Abstract: Some aspects of this disclosure provide methods for phage-assisted continuous evolution (PACE) of proteases. Some aspects of this invention provide methods for evaluating and selecting protease inhibitors based on the likelihood of the emergence of resistant proteases as determined by the protease PACE methods provided herein. Some aspects of this disclosure provide strategies, methods, and reagents for protease PACE, including fusion proteins for translating a desired protease activity into a selective advantage for phage particles encoding a protease exhibiting such an activity and improved mutagenesis-promoting expression constructs. Evolved proteases that recognize target cleavage sites which differ from their canonical cleavage site are also provided herein.

Abstract: Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5? nucleotide of the target is specific for thymine (T). TALE domains with alternative 5? nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. Another drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities.

Abstract: Some aspects of this disclosure provide methods for phage-assisted continuous evolution (PACE) of proteases. Some aspects of this invention provide methods for evaluating and selecting protease inhibitors based on the likelihood of the emergence of resistant proteases as determined by the protease PACE methods provided herein. Some aspects of this disclosure provide strategies, methods, and reagents for protease PACE, including fusion proteins for translating a desired protease activity into a selective advantage for phage particles encoding a protease exhibiting such an activity and improved mutagenesis-promoting expression constructs. Evolved proteases that recognize target cleavage sites which differ from their canonical cleavage site are also provided herein.

Abstract: The disclosure provides amino acid sequence variants of Bacillus thuringiensis (Bt) toxins and methods of producing the same. Some aspects of this disclosure provide methods for generating Bt toxin variants by continuous directed evolution. Some aspects of this disclosure provide compositions and methods for pest control using the disclosed variant Bt toxins.

Abstract: Some aspects of this disclosure relate to systems, apparatuses, compositions (e.g., isolated nucleic acids and vectors), and methods for improving the stability and/or solubility of proteins evolved using phage-assisted continuous evolution (PACE). In some embodiments, vectors described herein comprise nucleic acids encoding selection systems (e.g., positive and/or negative selection systems) that link expression of genes required for production of infectious phage particles to a desirable physiochemical (e.g., stability or solubility) and/or desired function of an evolved protein.

Abstract: The disclosure provides amino acid sequence variants of Bacillus thuringiensis (Bt) toxins and methods of producing the same. Some aspects of this disclosure provide methods for generating Bt toxin variants by continuous directed evolution. Some aspects of this disclosure provide compositions and methods for pest control using the disclosed variant Bt toxins.

Abstract: Strategies, reagents, methods, and systems for modulating the mutation rate in cells are provided herein. The strategies, reagents, methods, and systems are broadly applicable for the modulation of mutation rates in cells where high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products.

Abstract: Strategies, reagents, methods, and systems for modulating the mutation rate in cells are provided herein. The strategies, reagents, methods, and systems are broadly applicable for the modulation of mutation rates in cells where high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products.

Abstract: Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5? nucleotide of the target is specific for thymine (T). TALE domains with alternative 5? nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. Another drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities.

Abstract: Some aspects of this disclosure provide methods for phage-assisted continuous evolution (PACE) of proteases. Some aspects of this invention provide methods for evaluating and selecting protease inhibitors based on the likelihood of the emergence of resistant proteases as determined by the protease PACE methods provided herein. Some aspects of this disclosure provide strategies, methods, and reagents for protease PACE, including fusion proteins for translating a desired protease activity into a selective advantage for phage particles encoding a protease exhibiting such an activity and improved mutagenesis-promoting expression constructs. Evolved proteases that recognize target cleavage sites which differ from their canonical cleavage site are also provided herein.

Abstract: Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5? nucleotide of the target is specific for thymine (T). TALE domains with alternative 5? nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. Another drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities.

Abstract: Some aspects of this disclosure provide methods for phage-assisted continuous evolution (PACE) of proteases. Some aspects of this invention provide methods for evaluating and selecting protease inhibitors based on the likelihood of the emergence of resistant proteases as determined by the protease PACE methods provided herein. Some aspects of this disclosure provide strategies, methods, and reagents for protease PACE, including fusion proteins for translating a desired protease activity into a selective advantage for phage particles encoding a protease exhibiting such an activity and improved mutagenesis-promoting expression constructs. Evolved proteases that recognize target cleavage sites which differ from their canonical cleavage site are also provided herein.

Abstract: The invention, in part, includes compositions comprising quadruplet decoding tRNAs and their encoding sequences. The invention also includes assay methods to assess quadruplet decoding as well as methods of preparing quadruplet decoding suppression tRNAs.

Abstract: Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5? nucleotide of the target is specific for thymine (T). TALE domains with alternative 5? nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. Another drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities.

Abstract: Some aspects of this disclosure relate to systems, apparatuses, compositions (e.g., isolated nucleic acids and vectors), and methods for improving the stability and/or solubility of proteins evolved using phage-assisted continuous evolution (PACE). In some embodiments, vectors described herein comprise nucleic acids encoding selection systems (e.g., positive and/or negative selection systems) that link expression of genes required for production of infectious phage particles to a desirable physiochemical (e.g., stability or solubility) and/or desired function of an evolved protein.

Abstract: Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5? nucleotide of the target is specific for thymine (T). TALE domains with alternative 5? nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities. Some aspects of this disclosure provide engineered TALE domains and TALEs comprising such engineered domains, e.g.

Abstract: Strategies, systems, methods, reagents, and kits for phage-assisted continuous evolution are provided herein. These include strategies, systems, methods, reagents, and kits allowing for stringency modulation to evolve weakly active or inactive biomolecule variants, negative selection of undesired properties, and/or positive selection of desired properties.

Abstract: Strategies, systems, methods, reagents, and kits for phage-assisted continuous evolution are provided herein. These include strategies, systems, methods, reagents, and kits allowing for stringency modulation to evolve weakly active or inactive biomolecule variants, negative selection of undesired properties, and/or positive selection of desired properties.

Abstract: Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5? nucleotide of the target is specific for thymine (T). TALE domains with alternative 5? nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities. Some aspects of this disclosure provide engineered TALE domains and TALEs comprising such engineered domains, e.g.

Abstract: Strategies, reagents, methods, and systems for modulating the mutation rate in cells are provided herein. The strategies, reagents, methods, and systems are broadly applicable for the modulation of mutation rates in cells where high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products.