Project Goals: Engineering of strains for industrial production requires the targeted improvement... more Project Goals: Engineering of strains for industrial production requires the targeted improvement of multiple complex traits ranging from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for generating targeted genomic modifications at high efficiency along with high throughput phenotypic screening and growth strategies to rapidly engineer multiple traits in Escherichia coli.
Genome engineering methodologies are transforming biological research and discovery. Approaches b... more Genome engineering methodologies are transforming biological research and discovery. Approaches based on CRISPR technology have been broadly adopted and there is growing interest in the generation of massively parallel edited cell libraries. Comparing the libraries generated by these varying approaches is challenging and researchers lack a common framework for defining and assessing the characteristics of these libraries. Here we describe a framework for evaluating massively parallel libraries of edited genomes based on established methods for sampling complex populations. We define specific attributes and metrics that are informative for describing a complex cell library and provide examples for estimating these values. We also connect this analysis to generic phenotyping approaches, using either pooled (typically via a selection assay) or isolate (often referred to as screening) phenotyping approaches. We approach this from the context of creating massively parallel, precisely edi...
CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To fu... more CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.
CRISPR–Cas systems have revolutionized genome editing across a broad range of biotechnological en... more CRISPR–Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demon...
Sequence to activity mapping technologies are rapidly developing, enabling the isolation of mutat... more Sequence to activity mapping technologies are rapidly developing, enabling the isolation of mutations that confer novel phenotypes. Here we used the CRISPR EnAbled Trackable genome Engineering (CREATE) technology to investigate the inhibition of the essential IspC gene in Escherichia coli. IspC gene product, Deoxyxylulose Phosphate Reductoisomerase (DXR), converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the DXP pathway. Since this pathway is shared with many pathogenic bacteria and protozoa and is missing in humans, it is an appealing target for inhibition. We created a full saturation library of 33 sites proximal to ligand binding and other sites and challenged it with the DXR-specific inhibitor, fosmidomycin. We identified several mutations that confer fosmidomycin resistance. All sites are highly conserved and also exist in pathogens including the malaria-inducing Plasmodium falciparum. These findings may have general implications on the isolation...
Isopropanol is an important target molecule for sustainable production of fuels and chemicals. In... more Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these va...
Methods for importing heterologous genes into genetically tractable hosts are among the most desi... more Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.
Since the 1970s technological advancements in the fields of synthetic biology and metabolic engin... more Since the 1970s technological advancements in the fields of synthetic biology and metabolic engineering have led to a dramatic reduction in both time and cost required for generating genomic mutations in a variety of organisms. The union of genomic editing machinery, DNA inkjet printers, and bioinformatics algorithms allows engineers to design a library of thousands of unique oligos as well as build and test these designs on a ∼2 months time-scale and at a cost of roughly ∼0.3 cents per base pair. The implications of these capabilities for a variety of fields are far-reaching, with potential impacts in defense, agricultural, human health, and environmental research. The explosion of synthetic biology applications over the past two decades have led many to draw parallels between biological engineering and the computer sciences. In this review, we highlight some important parallels between these fields and emphasize the importance of engineering design strategies.
A critical feature of the hypothesized RNA world would have been the ability to control chemical ... more A critical feature of the hypothesized RNA world would have been the ability to control chemical processes in response to environmental cues. Riboswitches present themselves as viable candidates for a sophisticated mechanism of regulatory control in RNA-based life. These regulatory elements in the modern world are most commonly found in the 5'-untranslated regions of bacterial mRNAs, directly interacting with metabolites as a means of regulating expression of the coding region via a secondary structural switch. In this review, we focus on recent insights into how these RNAs fold into complex architectures capable of both recognizing a specific small molecule compound and exerting regulatory control over downstream sequences, with an emphasis on transcriptional regulation.
Methods in molecular biology (Clifton, N.J.), 2009
Structural biology plays a central role in gaining a full understanding of the myriad roles of RN... more Structural biology plays a central role in gaining a full understanding of the myriad roles of RNA in biology. In recent years, innovative approaches in RNA purification and crystallographic methods have lead to the visualization of an increasing number of unique structures, providing new insights into its function at the atomic level. This article presents general protocols which have streamlined the process of obtaining a homogeneous sample of properly folded and active RNA in high concentrations that crystallizes well in the presence of a suitable heavy-atom for phasing. Of particular importance are approaches toward RNA crystallography that include exploring "construct space" as opposed to "condition space". Moreover, development of a highly flexible method for experimentally phasing RNA crystals may open the door to a relatively simple means of solving these structures.
Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part B, 2009
A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have ... more A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have been solved to atomic resolution using X-ray crystallography. As with proteins, RNA crystallography involves a number of screening trials in which the concentration of macromolecule, precipitant, salt, and temperature are varied, an approach known as searching "condition space." In contrast to proteins, the nature of base pairing in nucleic acids creates predictable secondary structure that facilitates the rational design of RNA variants, allowing "sequence space" to be screened in parallel. This chapter reviews RNA-specific techniques and considerations for RNA crystallography and presents a complete workflow used by our laboratory for solving RNA structures starting with initial library construction, methods to investigate and improve RNA crystal quality, and finally phase determination and structure solution.
Rewiring and optimization of metabolic networks to enable the production of commercially valuable... more Rewiring and optimization of metabolic networks to enable the production of commercially valuable chemicals is a central goal of metabolic engineering. This prospect is challenged by the complexity of metabolic networks, lack of complete knowledge of gene function(s), and the vast combinatorial genotype space that is available for exploration and optimization. Various approaches have thus been developed to aid in the efficient identification of genes that contribute to a variety of different phenotypes, allowing more rapid design and engineering of traits desired for industrial applications. This review will highlight recent technologies that have enhanced capabilities to map genotype-phenotype relationships on a genome wide scale and emphasize how such approaches enable more efficient design and engineering of complex phenotypes.
Project Goals: Engineering of strains for industrial production requires the targeted improvement... more Project Goals: Engineering of strains for industrial production requires the targeted improvement of multiple complex traits ranging from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for generating targeted genomic modifications at high efficiency along with high throughput phenotypic screening and growth strategies to rapidly engineer multiple traits in Escherichia coli.
Genome engineering methodologies are transforming biological research and discovery. Approaches b... more Genome engineering methodologies are transforming biological research and discovery. Approaches based on CRISPR technology have been broadly adopted and there is growing interest in the generation of massively parallel edited cell libraries. Comparing the libraries generated by these varying approaches is challenging and researchers lack a common framework for defining and assessing the characteristics of these libraries. Here we describe a framework for evaluating massively parallel libraries of edited genomes based on established methods for sampling complex populations. We define specific attributes and metrics that are informative for describing a complex cell library and provide examples for estimating these values. We also connect this analysis to generic phenotyping approaches, using either pooled (typically via a selection assay) or isolate (often referred to as screening) phenotyping approaches. We approach this from the context of creating massively parallel, precisely edi...
CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To fu... more CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.
CRISPR–Cas systems have revolutionized genome editing across a broad range of biotechnological en... more CRISPR–Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demon...
Sequence to activity mapping technologies are rapidly developing, enabling the isolation of mutat... more Sequence to activity mapping technologies are rapidly developing, enabling the isolation of mutations that confer novel phenotypes. Here we used the CRISPR EnAbled Trackable genome Engineering (CREATE) technology to investigate the inhibition of the essential IspC gene in Escherichia coli. IspC gene product, Deoxyxylulose Phosphate Reductoisomerase (DXR), converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the DXP pathway. Since this pathway is shared with many pathogenic bacteria and protozoa and is missing in humans, it is an appealing target for inhibition. We created a full saturation library of 33 sites proximal to ligand binding and other sites and challenged it with the DXR-specific inhibitor, fosmidomycin. We identified several mutations that confer fosmidomycin resistance. All sites are highly conserved and also exist in pathogens including the malaria-inducing Plasmodium falciparum. These findings may have general implications on the isolation...
Isopropanol is an important target molecule for sustainable production of fuels and chemicals. In... more Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli. We first constructed variants to explore the effect of codon optimization, copy number, and translation initiation rates on isopropanol production. The best strain (PA06) produced isopropanol at titers of 17.5g/L, with a yield of 0.62 (mol/mol), and maximum productivity of 0.40g/L/h. We next integrated the isopropanol synthetic pathway into the genome and then used the CRISPR EnAbled Trackable genome Engineering (CREATE) strategy to generate an additional 640 individual RBS library variants for further evaluation. After testing each of these va...
Methods for importing heterologous genes into genetically tractable hosts are among the most desi... more Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E. coli genome as a stable testing platform.
Since the 1970s technological advancements in the fields of synthetic biology and metabolic engin... more Since the 1970s technological advancements in the fields of synthetic biology and metabolic engineering have led to a dramatic reduction in both time and cost required for generating genomic mutations in a variety of organisms. The union of genomic editing machinery, DNA inkjet printers, and bioinformatics algorithms allows engineers to design a library of thousands of unique oligos as well as build and test these designs on a ∼2 months time-scale and at a cost of roughly ∼0.3 cents per base pair. The implications of these capabilities for a variety of fields are far-reaching, with potential impacts in defense, agricultural, human health, and environmental research. The explosion of synthetic biology applications over the past two decades have led many to draw parallels between biological engineering and the computer sciences. In this review, we highlight some important parallels between these fields and emphasize the importance of engineering design strategies.
A critical feature of the hypothesized RNA world would have been the ability to control chemical ... more A critical feature of the hypothesized RNA world would have been the ability to control chemical processes in response to environmental cues. Riboswitches present themselves as viable candidates for a sophisticated mechanism of regulatory control in RNA-based life. These regulatory elements in the modern world are most commonly found in the 5'-untranslated regions of bacterial mRNAs, directly interacting with metabolites as a means of regulating expression of the coding region via a secondary structural switch. In this review, we focus on recent insights into how these RNAs fold into complex architectures capable of both recognizing a specific small molecule compound and exerting regulatory control over downstream sequences, with an emphasis on transcriptional regulation.
Methods in molecular biology (Clifton, N.J.), 2009
Structural biology plays a central role in gaining a full understanding of the myriad roles of RN... more Structural biology plays a central role in gaining a full understanding of the myriad roles of RNA in biology. In recent years, innovative approaches in RNA purification and crystallographic methods have lead to the visualization of an increasing number of unique structures, providing new insights into its function at the atomic level. This article presents general protocols which have streamlined the process of obtaining a homogeneous sample of properly folded and active RNA in high concentrations that crystallizes well in the presence of a suitable heavy-atom for phasing. Of particular importance are approaches toward RNA crystallography that include exploring "construct space" as opposed to "condition space". Moreover, development of a highly flexible method for experimentally phasing RNA crystals may open the door to a relatively simple means of solving these structures.
Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part B, 2009
A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have ... more A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have been solved to atomic resolution using X-ray crystallography. As with proteins, RNA crystallography involves a number of screening trials in which the concentration of macromolecule, precipitant, salt, and temperature are varied, an approach known as searching "condition space." In contrast to proteins, the nature of base pairing in nucleic acids creates predictable secondary structure that facilitates the rational design of RNA variants, allowing "sequence space" to be screened in parallel. This chapter reviews RNA-specific techniques and considerations for RNA crystallography and presents a complete workflow used by our laboratory for solving RNA structures starting with initial library construction, methods to investigate and improve RNA crystal quality, and finally phase determination and structure solution.
Rewiring and optimization of metabolic networks to enable the production of commercially valuable... more Rewiring and optimization of metabolic networks to enable the production of commercially valuable chemicals is a central goal of metabolic engineering. This prospect is challenged by the complexity of metabolic networks, lack of complete knowledge of gene function(s), and the vast combinatorial genotype space that is available for exploration and optimization. Various approaches have thus been developed to aid in the efficient identification of genes that contribute to a variety of different phenotypes, allowing more rapid design and engineering of traits desired for industrial applications. This review will highlight recent technologies that have enhanced capabilities to map genotype-phenotype relationships on a genome wide scale and emphasize how such approaches enable more efficient design and engineering of complex phenotypes.
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