Original Research ArticleCRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002
Graphical abstract
Introduction
The ability to manipulate and predictably control gene expression is an essential tool for engineering metabolism and biology. Modern gene expression toolboxes include promoter libraries for initiating transcription at desired rates (Alper et al., 2005, Markley et al., 2015), transcriptional regulator/operator pairs for creating dynamic switches and circuits (Stanton et al., 2013, Zhang et al., 2012), transcription terminators for insulating neighboring expression cassettes (Chen et al., 2013) models of translation initiation for engineering ribosome binding sites (Espah Borujeni et al., 2014, Salis et al., 2010), codon optimization algorithms for optimizing gene sequences (Puigbò et al., 2007), and RNA structures that tune mRNA turnover, termination, and translation initiation (Pfleger et al., 2006). These tools have enhanced the study of natural systems and enabled the creation of engineered microbes for addressing societal challenges such as sustainable chemical production (Chubukov et al., 2014, Hara et al., 2014, Liao et al., 2016, Lynch and Gill, 2012, Smanski et al., 2016). Unfortunately, differences in how microbes recognize promoters, regulate gene expression, and translate protein do not allow genetic circuits and tools to be universally moved between organisms with the desired outcome. Instead synthetic biology tools must be validated in new hosts and then frequently adapted and refined for optimal functionality (Keasling, 2012).
The majority of established tools regulate genes in cis and require replacement of native expression cassettes with heterologous sequences to alter the level of gene expression. Replacing sequences has the unfortunate side-effect of removing native regulation that has often evolved to optimize the protein abundance needed for a given set of natural physiological states. In many instances, the native context is ideal for one state but needs to be altered for a new unnatural state (e.g. chemical production instead of growth). Trans acting tools that supersede native regulation in specific environments are therefore desirable additions to the synthetic biology toolbox (Copeland et al., 2014). One such tool, termed CRISPR interference (CRISPRi), takes advantage of the adaptive RNA-based defense system that in many bacteria targets and cleaves foreign nucleic acids such as viruses and plasmids (Qi et al., 2013). Diverse CRISPR-Cas systems exist in bacteria with altered locus architecture, components, maturation processes, and functions (Makarova et al., 2015). The type II system from Streptococcus pyogenes has been the most widely adapted to manipulate gene expression. A complex of a single guide RNA (sgRNA) containing a sequence complementary to the target and the Cas9 DNA nuclease protein can initiate double-stranded breaks. Point mutations in the two active sites can be used to create a nuclease deficient or dead Cas9 (dCas9). The complex of the sgRNA and dCas9 is still able to bind target DNA and can be used to either repress or activate gene expression (Bikard et al., 2013). There is strong evidence that repression is caused by the dCas9-sgRNA complex sterically blocking RNA polymerase elongation (Gilbert et al., 2013). The use of CRISPRi in Escherichia coli has led to efficient repression of targets as high as 300-fold when the sgRNA was targeted to the non-template strand near the 5′ end of the gene with no detectable off-target affects (Qi et al., 2013). This repression was reversible and multiplexed to target several genes simultaneously.
CRISPRi has become an increasingly attractive alternative to other trans-acting regulators repressor proteins such as trans-activator-like effectors (TALEs) or zinc fingers, due to the simplicity of design and ease of synthesis. By altering just 20 nucleotides of the sgRNA, the system can be designed to repress any gene of interest. However, it is necessary to choose a target site with a protospacer adjacent motif (PAM) which is 5′-NGG-3′ for S. pyogenes Cas9, but engineered Cas9 nucleases are reducing this constraint (Kleinstiver et al., 2015). Despite this limitation this technology has been used to repress reporter genes as well as enhance titers of products in various organisms: flavonoids in E. coli (Wu et al., 2015) and l-lysine and l-glutamate in Corneybacterium glutamicum (Cleto et al., 2016).
Here, we adapted this technology to an industrially relevant cyanobacterial strain, Synechococcus sp. strain PCC 7002 (PCC 7002) (Devroe et al., 2010, Reppas and Ridley, 2011). Cyanobacteria are an attractive chassis for chemical production that enables direct conversion of carbon dioxide and sunlight into useful products (Oliver and Atsumi, 2014). PCC 7002 is a promising strain of cyanobacteria because it grows rapidly, is halotolerant, is naturally transformable, can tolerate high light conditions, and has a growing synthetic biology toolbox (Markley et al., 2015, Zess et al., 2016). To add to the PCC 7002 toolbox, we developed a tunable gene repression system using CRISPRi. The system is novel because targets can be repressed to varying degrees by controlling the expression of CRISPRi components with an inducer, anhydrotetracycline (aTc). Most CRISPRi systems including one developed for another cyanobacterium Synechocystis sp. PCC 6803, can be turned on and off but the ability to titrate the levels of expression was not reported (Yao et al., 2015). Attempts have been made to tune repression by introducing mismatches in the sgRNA, but this involves making numerous strains to reach a variety of repression levels (Qi et al., 2013).
The ability to finely tune native gene expression will be critical for manipulating essential genes and to achieve growth regimes that decouple biomass and chemical production for maximum yield (Xu et al., 2014). In many cases, intermediate levels of enzyme expression result in maximum product titer (Freed et al., 2015, Pitera et al., 2007). With the described CRISPRi technology we can lower expression of essential cyanobacterial genes, decrease but not abolish flux towards competing products, and manipulate cellular processes to varying degrees. Here, we demonstrate the utility of finely tuning native gene expression by downregulating the abundance of phycobilisomes. In addition, we create a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrate a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation pathways. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date.
Section snippets
Chemicals, reagents, and media
Strains were grown and maintained on media A+ (Stevens et al., 1973) with 1.5% (w/v) Bacto-Agar (Fisher). Strains with antibiotic resistance markers were selected on media with antibiotics (kanamycin, 100 μg/mL; gentamicin, 30 μg/mL) and strains with cassettes introduced in the acsA locus were plated on 100 μM acrylic acid. Strains were grown in glass culture tubes (2×15 cm) with 20 mL media A+ and bubbled with either air (0.04% CO2) or high CO2 (10% CO2). Temperature was maintained at 38 °C and
Optimization of YFP repression with CRISPRi
To demonstrate a functional CRISPRi system in PCC 7002 we integrated three expression cassettes into the chromosome: 1) a fluorescent reporter, 2) an inducible dCas9, and 3) a constitutively expressed sgRNA (Fig. 1a). We constructed a reporter cassette consisting of a heterologous yellow fluorescence protein (EYFP) expressed from a strong constitutive promoter and a gentamicin resistance marker. The resulting cassette was integrated into a neutral site in the genome by replacing the glpK
Conclusions
CRISPRi provides a straightforward method to repress native genes of interest. We showed the functionality of this system genes in the fast-growing cyanobacterium, Synechococcus sp. strain PCC 7002 by repressing heterologous YFP and three native genes. We found that leaky expression of the system components was sufficient to achieve strong repression of its target and therefore the system required significant optimization to achieve the desired dynamic range of repression. We found that
Author information
Department of Chemical and Biological Engineering, University of Wisconsin-Madison Engineering Dr., Madison, WI 53706, United States.
Acknowledgements
This work was supported by the US Department of Energy through grant DE-SC0010329; the National Science Foundation grant EFRI-1240268; and the William F. Vilas Trust. GCG and TCK are recipients of NIH Biotechnology Training Fellowships (NIGMS - 5 T32 GM08349).
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2022, Bioresource TechnologyCitation Excerpt :Lactate production by Synechococcus sp. PCC 7002 was enhanced by CRISPRi-tunable gene repression of glutamine synthetase I (glnA) gene (Gordon et al., 2016). The genes for the biosynthesis of polyhydroxy butyrate (PHB) like polyhydroxyalkanoate (PHA) synthase (phaE and phaC) genes, glycogen, and aldehyde reductase/dehydrogenase like slr0942, sll0990, slr1192, and slr0091 were also silenced using this method (Yao et al., 2016).