can rapidly toggle between normal and altered G1 progression. By integrating OPTO-Cln2 with a readout of nutrient-sensing pathways (TORC1 and PKA), we show that the oscillatory activity of these pathways is tightly coordinated with G1 progression. Finally, we demonstrate that the rapid reversibility of OPTO-Cln2 facilitates multiple cycles of synchronous arrest and release of liquid cell cultures. Our work provides a powerful new approach for studying cell cycle dynamics and the coordination of growth- with division-related processes.

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Automated plasmid design for marker-free genome editing in budding yeast

Stojković,

Gligorovski,

Geramimanesh

et al. 2024

Preprint

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The ease of genome editing has contributed to the popularity of budding yeast as a model organism. However, the palette of selectable markers is in principle limited as most can only be used once. Some markers such as URA3 and TRP1 can be recycled through counterselection. This permits seamless genome modification with pop-in/pop-out (PIPO), in which a DNA construct first integrates in the genome and, subsequently, homologous regions recombine and excise undesired sequences. Popular approaches for creating such constructs use oligonucleotides and polymerase chain reaction (PCR). The drawbacks are that long oligonucleotides are unstable, can form secondary structures that interfere with PCR, cannot be regenerated in a typical biological laboratory, and are only widely available for lengths less than about 120, which limits the homology and efficiency that can be attained. With the rapid reduction in price, synthesizing custom DNA sequences in specific plasmid backbones has become an appealing alternative. For designing plasmids for seamless PIPO gene tagging or deletion, there are a number of factors to consider. To create only the shortest DNA sequences necessary, avoid errors in manual design, specify the amount of homology desired, and customize restriction sites, we created the computational tool PIPOline. Using it, we tested the ratios of homology that improve pop-out efficiency when targeting the genes HTB2 or WHI5. We supply optimal PIPO plasmid sequences for tagging or deleting almost all S288C budding yeast open reading frames (ORFs). Finally, we demonstrate how the histone variant Htb2 marked with a red fluorescent protein can be used as a cell-cycle stage marker, alternative to superfolder GFP (sfGPF), reducing light toxicity. We expect PIPOline to streamline genome editing in budding yeast.

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A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast

Cited by 4 publications

References 18 publications

Quantifying intracellular glucose levels when yeast is grown in glucose media

Quantifying intracellular glucose levels when yeast is grown in glucose media

Rapid and reversible regulation of cell cycle progression in budding yeast using optogenetics

Automated plasmid design for marker-free genome editing in budding yeast

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