PeerJ Preprints: Synthetic Biologyhttps://peerj.com/preprints/index.atom?journal=peerj&subject=2650Synthetic Biology articles published in PeerJ PreprintsIntegrating the extracellular, intracellular, and intercellular metabolic processes of Escherichia coli through glucose saturation, inhibition of the acetyl-CoA carboxylase subunit accA with asRNA, and through quantifying cell to cell quorum-sensinghttps://peerj.com/preprints/274592019-07-032019-07-03Tatiana HillmanCory Tobin
The study aims to demonstrate the link between bacterial cell metabolism and virulence through integrating the environmental, genetic, and cell to cell signaling molecular processes. Dietary fiber metabolized into glucose, increases the proliferation of intestinal microflora, which augments the outputof the Short Chain Fatty Acids. Bacteria ferment the glucose, from fiber, into Short Chain Fatty Acids, which help regulate many biochemical processes and pathways. Each SCFA maintains colonic pH, promotes cell differentiation, and the apoptosis of colonocytes. To model a high-fiber diet, increasing the synthesis of Acetyl-CoA carboxylase, an enzyme that catabolizes glucose into SCFAs, Escherichia coli was cultured in Luria Broth enhanced with a high to low concentration of glucose. The 15mM, a high concentration of glucose, yielded qPCR products measured, for the target gene accA, which was 4,210ng/µL. The 7.5mM sample produced a concentration equaled to 375 ng/µL, and the 0µM sample measured an accA concentration of 196 ng/µL. The gene accA, 1 of 4 subunits for the Acetyl-CoA Carboxylase enzyme, was suppressed by asRNA, producing a qPCR concentration of 63ng/µL. Antisense RNA for accA reduced the amount of Lux-S, a vital gene needed for propagating quorum-sensing signal molecules. The Lux-S gene, responsible for releasing autoinducer 2 for cell to cell quorum sensing, was reduced by the gene inhibition of accA with asRNA. The increase in Lux-S transcription increases biofilm production for spreading virulence. The further implications of the study propose designing antibiotics that target bacterial cell metabolic processes to block bacterial antibiotic resistance.
The study aims to demonstrate the link between bacterial cell metabolism and virulence through integrating the environmental, genetic, and cell to cell signaling molecular processes. Dietary fiber metabolized into glucose, increases the proliferation of intestinal microflora, which augments the outputof the Short Chain Fatty Acids. Bacteria ferment the glucose, from fiber, into Short Chain Fatty Acids, which help regulate many biochemical processes and pathways. Each SCFA maintains colonic pH, promotes cell differentiation, and the apoptosis of colonocytes. To model a high-fiber diet, increasing the synthesis of Acetyl-CoA carboxylase, an enzyme that catabolizes glucose into SCFAs, Escherichia coli was cultured in Luria Broth enhanced with a high to low concentration of glucose. The 15mM, a high concentration of glucose, yielded qPCR products measured, for the target gene accA, which was 4,210ng/µL. The 7.5mM sample produced a concentration equaled to 375 ng/µL, and the 0µM sample measured an accA concentration of 196 ng/µL. The gene accA, 1 of 4 subunits for the Acetyl-CoA Carboxylase enzyme, was suppressed by asRNA, producing a qPCR concentration of 63ng/µL. Antisense RNA for accA reduced the amount of Lux-S, a vital gene needed for propagating quorum-sensing signal molecules. The Lux-S gene, responsible for releasing autoinducer 2 for cell to cell quorum sensing, was reduced by the gene inhibition of accA with asRNA. The increase in Lux-S transcription increases biofilm production for spreading virulence. The further implications of the study propose designing antibiotics that target bacterial cell metabolic processes to block bacterial antibiotic resistance.CRISPR-Cas9 may restore the balance of hormones affected by the frequency of DNA methylation sites and a decrease of commensal bacteriahttps://peerj.com/preprints/275092019-06-212019-06-21Tatiana Hillman
In this review, it is suggested that there are connections between hormonal changes, the frequency of DNA methylation, and disease. The commensal microbes of the gut may also affect the production of those hormones. Short Chain Fatty Acids, produced from gut microbiota glucose metabolism, like butyrate, propionate, folate, and acetate act as ligands that bind to G-coupled protein receptors. For example, folate from Bifidobacterium donates a methyl for synthesizing S-adenosylmethionine or SAM, which then donates a methyl to the enzymes of DNA methylation, acting as a substrate. The effects of hormones on DNA methylation was reviewed. Increased progesterone can produce breast cancer by lessening DNA methylation allowing progesterone molecules to bind DNA, amplifying gene expression. Through measuring the frequency of DNA methylation perhaps breast cancer can be more readily identified, diagnosed, and treated. The intended purpose for this review is to propose the possibility of applying CRISPR-Cas9 methods to correct and restore the balance of hormones through epigenetic means. In this review, 1) the effects of microbes on hormonal balance, 2) the connection between hormones and DNA Methylation, 3) cancer and DNA methylation, 4) measuring DNA methylation, and 5) applying CRISPR methods will be discussed.
In this review, it is suggested that there are connections between hormonal changes, the frequency of DNA methylation, and disease. The commensal microbes of the gut may also affect the production of those hormones. Short Chain Fatty Acids, produced from gut microbiota glucose metabolism, like butyrate, propionate, folate, and acetate act as ligands that bind to G-coupled protein receptors. For example, folate from Bifidobacterium donates a methyl for synthesizing S-adenosylmethionine or SAM, which then donates a methyl to the enzymes of DNA methylation, acting as a substrate. The effects of hormones on DNA methylation was reviewed. Increased progesterone can produce breast cancer by lessening DNA methylation allowing progesterone molecules to bind DNA, amplifying gene expression. Through measuring the frequency of DNA methylation perhaps breast cancer can be more readily identified, diagnosed, and treated. The intended purpose for this review is to propose the possibility of applying CRISPR-Cas9 methods to correct and restore the balance of hormones through epigenetic means. In this review, 1) the effects of microbes on hormonal balance, 2) the connection between hormones and DNA Methylation, 3) cancer and DNA methylation, 4) measuring DNA methylation, and 5) applying CRISPR methods will be discussed.A review: Possible optimization of Cas9-sgRNA nuclease delivery via ingested lipid nanoparticles bioencapsulated within plant cell-based enfoldinghttps://peerj.com/preprints/277092019-06-072019-06-07Tatiana Hillman
The possibility of gene editing to correct disorders is one of the most impactful therapeutic agents, currently. CRISPR Cas9-sgRNA nucleases can be used to cleave and to delete harmful or pathogenic DNA sequences, which cause genetic disorders. Cas9 nuclease with palindromic repeats can cut and delete a single point mutation or multiple DNA target site sequences. The Cas9, attached to a sgRNA or a guiding RNA, finds and then cleaves the target DNA sequence. The Cas9-sgRNA method of cleavage has corrected DNA mutations that cause cataracts in the eyes, cystic fibrosis, and chronic granulomatous disease. However, there are issues for producing a less strenuous delivery of Cas9-sgRA to target DNA sequences. Delivering Cas-9 nucleases are negatively affected by off-target DNA sites, sgRNA design, off-target cleavage, Cas9 activation, and the method of delivery. This review focuses on oral and ingested delivery methods to effectively guide the transport of Cas9-sgRNA nucleases in vivo. A review of Cas9 delivery will present possible alternatives for nuclease delivery within optimized lipid-nanoparticles, plant, algae, and bacterial-based orally ingested edibles. This review will attempt to provide evidence in support of enhancing the Cas9 delivery through therapeutic bioencapsulated ingestion. In this review, it is suggested that the ingestion of encapsulated edibles carrying the nuclease can more directly target cells within the gastrointestinal tract for blood or lymph circulation.
The possibility of gene editing to correct disorders is one of the most impactful therapeutic agents, currently. CRISPR Cas9-sgRNA nucleases can be used to cleave and to delete harmful or pathogenic DNA sequences, which cause genetic disorders. Cas9 nuclease with palindromic repeats can cut and delete a single point mutation or multiple DNA target site sequences. The Cas9, attached to a sgRNA or a guiding RNA, finds and then cleaves the target DNA sequence. The Cas9-sgRNA method of cleavage has corrected DNA mutations that cause cataracts in the eyes, cystic fibrosis, and chronic granulomatous disease. However, there are issues for producing a less strenuous delivery of Cas9-sgRA to target DNA sequences. Delivering Cas-9 nucleases are negatively affected by off-target DNA sites, sgRNA design, off-target cleavage, Cas9 activation, and the method of delivery. This review focuses on oral and ingested delivery methods to effectively guide the transport of Cas9-sgRNA nucleases in vivo. A review of Cas9 delivery will present possible alternatives for nuclease delivery within optimized lipid-nanoparticles, plant, algae, and bacterial-based orally ingested edibles. This review will attempt to provide evidence in support of enhancing the Cas9 delivery through therapeutic bioencapsulated ingestion. In this review, it is suggested that the ingestion of encapsulated edibles carrying the nuclease can more directly target cells within the gastrointestinal tract for blood or lymph circulation.Geobacter protein nanowireshttps://peerj.com/preprints/277732019-06-022019-06-02Derek R LovleyDavid J F Walker
The study of electrically conductive protein nanowires in Geobacter sulfurreducens has led to new concepts for long-range electron extracellular transport, as well as the development of sustainable conductive materials and electronic devices with novel functions. Until recently, electrically conductive pili (e-pili), assembled from the PilA pilin monomer, were the only known Geobacter protein nanowires. However, filaments comprised of the multi-heme c-type cytochrome, OmcS, are present in some preparations of G. sulfurreducens outer-surface proteins. The purpose of this review is to evaluate the available evidence on the in vivo expression of e-pili and OmcS filaments and their biological function. Abundant literature demonstrates that G. sulfurreducens expresses e-pili, which are required for long-range electron transport to Fe(III) oxides and through conductive biofilms. In contrast, there is no definitive evidence yet that wild-type G. sulfurreducens express long filaments of OmcS extending from the cells, and deleting the gene for OmcS actually increases biofilm conductivity. The literature does not support the concern that many previous studies on e-pili were mistakenly studying OmcS filaments. For example, heterologous expression of the aromatic-rich pilin monomer of G. metallireducens in G. sulfurreducens increases the conductivity of individual nanowires more than 5000-fold, whereas expression of an aromatic-poor pilin reduced conductivity more than 1000-fold. This more than million-fold range in nanowire conductivity was achieved while maintaining the 3 nm diameter consistent with e-pili, not OmcS. Purification methods that eliminate all traces of OmcS yield highly conductive e-pili. Substantial evidence suggests that OmcS is often associated with the outer cell surface and intermittently localized along e-pili in vivo. Future studies of G. sulfurreducens expression of protein nanowires need to be cognizant of the importance of maintaining environmentally relevant growth conditions because artificial laboratory culture conditions can rapidly select against e-pili expression. Principles derived from the study of e-pili have enabled identification of non-cytochrome protein nanowires in diverse bacteria and archaea. A similar search for cytochrome appendages is warranted. Both e-pili and OmcS filaments offer design options for the synthesis of protein-based ‘green’ electronics, which may be the primary driving force for the study of these structures in the near future.
The study of electrically conductive protein nanowires in Geobacter sulfurreducens has led to new concepts for long-range electron extracellular transport, as well as the development of sustainable conductive materials and electronic devices with novel functions. Until recently, electrically conductive pili (e-pili), assembled from the PilA pilin monomer, were the only known Geobacter protein nanowires. However, filaments comprised of the multi-heme c-type cytochrome, OmcS, are present in some preparations of G. sulfurreducens outer-surface proteins. The purpose of this review is to evaluate the available evidence on the in vivo expression of e-pili and OmcS filaments and their biological function. Abundant literature demonstrates that G. sulfurreducens expresses e-pili, which are required for long-range electron transport to Fe(III) oxides and through conductive biofilms. In contrast, there is no definitive evidence yet that wild-type G. sulfurreducens express long filaments of OmcS extending from the cells, and deleting the gene for OmcS actually increases biofilm conductivity. The literature does not support the concern that many previous studies on e-pili were mistakenly studying OmcS filaments. For example, heterologous expression of the aromatic-rich pilin monomer of G. metallireducens in G. sulfurreducens increases the conductivity of individual nanowires more than 5000-fold, whereas expression of an aromatic-poor pilin reduced conductivity more than 1000-fold. This more than million-fold range in nanowire conductivity was achieved while maintaining the 3 nm diameter consistent with e-pili, not OmcS. Purification methods that eliminate all traces of OmcS yield highly conductive e-pili. Substantial evidence suggests that OmcS is often associated with the outer cell surface and intermittently localized along e-pili in vivo. Future studies of G. sulfurreducens expression of protein nanowires need to be cognizant of the importance of maintaining environmentally relevant growth conditions because artificial laboratory culture conditions can rapidly select against e-pili expression. Principles derived from the study of e-pili have enabled identification of non-cytochrome protein nanowires in diverse bacteria and archaea. A similar search for cytochrome appendages is warranted. Both e-pili and OmcS filaments offer design options for the synthesis of protein-based ‘green’ electronics, which may be the primary driving force for the study of these structures in the near future.Possibility of tuning the differentiation state of tumor associated macrophages towards tumor controlling phenotypeshttps://peerj.com/preprints/277472019-05-202019-05-20Wenfa Ng
Although various immune cells could infiltrate the cellular and tissue environment surrounding a tumor, the tumor microenvironment nevertheless presents immunosuppressive conditions unfavorable for immune cells to conduct large scale attack on cancer cells. For example, T-cells that make it to the tumor microenvironment are typically non-functional in containing tumor growth. On the other hand, macrophages could infiltrate the tumor microenvironment and is an important cell type modulated by and which also modulates the tumor. Specifically, two variants of macrophages with different phenotypes are known to exhibit close interactions with tumors. Known as M1 and M2 macrophages, they present dichotomously different signals to the tumor. Specifically, M1 macrophages control tumor growth while M2 macrophages promote tumor growth. Thus, from a treatment perspective, it would be desirable to tune the phenotypes and cell differentiation program of macrophages towards the M1 subset. To do that, differential gene expression of macrophages in the M1 and M2 lineages must be understood. Such a goal could be achieved with the profiling of tumor associated macrophages from tumor biopsy samples for gene expression patterns characteristic of the two dominant macrophage lineages. Single cell RNA-sequencing conducted after flow cytometry sorting of M1 and M2 macrophages would highlight gene expression patterns associated with each lineage, and the cellular differentiation programs that prompted entry into particular macrophage subtype. Knowledge of gene expression pattern associated with each macrophage lineage is not useful for tuning their differentiation state unless specific transcription factor that trigger the regulon could be identified. To this end, transcription factors that have been upregulated in the differentiation program could be profiled from the transcriptome data, and help inform the design of vectors for targeted overexpression of specific transcription factor for modulating cellular differentiation of macrophage. Given their low immunogenicity, adeno-associated virus (AAV) could serve as vectors for ferrying the gene cassette containing specific transcription factors into macrophages. Delivery methods for the AAV could be via targeted local infusion of vectors to tumors or through the systemic circulation, but the latter approach would result in lower transfection efficiency. Collectively, possibility exists of tuning the differentiation state of macrophage associated with tumors for enabling tumor controlling lineage to be dominant. Such immuno-targeted therapy would harness the body’s macrophages for controlling tumor growth and represents a treatment option that may yield fewer side effects compared to conventional chemotherapy. But, identification of genes that control lineage-specific differentiation program and the delivery of gene cassette to macrophages for modulating their differentiation remain key challenges.
Although various immune cells could infiltrate the cellular and tissue environment surrounding a tumor, the tumor microenvironment nevertheless presents immunosuppressive conditions unfavorable for immune cells to conduct large scale attack on cancer cells. For example, T-cells that make it to the tumor microenvironment are typically non-functional in containing tumor growth. On the other hand, macrophages could infiltrate the tumor microenvironment and is an important cell type modulated by and which also modulates the tumor. Specifically, two variants of macrophages with different phenotypes are known to exhibit close interactions with tumors. Known as M1 and M2 macrophages, they present dichotomously different signals to the tumor. Specifically, M1 macrophages control tumor growth while M2 macrophages promote tumor growth. Thus, from a treatment perspective, it would be desirable to tune the phenotypes and cell differentiation program of macrophages towards the M1 subset. To do that, differential gene expression of macrophages in the M1 and M2 lineages must be understood. Such a goal could be achieved with the profiling of tumor associated macrophages from tumor biopsy samples for gene expression patterns characteristic of the two dominant macrophage lineages. Single cell RNA-sequencing conducted after flow cytometry sorting of M1 and M2 macrophages would highlight gene expression patterns associated with each lineage, and the cellular differentiation programs that prompted entry into particular macrophage subtype. Knowledge of gene expression pattern associated with each macrophage lineage is not useful for tuning their differentiation state unless specific transcription factor that trigger the regulon could be identified. To this end, transcription factors that have been upregulated in the differentiation program could be profiled from the transcriptome data, and help inform the design of vectors for targeted overexpression of specific transcription factor for modulating cellular differentiation of macrophage. Given their low immunogenicity, adeno-associated virus (AAV) could serve as vectors for ferrying the gene cassette containing specific transcription factors into macrophages. Delivery methods for the AAV could be via targeted local infusion of vectors to tumors or through the systemic circulation, but the latter approach would result in lower transfection efficiency. Collectively, possibility exists of tuning the differentiation state of macrophage associated with tumors for enabling tumor controlling lineage to be dominant. Such immuno-targeted therapy would harness the body’s macrophages for controlling tumor growth and represents a treatment option that may yield fewer side effects compared to conventional chemotherapy. But, identification of genes that control lineage-specific differentiation program and the delivery of gene cassette to macrophages for modulating their differentiation remain key challenges.Exploring lowering the optimal growth temperature of Escherichia coli in biotechnology applicationshttps://peerj.com/preprints/277232019-05-122019-05-12Wenfa Ng
Different microbes grow at different optimal growth temperature. But, what defines this metabolic adaptation at the molecular and genetic level? And, more importantly, how different metabolic and signalling networks interact to yield a cellular system able to achieve maximal growth rate at a specific temperature? Molecular knowledge of such interacting components could provide a template on which modifications could be made to help adapt a microbe to another optimal growth temperature. However, given the large number of genes, proteins and pathways involved, efforts to re-adapt a microbe to another optimal growth temperature is likely difficult through a rational design approach. On the other hand, laboratory evolution approach might do the trick, but significant efforts are needed to understand the biochemical and physiological logic of the re-adaption. Using the genetically tractable Escherichia coli as model organism, this work aims to explore the possibility of using a rational approach at lowering the optimal growth temperature of the bacterium from 37 oC to 25 oC to help reduce energy costs and carbon emissions of fermentation. To this end, population level RNA-seq would be used to understand the global transcriptome of E. coli cultivated at 25, 30 and 37 oC in LB medium. Highly transcribed genes at 37 oC would represent those that need to be activated during growth at 25 oC. On the other hand, genes transcribed at a low level at 37 oC should remain poorly expressed at 25 oC. While modern genetic engineering tools such as use of promoters and terminators with differentiated strength would allow the targeted tuning of expression of specific genes, potential need for re-engineering the expression of large number of genes might present difficulties. Thus, answers to what tune a microbe to operate optimally at a specific temperature might come from the signalling and gene regulation level where genes and proteins occupying particular nodes in the biochemical network hold sway on the expression of large number of downstream genes. Knowledge such as these could accrue from the feeding of transcriptome data into genome-scale metabolic models able to help identify critical nodes in metabolic pathways whose modulation would change cellular physiology. Given the importance of regulons governed by specific sigma factors, their modulation through altering sigma factor expression might be critical to gaining more widespread control of global gene expression at particular temperature. Collectively, developing rational approaches for tuning the optimal growth temperature of E. coli present critical challenges compared to laboratory evolution methods. As gene expression is regulated at multiple levels using a variety of mechanisms, transposing expression levels of highly transcribed genes at 37 oC to 25 oC would require the simultaneous modulation of different regulatory nodes belonging to both metabolic and signalling pathways.
Different microbes grow at different optimal growth temperature. But, what defines this metabolic adaptation at the molecular and genetic level? And, more importantly, how different metabolic and signalling networks interact to yield a cellular system able to achieve maximal growth rate at a specific temperature? Molecular knowledge of such interacting components could provide a template on which modifications could be made to help adapt a microbe to another optimal growth temperature. However, given the large number of genes, proteins and pathways involved, efforts to re-adapt a microbe to another optimal growth temperature is likely difficult through a rational design approach. On the other hand, laboratory evolution approach might do the trick, but significant efforts are needed to understand the biochemical and physiological logic of the re-adaption. Using the genetically tractable Escherichia coli as model organism, this work aims to explore the possibility of using a rational approach at lowering the optimal growth temperature of the bacterium from 37 oC to 25 oC to help reduce energy costs and carbon emissions of fermentation. To this end, population level RNA-seq would be used to understand the global transcriptome of E. coli cultivated at 25, 30 and 37 oC in LB medium. Highly transcribed genes at 37 oC would represent those that need to be activated during growth at 25 oC. On the other hand, genes transcribed at a low level at 37 oC should remain poorly expressed at 25 oC. While modern genetic engineering tools such as use of promoters and terminators with differentiated strength would allow the targeted tuning of expression of specific genes, potential need for re-engineering the expression of large number of genes might present difficulties. Thus, answers to what tune a microbe to operate optimally at a specific temperature might come from the signalling and gene regulation level where genes and proteins occupying particular nodes in the biochemical network hold sway on the expression of large number of downstream genes. Knowledge such as these could accrue from the feeding of transcriptome data into genome-scale metabolic models able to help identify critical nodes in metabolic pathways whose modulation would change cellular physiology. Given the importance of regulons governed by specific sigma factors, their modulation through altering sigma factor expression might be critical to gaining more widespread control of global gene expression at particular temperature. Collectively, developing rational approaches for tuning the optimal growth temperature of E. coli present critical challenges compared to laboratory evolution methods. As gene expression is regulated at multiple levels using a variety of mechanisms, transposing expression levels of highly transcribed genes at 37 oC to 25 oC would require the simultaneous modulation of different regulatory nodes belonging to both metabolic and signalling pathways.In silico experiments validate that twenty nucleotide spacer sequence provides precise targeting of bacterial genes in CRISPR-Cas9https://peerj.com/preprints/277192019-05-102019-05-10Wenfa Ng
As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.
As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.Possible differences in efficiency of guide RNA with different spacer sequence in CRISPR knock-down or knock-out of particular genehttps://peerj.com/preprints/277012019-05-022019-05-02Wenfa Ng
Cluster regularly interspersed short palindromic repeats (CRISPR) mediated genome editing has emerged as the dominant technique for modulating the expression of target genes. Specifically, when coupled with different effectors, CRISPR could be utilized to either activate or repress gene expression. Specificity of the CRISPR gene editing method arises from the unique spacer sequence in guide RNA that mediates the specific localization of Cas9 endonuclease to particular stretches of DNA. However, complementary base pairing between the guide RNA and template DNA depends critically on existence of protospacer adjacent motif (PAM) sequence immediately downstream of the spacer sequence. Such three nucleotide PAM sequence could be present at multiple loci in a given gene, which meant that different spacer sequence could be incorporated in guide RNA design to target the same gene. Given that different spacer sequences have different binding affinities to template DNA, differences could exist in the efficiency in which CRISPR-Cas9 could be guided to generate a double strand break in a particular gene locus. Using green fluorescent protein (GFP) reporter gene expressed in recombinant Escherichia coli as experimental system, this study sought to understand if differences in targeting efficiency exist between guide RNA with different spacer sequence that could target the same gene. Fluorescent intensity of cells at the population level would serve as readout of the targeting efficiency. For example, spacer sequence in guide RNA that could better activate the endonuclease activity of Cas9 would result in lower fluorescent intensity of GFP. To check for the effect of expression mode on targeting efficiency of guide RNA, GFP gene would be expressed on a plasmid in E. coli as well as integrated into the genome of the bacterium. Doing so would provide critical information on whether the CRISPR-Cas9 system has differentiated efficacy in generating double strand breaks in genomic versus plasmid DNA. Such information would inform future experimental design involving CRISPR-Cas9 genome editing technology as well as hold implications on how CRISPR evolved as an adaptive immune system in defending bacterial cells against foreign DNA. Given the goal of the study to understand the relative extent in which a target gene would be disrupted by CRISPR-Cas9 guided by different spacer sequence on guide RNA, no repair module for the target gene would be provided. Collectively, multiple occurrence of PAM sequence in a target gene meant that different spacer sequences could be used in CRISPR-Cas9 to downregulate gene expression. Relative efficacies of different spacer sequence in guide RNA in achieving targeted gene inactivation remain poorly understood and constitutes the basis of this study, which hopefully would provide guidance on the selection of specific spacer sequence that would yield the most efficacious disruption of gene expression at the genome and plasmid level.
Cluster regularly interspersed short palindromic repeats (CRISPR) mediated genome editing has emerged as the dominant technique for modulating the expression of target genes. Specifically, when coupled with different effectors, CRISPR could be utilized to either activate or repress gene expression. Specificity of the CRISPR gene editing method arises from the unique spacer sequence in guide RNA that mediates the specific localization of Cas9 endonuclease to particular stretches of DNA. However, complementary base pairing between the guide RNA and template DNA depends critically on existence of protospacer adjacent motif (PAM) sequence immediately downstream of the spacer sequence. Such three nucleotide PAM sequence could be present at multiple loci in a given gene, which meant that different spacer sequence could be incorporated in guide RNA design to target the same gene. Given that different spacer sequences have different binding affinities to template DNA, differences could exist in the efficiency in which CRISPR-Cas9 could be guided to generate a double strand break in a particular gene locus. Using green fluorescent protein (GFP) reporter gene expressed in recombinant Escherichia coli as experimental system, this study sought to understand if differences in targeting efficiency exist between guide RNA with different spacer sequence that could target the same gene. Fluorescent intensity of cells at the population level would serve as readout of the targeting efficiency. For example, spacer sequence in guide RNA that could better activate the endonuclease activity of Cas9 would result in lower fluorescent intensity of GFP. To check for the effect of expression mode on targeting efficiency of guide RNA, GFP gene would be expressed on a plasmid in E. coli as well as integrated into the genome of the bacterium. Doing so would provide critical information on whether the CRISPR-Cas9 system has differentiated efficacy in generating double strand breaks in genomic versus plasmid DNA. Such information would inform future experimental design involving CRISPR-Cas9 genome editing technology as well as hold implications on how CRISPR evolved as an adaptive immune system in defending bacterial cells against foreign DNA. Given the goal of the study to understand the relative extent in which a target gene would be disrupted by CRISPR-Cas9 guided by different spacer sequence on guide RNA, no repair module for the target gene would be provided. Collectively, multiple occurrence of PAM sequence in a target gene meant that different spacer sequences could be used in CRISPR-Cas9 to downregulate gene expression. Relative efficacies of different spacer sequence in guide RNA in achieving targeted gene inactivation remain poorly understood and constitutes the basis of this study, which hopefully would provide guidance on the selection of specific spacer sequence that would yield the most efficacious disruption of gene expression at the genome and plasmid level.A sustainable synthetic biology approach for the control of the invasive golden mussel (Limnoperna fortunei)https://peerj.com/preprints/271642018-09-122018-09-12Mauro F RebeloLuana F AfonsoJuliana A AmericoLucas da SilvaJosé L B NetoFrancesco DonderoQiaoying Zhang
The recent development of the CRISPR-Cas9-based gene drive has created the conditions to seriously consider this technology to solve one of the major environmental challenges in biodiversity conservation i.e. the control of invasive species. There is no efficient control method for golden mussel infestation available so far. Here we discuss the technical and economic feasibility of using a synthetic biology based approach to fight and control the invasive mussel Limnoperna fortunei in South American rivers and reservoirs.
The recent development of the CRISPR-Cas9-based gene drive has created the conditions to seriously consider this technology to solve one of the major environmental challenges in biodiversity conservation i.e. the control of invasive species. There is no efficient control method for golden mussel infestation available so far. Here we discuss the technical and economic feasibility of using a synthetic biology based approach to fight and control the invasive mussel Limnoperna fortunei in South American rivers and reservoirs.CRSeek: a Python module for facilitating complicated CRISPR design strategieshttps://peerj.com/preprints/270942018-08-062018-08-06Will DampierCheng-Han ChungNeil T SullivanAndrew J AtkinsMichael R NonnemacherBrian Wigdahl
With the popularization of the CRISPR-Cas gene editing system there has been an explosion of new techniques made possible by this versatile technology. However, the computational field has lagged behind with a current lack of computational tools for developing complicated CRISPR-Cas gene editing strategies. We present crseek, a Python package that provides a consistent application programming interface (API) for multiple cleavage prediction algorithms. Four popular cleavage prediction algorithms were implemented and further adapted to work on draft-quality genomes. Furthermore, since crseek mirrors the popular scikit-learn API, the package can be easily integrated as an upstream processing module for facilitating further CRISPR-Cas machine learning research. The package is fully integrated with the biopython package facilitating simple import, export, and manipulation of sequences before and after gene editing. This manuscript presents four common gene editing tasks that would be difficult with current tools but are easily performed with the crseek package. We believe this package will help bioinformaticians rapidly design complex CRISPR-Cas gene editing strategies and will be a useful addition to the field.
With the popularization of the CRISPR-Cas gene editing system there has been an explosion of new techniques made possible by this versatile technology. However, the computational field has lagged behind with a current lack of computational tools for developing complicated CRISPR-Cas gene editing strategies. We present crseek, a Python package that provides a consistent application programming interface (API) for multiple cleavage prediction algorithms. Four popular cleavage prediction algorithms were implemented and further adapted to work on draft-quality genomes. Furthermore, since crseek mirrors the popular scikit-learn API, the package can be easily integrated as an upstream processing module for facilitating further CRISPR-Cas machine learning research. The package is fully integrated with the biopython package facilitating simple import, export, and manipulation of sequences before and after gene editing. This manuscript presents four common gene editing tasks that would be difficult with current tools but are easily performed with the crseek package. We believe this package will help bioinformaticians rapidly design complex CRISPR-Cas gene editing strategies and will be a useful addition to the field.