Synthetic Gene Circuits Combining CRISPR Interference and CRISPR Activation in <i>E. coli</i>: Importance of Equal Guide RNA Binding Affinities to Avoid Context-Dependent Effects

Barbier, Içvara; Kusumawardhani, Hadiastri; Chauhan, Lakshya; Harlapur, Pradyumna Vinod; Jolly, Mohit Kumar; Schaerli, Yolanda

doi:10.1021/acssynbio.3c00375

Cited by 6 publications

(3 citation statements)

References 55 publications

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“…The use of orthogonal dCas9 genes has been previously reported in yeast and mammalian cells to engineer repression, activation, and deletion; , however, to our knowledge, the evaluation of orthogonality between different dCas9 systems is still unreported in bacteria, in which multitarget or multifunctional regulations (i.e., repression and induction in the same cell) have been successfully engineered using a single dCas9 gene. , The described data demonstrate the orthogonality between two CRISPRi repression systems to expand bacterial synthetic circuit design toolboxes.…”

Section: Resultsmentioning

confidence: 68%

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

De Marchi,

Shaposhnikov,

Gobaa

et al. 2024

ACS Synth. Biol.

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Synthetic circuit design is crucial for engineering microbes that process environmental cues and provide biologically relevant outputs. To reliably scale-up circuit complexity, the availability of parts toolkits is central. Streptococcus pyogenes (sp)-derived CRISPR interference/dead-Cas9 (CRISPRi/spdCas9) is widely adopted for implementing programmable regulations in synthetic circuits, and alternative CRISPRi systems will further expand our toolkits of orthogonal components. Here, we showcase the potential of CRISPRi using the engineered dCas9 from Staphylococcus aureus (sadCas9), not previously used in bacterial circuits, that is attractive for its low size and high specificity. We designed a collection of ∼20 increasingly complex circuits and variants in Escherichia coli, including circuits with static function like one-/two-input logic gates (NOT, NAND), circuits with dynamic behavior like incoherent feedforward loops (iFFLs), and applied sadCas9 to fix a T7 polymerase-based cascade. Data demonstrated specific and efficient target repression (100-fold) and qualitatively successful functioning for all circuits. Other advantageous features included low sadCas9-borne cell load and orthogonality with spdCas9. However, different circuit variants showed quantitatively unexpected and previously unreported steady-state responses: the dynamic range, switch point, and slope of NOT/NAND gates changed for different output promoters, and a multiphasic behavior was observed in iFFLs, differing from the expected bell-shaped or sigmoidal curves. Model analysis explained the observed curves by complex interplays among components, due to reporter geneborne cell load and regulator competition. Overall, CRISPRi/sadCas9 successfully expanded the available toolkit for bacterial engineering. Analysis of our circuit collection depicted the impact of generally neglected effects modulating the shape of component dose−response curves, to avoid drawing wrong conclusions on circuit functioning.

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Section: Resultsmentioning

confidence: 68%

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

De Marchi,

Shaposhnikov,

Gobaa

et al. 2024

ACS Synth. Biol.

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“…Still, we found that txCRISPRi resulted in no differential LNT production relative to the txCRISPRa-only strain ( Supplementary Figure S10 ). One possible explanation for the lack of improved LNT production from these CRISPRi targets is that the expression of additional gRNAs leads to competitive effects, in which gRNAs with varying binding affinities are competing for a limited pool of dCas9 ( 57 , 58 ). However, the LNT titers are similar between the conditions with no CRISPRi gRNA and the non-targeting gRNA.…”

Section: Resultsmentioning

confidence: 99%

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria

Cardiff,

Faulkner,

Beall

et al. 2024

Nucleic Acids Research

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Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. In Escherichia coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of other genes in the same operon. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens.

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“…We recently developed an approach for the construction of multi-gene CRISPR transcriptional control programs in bacteria, with activation (CRISPRa) or repression (CRISPRi) functions specified through the regulated expression of multiple guide RNAs (gRNAs) 11,12 . Recent demonstrations of dynamic multi-layer CRISPRa/i gene regulatory network designs in E. coli 13,14 and CRISPR-based metabolic pathway engineering in the soil microbe Pseudomonas putida [15][16][17] highlight the versatility of these systems for programmable multi-gene control. However, gaps in knowledge and technique continue to prevent the routine design of CRISPRa/i programs capable of quantitatively tuning activated expression from multiple bacterial genes at the same time 9,18 .…”

Section: Introductionmentioning

confidence: 99%

Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

Fontana,

Sparkman-Yager,

Faulkner

et al. 2023

Preprint

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Engineering bacterial metabolism to efficiently produce chemicals and materials from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important metabolic engineering tools for programming multi-gene expression regulation. However, poor predictability of guide RNA folding can disrupt enzyme balance through unreliable expression control. We devised a set of computational parameters that can describe guide RNA folding, and we expect them to be broadly applicable across CRISPR-Cas9 systems. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) inE. coliwith a kinetic parameter describing folding rate into the active structure. This parameter also enables forward design of new scRNAs, with no observed failures in our screen. We use CRISPRa target sequences from this set to design a system of three synthetic promoters that can orthogonally activate and tune expression of chosen outputs over a >35-fold dynamic range. Independent activation tuning allows experimental exploration of a three-dimensional expression design spaceviaa 64-member combinatorial triple-scRNA library. We apply these CRISPRa programs to two biosynthetic pathways, demonstrating production of valuable pteridine and human milk oligosaccharide products inE. coli. Profiling these design spaces indicated expression combinations producing up to 2.3-fold higher titer than that produced by maximal expression. Mapping production can also identify bottlenecks as targets for pathway redesign, improving titer of the oligosaccharide lacto-N-tetraose by 6-fold. Aided by computational scRNA efficacy prediction, the combinatorial CRISPRa strategy enables effective optimization of multi-step metabolic pathways. More broadly, the guide RNA design rules uncovered here may enable the routine design of effective multi-guide programs for a wide range of model- and data-driven applications of CRISPR gene regulation in bacterial hosts.

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Synthetic Gene Circuits Combining CRISPR Interference and CRISPR Activation in E. coli: Importance of Equal Guide RNA Binding Affinities to Avoid Context-Dependent Effects

Cited by 6 publications

References 55 publications

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria

Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

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