Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific nucleic acid modification using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a nuclease catalytic domain or a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.

Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific nucleic acid modification using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a nuclease catalytic domain or a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.

A method for directed exaptation includes dividing an original microorganism monoculture into subcultures that are subjected to different exaptation agents to obtain diversified substrains. At least one of the exaptation agents is selected to favor survival of sub strains exhibiting desired traits. The steps of dividing and subjecting may be iterated using at least some of the diversified substrains. Performance of diversified substrains is assessed and those that meet performance criteria for at least one desired trait are selected. Exaptation agents may include mutagenesis agents, training, horizontal gene transfer opportunities, and stressors. Substrains may be co-incubated with other living or dead microorganisms known to be preferentially adapted to have the desired trait. Diversified substrains may be combined into a multiculture microorganism population, to which microorganisms from the original monoculture may be added. The method may be used to create a treatment for a Multiple-Antibiotic Resistant Infection, preferably including a kill switch.

A method for directed exaptation includes dividing an original microorganism monoculture into subcultures that are subjected to different exaptation agents to obtain diversified substrains. At least one of the exaptation agents is selected to favor survival of sub strains exhibiting desired traits. The steps of dividing and subjecting may be iterated using at least some of the diversified substrains. Performance of diversified substrains is assessed and those that meet performance criteria for at least one desired trait are selected. Exaptation agents may include mutagenesis agents, training, horizontal gene transfer opportunities, and stressors. Substrains may be co-incubated with other living or dead microorganisms known to be preferentially adapted to have the desired trait. Diversified substrains may be combined into a multiculture microorganism population, to which microorganisms from the original monoculture may be added. The method may be used to create a treatment for a Multiple-Antibiotic Resistant Infection, preferably including a kill switch.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide mRNA-sensing gRNAs that modulate the activity of RNA-programmable endonucleases based on the presence or absence of a target mRNA. Some aspects of this disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of an extended DNA (xDNA).

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide mRNA-sensing gRNAs that modulate the activity of RNA-programmable endonucleases based on the presence or absence of a target mRNA. Some aspects of this disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of an extended DNA (xDNA).

A method for improving the production of Streptomyces polyketide compounds is provided. The method greatly improves the capability of the Streptomyces polyketide compounds by strengthening a triacylglycerol decomposition pathway in Streptomyces during the stationary phase. A method for switching the primary metabolism of Streptomyces to the secondary metabolism, Streptomyces producing polyketide compounds, and use thereof are also provided.

A method for improving the production of Streptomyces polyketide compounds is provided. The method greatly improves the capability of the Streptomyces polyketide compounds by strengthening a triacylglycerol decomposition pathway in Streptomyces during the stationary phase. A method for switching the primary metabolism of Streptomyces to the secondary metabolism, Streptomyces producing polyketide compounds, and use thereof are also provided.

Plant metabolism and alkaloid levels can be regulated by transcription factors that regulate the nicotinic alkaloid biosynthetic pathway. In one embodiment, the disclosure provides a transcription factor that negatively regulates alkaloid biosynthesis, such as nicotine biosynthesis.

A genetically modified microorganism that can produce 3-hydroxyadipic acid and/or α-hydromuconic acid with a high yield; and a method of producing 3-hydroxyadipic acid and/or α-hydromuconic acid using the genetically modified microorganism, are disclosed. The genetically modified microorganism has an ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, and has an enhanced enzymatic activity to catalyze a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, wherein, in the genetically modified microorganism, a dicarboxylic acid excretion carrier function is deleted or decreased.