Abstract:BackgroundIn bacteria, transcription units can be insulated by placing a terminator in front of a promoter. In this way promoter leakage due to the read-through from an upstream gene or RNA polymerase unspecific binding to the DNA is, in principle, removed. Differently from bacterial terminators, yeast S. cerevisiae terminators contain a hexamer sequence, the efficiency element, that strongly resembles the eukaryotic TATA box i.e. the promoter sequence recognized and bound by RNA polymerase II.ResultsBy placin… Show more
“…The output of our biosensing circuits, which were based on dSpCas9:gRNA–AcrAII (anti-CRISPR) interactions, was yEGFP. This reporter protein is expressed by the synthetic promoter Tsynth8_pCYC1noTATA, produced by fusing a synthetic terminator (Tsynth8 [ 5 ]) to the minimal CYC1 promoter stripped of its TATA boxes (pCYC1noTATA [ 35 ]). Thus, the efficiency element of Tsynth8 became the TATA box of the synthetic promoter.…”
Section: Resultsmentioning
confidence: 99%
“…As depicted in Fig. 4 a , the chimeric protein LmAcrIIA2–HBD(ER) was placed downstream of the yeast TEF2 promoter (47% as strong as pGPD [ 35 ]). Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The vector targeted by the CRISPR-dSpCas9 system (pMM260) contains the transcription unit Tsynth8_pCYC1noTATA-yEGFP-CYC1t. The assembly of this plasmid is described in our previous work [ 35 ].…”
Section: Methodsmentioning
confidence: 99%
“…After identifying the best solution for repressing the production of yEGFP from the synthetic promoter Tsynth8_pCYC1noTATA [ 35 ] employed in our circuits, we constructed several small networks (four genes overall, as mentioned above), each containing one of the anti-CRISPR proteins LmAcrIIA2, LmAcrIIA4, and StAcrIIA5. They were expressed upon induction with galactose.…”
BackgroundAnti-CRISPR proteins are expressed by phages as a reaction to the bacterial CRISPR–Cas defense system. Recently, the structures of anti-CRISPR proteins have been determined, and their diverse functions have been clarified. Anti-CRISPR proteins such as LmAcrIIA2 and LmAcrIIA4 interact with the SpCas9:gRNA system and occlude the protospacer adjacent motif (PAM) recognition site, thereby preventing SpCas9:gRNA from binding to the DNA. Hence, anti-CRISPR proteins represent a powerful means to control and modulate the activity of SpCas9 and its nuclease-deficient version dSpCas9. LmAcrIIA2 and LmAcrIIA4 have been shown to be efficient inhibitors of SpCas9 in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells. To date, there have been no reports of anti-CRISPR-based synthetic gene circuits engineered into yeast cells.ResultsWe constructed in the yeast S. cerevisiae synthetic biosensors based on the anti-CRISPR–dSpCas9:gRNA interaction. Upon induction with galactose or β-estradiol, anti-CRISPR proteins (LmAcrIIA4, LmAcrIIA2, and StAcrIIA5) produced an enhancement in fluorescence expression by preventing the dSpCas9–Mxi1:gRNA complex from binding to the DNA. We found that LmAcrIIA2 performed as well as LmAcrIIA4 in S. cerevisiae, whereas StAcrIIA5, which had previously been tested in bacteria only, had non-negligible negative effects on yeast cell growth. The efficiency of anti-CRISPR-based biosensors was strongly dependent on the means by which the guide RNAs were produced. The best performance, as measured by the increase in fluorescence, was achieved using a “ribozyme–gRNA–ribozyme” expression cassette under the control of the yeast constitutive ADH1 promoter.ConclusionsThis work demonstrates that anti-CRISPR proteins are effective dSpCas9 suppressors in yeast cells. In particular, LmAcrIIA2 and LmAcrIIA4 could be employed as new components of yeast synthetic gene circuits.Electronic supplementary materialThe online version of this article (10.1186/s13036-018-0101-z) contains supplementary material, which is available to authorized users.
“…The output of our biosensing circuits, which were based on dSpCas9:gRNA–AcrAII (anti-CRISPR) interactions, was yEGFP. This reporter protein is expressed by the synthetic promoter Tsynth8_pCYC1noTATA, produced by fusing a synthetic terminator (Tsynth8 [ 5 ]) to the minimal CYC1 promoter stripped of its TATA boxes (pCYC1noTATA [ 35 ]). Thus, the efficiency element of Tsynth8 became the TATA box of the synthetic promoter.…”
Section: Resultsmentioning
confidence: 99%
“…As depicted in Fig. 4 a , the chimeric protein LmAcrIIA2–HBD(ER) was placed downstream of the yeast TEF2 promoter (47% as strong as pGPD [ 35 ]). Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The vector targeted by the CRISPR-dSpCas9 system (pMM260) contains the transcription unit Tsynth8_pCYC1noTATA-yEGFP-CYC1t. The assembly of this plasmid is described in our previous work [ 35 ].…”
Section: Methodsmentioning
confidence: 99%
“…After identifying the best solution for repressing the production of yEGFP from the synthetic promoter Tsynth8_pCYC1noTATA [ 35 ] employed in our circuits, we constructed several small networks (four genes overall, as mentioned above), each containing one of the anti-CRISPR proteins LmAcrIIA2, LmAcrIIA4, and StAcrIIA5. They were expressed upon induction with galactose.…”
BackgroundAnti-CRISPR proteins are expressed by phages as a reaction to the bacterial CRISPR–Cas defense system. Recently, the structures of anti-CRISPR proteins have been determined, and their diverse functions have been clarified. Anti-CRISPR proteins such as LmAcrIIA2 and LmAcrIIA4 interact with the SpCas9:gRNA system and occlude the protospacer adjacent motif (PAM) recognition site, thereby preventing SpCas9:gRNA from binding to the DNA. Hence, anti-CRISPR proteins represent a powerful means to control and modulate the activity of SpCas9 and its nuclease-deficient version dSpCas9. LmAcrIIA2 and LmAcrIIA4 have been shown to be efficient inhibitors of SpCas9 in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells. To date, there have been no reports of anti-CRISPR-based synthetic gene circuits engineered into yeast cells.ResultsWe constructed in the yeast S. cerevisiae synthetic biosensors based on the anti-CRISPR–dSpCas9:gRNA interaction. Upon induction with galactose or β-estradiol, anti-CRISPR proteins (LmAcrIIA4, LmAcrIIA2, and StAcrIIA5) produced an enhancement in fluorescence expression by preventing the dSpCas9–Mxi1:gRNA complex from binding to the DNA. We found that LmAcrIIA2 performed as well as LmAcrIIA4 in S. cerevisiae, whereas StAcrIIA5, which had previously been tested in bacteria only, had non-negligible negative effects on yeast cell growth. The efficiency of anti-CRISPR-based biosensors was strongly dependent on the means by which the guide RNAs were produced. The best performance, as measured by the increase in fluorescence, was achieved using a “ribozyme–gRNA–ribozyme” expression cassette under the control of the yeast constitutive ADH1 promoter.ConclusionsThis work demonstrates that anti-CRISPR proteins are effective dSpCas9 suppressors in yeast cells. In particular, LmAcrIIA2 and LmAcrIIA4 could be employed as new components of yeast synthetic gene circuits.Electronic supplementary materialThe online version of this article (10.1186/s13036-018-0101-z) contains supplementary material, which is available to authorized users.
“…Taking inspiration from the paper by Ede et al [ 25 ], we constructed, in this work, a new library of S. cerevisiae constitutive promoters based on foreign core promoters—mainly from viruses plus one from human cells and another from the yeast Schizosaccharomyces pombe . To make them functional in S. cerevisiae , we extended them with an 87 nt-long sequence, termed pCYC1noTATA [ 26 ], that contains the full 5′UTR of the yeast CYC1 promoter where at least six TSSs are present (see Figure 1 B). The pCYC1noTATA provided the necessary spacing for the TATA box of the foreign core promoters to activate transcription from one or more TSSs belonging to pCYC1.…”
Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.
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