Inducible dna binding proteins and genome perturbation tools and applications … — The Broad Institute, Inc. (US20190390204A1)

ABSTRACT

Abstract

The present invention generally relates to methods and compositions used for the spatial and temporal control of gene expression that may use inducible transcriptional effectors. The invention particularly relates to inducible methods of altering or perturbing expression of a genomic locus of interest in a cell wherein the genomic locus may be contacted with a non-naturally occurring or engineered composition comprising a deoxyribonucleic acid (DNA) binding polypeptide.

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No. 15/388,248 filed Dec. 22, 2016, which is a continuation of U.S. patent application Ser. No. 14/604,641 filed Jan. 23, 2015, which is a continuation-in part of international patent application Serial No. PCT/US13/51418 filed Jul. 21, 2013, which published as WO2014/018423 on Jan. 30, 2014 which claims priority to and claims benefit of U.S. provisional patent application Ser. Nos. 61/675,778 filed Jul. 25, 2012, 61/721,283 filed Nov. 1, 2012, 61/736,465 filed Dec. 12, 2012, 61/794,458 filed Mar. 15, 2013 and 61/835,973 filed Jun. 17, 2013 titled INDUCIBLE DNA BINDING PROTEINS AND GENOME PERTURBATION TOOLS AND APPLICATIONS THEREOF.

Reference is also made to U.S. Provisional Application No. 61/565,171 filed Nov. 30, 2011 and U.S. Application Nos. 13/554,922 filed Jul. 30, 2012 and 13/604,945 filed Sep. 6, 2012, titled NUCLEOTIDE-SPECIFIC RECOGNITION SEQUENCES FOR DESIGNER TAL EFFECTORS.

Reference is also made to U.S. Provisional Application Nos. 61/736,527 filed Dec. 12, 2012; 61/748,427 filed Jan. 2, 2013; 61/757,972 filed Jan. 29, 2013, 61/768,959, filed Feb. 25, 2013 and 61/791,409 filed Mar. 15, 2013, titled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION.

Reference is also made to U.S. Provisional Application Nos. 61/758,468 filed Jan. 30, 2013 and 61/769,046 filed Mar. 15, 2013, titled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION.

Reference is also made to U.S. Provisional Application Nos. 61/835,931; 61/835,936; 61/836,080; 61/836,101; 61/836,123 and 61/836,127 filed Jun. 17, 2013.

Reference is also made to U.S. Provisional Application No. 61/842,322, filed Jul. 2, 2013, titled CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS and US Provisional Application No. 61/847,537, filed Jul. 17, 2013, titled DELIVERY, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION AND APPLICATIONS.

The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was made with government support under R01NS073124 and Pioneer Award 1MH100706 awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to methods and compositions used for the spatial and temporal control of gene expression, such as genome perturbation, that may use inducible transcriptional effectors.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 16, 2015, is named 44790.04.2005_SL.txt and is 827,181 bytes in size.

BACKGROUND OF THE INVENTION

Normal gene expression is a dynamic process with carefully orchestrated temporal and spatial components, the precision of which are necessary for normal development, homeostasis, and advancement of the organism. In turn, the dysregulation of required gene expression patterns, either by increased, decreased, or altered function of a gene or set of genes, has been linked to a wide array of pathologies. Technologies capable of modulating gene expression in a spatiotemporally precise fashion will enable the elucidation of the genetic cues responsible for normal biological processes and disease mechanisms. To address this technological need, Applicants developed inducible molecular tools that may regulate gene expression, in particular, light-inducible transcriptional effectors (LITEs), which provide light-mediated control of endogenous gene expression.

Inducible gene expression systems have typically been designed to allow for chemically induced activation of an inserted open reading frame or shRNA sequence, resulting in gene overexpression or repression, respectively. Disadvantages of using open reading frames for overexpression include loss of splice variation and limitation of gene size. Gene repression via RNA interference, despite its transformative power in human biology, can be hindered by complicated off-target effects. Certain inducible systems including estrogen, ecdysone, and FKBP12/FRAP based systems are known to activate off-target endogenous genes. The potentially deleterious effects of long-term antibiotic treatment can complicate the use of tetracycline transactivator (TET) based systems. In vivo, the temporal precision of these chemically inducible systems is dependent upon the kinetics of inducing agent uptake and elimination. Further, because inducing agents are generally delivered systemically, the spatial precision of such systems is bounded by the precision of exogenous vector delivery.

US Patent Publication No. 20030049799 relates to engineered stimulus-responsive switches to cause a detectable output in response to a preselected stimulus.

There is an evident need for methods and compositions that allow for efficient and precise spatial and temporal control of a genomic locus of interest. These methods and compositions may provide for the regulation and modulation of genomic expression both in vivo and in vitro as well as provide for novel treatment methods for a number of disease pathologies.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

In one aspect the invention provides a non-naturally occurring or engineered TALE or CRISPR-Cas system which may comprise at least one switch wherein the activity of said TALE or CRISPR-Cas system is controlled by contact with at least one inducer energy source as to the switch. In an embodiment of the invention the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system may be activated, enhanced, terminated or repressed. The contact with the at least one inducer energy source may result in a first effect and a second effect. The first effect may be one or more of nuclear import, nuclear export, recruitment of a secondary component (such as an effector molecule), conformational change (of protein, DNA or RNA), cleavage, release of cargo (such as a caged molecule or a co-factor), association or dissociation. The second effect may be one or more of activation, enhancement, termination or repression of the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system. In one embodiment the first effect and the second effect may occur in a cascade.

In another aspect of the invention the TALE or CRISPR-Cas system may further comprise at least one nuclear localization signal (NLS), nuclear export signal (NES), functional domain, flexible linker, mutation, deletion, alteration or truncation. The one or more of the NLS, the NES or the functional domain may be conditionally activated or inactivated. In another embodiment, the mutation may be one or more of a mutation in a transcription factor homology region, a mutation in a DNA binding domain (such as mutating basic residues of a basic helix loop helix), a mutation in an endogenous NLS or a mutation in an endogenous NES. The invention comprehends that the inducer energy source may be heat, ultrasound, electromagnetic energy or chemical. In a preferred embodiment of the invention, the inducer energy source may be an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid or a steroid derivative. In a more preferred embodiment, the inducer energy source maybe abscisic acid (ABA), doxycyeline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. The invention provides that the at least one switch may be selected from the group consisting of antibiotic based inducible systems, electromagnetic energy based inducible systems, small molecule based inducible systems, nuclear receptor based inducible systems and hormone based inducible systems. In a more preferred embodiment the at least one switch may be selected from the group consisting of tetracycline (Tet)/DOX inducible systems, light inducible systems, ABA inducible systems, cumate repressor/operator systems, 4OHT/estrogen inducible systems, ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.

In one aspect of the invention the inducer energy source is electromagnetic energy. The electromagnetic energy may be a component of visible light having a wavelength in the range of 450 nm-700 nm. In a preferred embodiment the component of visible light may have a wavelength in the range of 450 nm-500 nm and may be blue light. The blue light may have an intensity of at least 0.2 mW/cm 2 , or more preferably at least 4 mW/cm 2 . In another embodiment, the component of visible light may have a wavelength in the range of 620-700 nm and is red light.

The invention comprehends systems wherein the at least one functional domain may be selected from the group consisting of: transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domains, transcription-regulatory protein (or transcription complex recruiting) domain, cellular uptake activity associated domain, nucleic acid binding domain, antibody presentation domain, histone modifying enzymes, recruiter of histone modifying enzymes; inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease.

The invention also provides for use of the system for perturbing a genomic or epigenomic locus of interest. Also provided are uses of the system for the preparation of a pharmaceutical compound.

In a further aspect, the invention provides a method of controlling a non-naturally occurring or engineered TALE or CRISPR-Cas system, comprising providing said TALE or CRISPR-Cas system comprising at least one switch wherein the activity of said TALE or CRISPR-Cas system is controlled by contact with at least one inducer energy source as to the switch.

In an embodiment of the invention, the invention provides methods wherein the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system may be activated, enhanced, terminated or repressed. The contact with the at least one inducer energy source may result in a first effect and a second effect. The first effect may be one or more of nuclear import, nuclear export, recruitment of a secondary component (such as an effector molecule), conformational change (of protein, DNA or RNA), cleavage, release of cargo (such as a caged molecule or a co-factor), association or dissociation. The second effect may be one or more of activation, enhancement, termination or repression of the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system. In one embodiment the first effect and the second effect may occur in a cascade.

In another aspect of the methods of the invention the TALE or CRISPR-Cas system may further comprise at least one nuclear localization signal (NLS), nuclear export signal (NES), functional domain, flexible linker, mutation, deletion, alteration or truncation. The one or more of the NLS, the NES or the functional domain may be conditionally activated or inactivated. In another embodiment, the mutation may be one or more of a mutation in a transcription factor homology region, a mutation in a DNA binding domain (such as mutating basic residues of a basic helix loop helix), a mutation in an endogenous NLS or a mutation in an endogenous NES. The invention comprehends that the inducer energy source may be heat, ultrasound, electromagnetic energy or chemical. In a preferred embodiment of the invention, the inducer energy source may be an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid or a steroid derivative. In a more preferred embodiment, the inducer energy source maybe abscisic acid (ABA), doxycyeline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. The invention provides that the at least one switch may be selected from the group consisting of antibiotic based inducible systems, electromagnetic energy based inducible systems, small molecule based inducible systems, nuclear receptor based inducible systems and hormone based inducible systems. In a more preferred embodiment the at least one switch may be selected from the group consisting of tetracycline (Tet)/DOX inducible systems, light inducible systems, ABA inducible systems, cumate repressor/operator systems, 4OHT/estrogen inducible systems, ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.

In one aspect of the methods of the invention the inducer energy source is electromagnetic energy. The electromagnetic energy may be a component of visible light having a wavelength in the range of 450 nm-700 nm. In a preferred embodiment the component of visible light may have a wavelength in the range of 450 nm-500 nm and may be blue light. The blue light may have an intensity of at least 0.2 mW/cm 2 , or more preferably at least 4 mW/cm 2 . In another embodiment, the component of visible light may have a wavelength in the range of 620-700 nm and is red light.

The invention comprehends methods wherein the at least one functional domain may be selected from the group consisting of: transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domains, transcription-regulatory protein (or transcription complex recruiting) domain, cellular uptake activity associated domain, nucleic acid binding domain, antibody presentation domain, histone modifying enzymes, recruiter of histone modifying enzymes; inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease.

Further aspects of the invention provides for systems or methods as described herein wherein the TALE system comprises a DNA binding polypeptide comprising:

(i) a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target a locus of interest or at least one or more effector domains linked to an energy sensitive protein or fragment thereof, wherein the energy sensitive protein or fragment th

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No. 15/388,248 filed Dec. 22, 2016, which is a continuation of U.S. patent application Ser. No. 14/604,641 filed Jan. 23, 2015, which is a continuation-in part of international patent application Serial No. PCT/US13/51418 filed Jul. 21, 2013, which published as WO2014/018423 on Jan. 30, 2014 which claims priority to and claims benefit of U.S. provisional patent application Ser. Nos. 61/675,778 filed Jul. 25, 2012, 61/721,283 filed Nov. 1, 2012, 61/736,465 filed Dec. 12, 2012, 61/794,458 filed Mar. 15, 2013 and 61/835,973 filed Jun. 17, 2013 titled INDUCIBLE DNA BINDING PROTEINS AND GENOME PERTURBATION TOOLS AND APPLICATIONS THEREOF.

Reference is also made to U.S. Provisional Application No. 61/565,171 filed Nov. 30, 2011 and U.S. Application Nos. 13/554,922 filed Jul. 30, 2012 and 13/604,945 filed Sep. 6, 2012, titled NUCLEOTIDE-SPECIFIC RECOGNITION SEQUENCES FOR DESIGNER TAL EFFECTORS.

Reference is also made to U.S. Provisional Application Nos. 61/736,527 filed Dec. 12, 2012; 61/748,427 filed Jan. 2, 2013; 61/757,972 filed Jan. 29, 2013, 61/768,959, filed Feb. 25, 2013 and 61/791,409 filed Mar. 15, 2013, titled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION.

Reference is also made to U.S. Provisional Application Nos. 61/758,468 filed Jan. 30, 2013 and 61/769,046 filed Mar. 15, 2013, titled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION.

Reference is also made to U.S. Provisional Application Nos. 61/835,931; 61/835,936; 61/836,080; 61/836,101; 61/836,123 and 61/836,127 filed Jun. 17, 2013.

Reference is also made to U.S. Provisional Application No. 61/842,322, filed Jul. 2, 2013, titled CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS and US Provisional Application No. 61/847,537, filed Jul. 17, 2013, titled DELIVERY, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION AND APPLICATIONS.

The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was made with government support under R01NS073124 and Pioneer Award 1MH100706 awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to methods and compositions used for the spatial and temporal control of gene expression, such as genome perturbation, that may use inducible transcriptional effectors.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 16, 2015, is named 44790.04.2005_SL.txt and is 827,181 bytes in size.

BACKGROUND OF THE INVENTION

Normal gene expression is a dynamic process with carefully orchestrated temporal and spatial components, the precision of which are necessary for normal development, homeostasis, and advancement of the organism. In turn, the dysregulation of required gene expression patterns, either by increased, decreased, or altered function of a gene or set of genes, has been linked to a wide array of pathologies. Technologies capable of modulating gene expression in a spatiotemporally precise fashion will enable the elucidation of the genetic cues responsible for normal biological processes and disease mechanisms. To address this technological need, Applicants developed inducible molecular tools that may regulate gene expression, in particular, light-inducible transcriptional effectors (LITEs), which provide light-mediated control of endogenous gene expression.

Inducible gene expression systems have typically been designed to allow for chemically induced activation of an inserted open reading frame or shRNA sequence, resulting in gene overexpression or repression, respectively. Disadvantages of using open reading frames for overexpression include loss of splice variation and limitation of gene size. Gene repression via RNA interference, despite its transformative power in human biology, can be hindered by complicated off-target effects. Certain inducible systems including estrogen, ecdysone, and FKBP12/FRAP based systems are known to activate off-target endogenous genes. The potentially deleterious effects of long-term antibiotic treatment can complicate the use of tetracycline transactivator (TET) based systems. In vivo, the temporal precision of these chemically inducible systems is dependent upon the kinetics of inducing agent uptake and elimination. Further, because inducing agents are generally delivered systemically, the spatial precision of such systems is bounded by the precision of exogenous vector delivery.

US Patent Publication No. 20030049799 relates to engineered stimulus-responsive switches to cause a detectable output in response to a preselected stimulus.

There is an evident need for methods and compositions that allow for efficient and precise spatial and temporal control of a genomic locus of interest. These methods and compositions may provide for the regulation and modulation of genomic expression both in vivo and in vitro as well as provide for novel treatment methods for a number of disease pathologies.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

In one aspect the invention provides a non-naturally occurring or engineered TALE or CRISPR-Cas system which may comprise at least one switch wherein the activity of said TALE or CRISPR-Cas system is controlled by contact with at least one inducer energy source as to the switch. In an embodiment of the invention the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system may be activated, enhanced, terminated or repressed. The contact with the at least one inducer energy source may result in a first effect and a second effect. The first effect may be one or more of nuclear import, nuclear export, recruitment of a secondary component (such as an effector molecule), conformational change (of protein, DNA or RNA), cleavage, release of cargo (such as a caged molecule or a co-factor), association or dissociation. The second effect may be one or more of activation, enhancement, termination or repression of the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system. In one embodiment the first effect and the second effect may occur in a cascade.

In another aspect of the invention the TALE or CRISPR-Cas system may further comprise at least one nuclear localization signal (NLS), nuclear export signal (NES), functional domain, flexible linker, mutation, deletion, alteration or truncation. The one or more of the NLS, the NES or the functional domain may be conditionally activated or inactivated. In another embodiment, the mutation may be one or more of a mutation in a transcription factor homology region, a mutation in a DNA binding domain (such as mutating basic residues of a basic helix loop helix), a mutation in an endogenous NLS or a mutation in an endogenous NES. The invention comprehends that the inducer energy source may be heat, ultrasound, electromagnetic energy or chemical. In a preferred embodiment of the invention, the inducer energy source may be an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid or a steroid derivative. In a more preferred embodiment, the inducer energy source maybe abscisic acid (ABA), doxycyeline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. The invention provides that the at least one switch may be selected from the group consisting of antibiotic based inducible systems, electromagnetic energy based inducible systems, small molecule based inducible systems, nuclear receptor based inducible systems and hormone based inducible systems. In a more preferred embodiment the at least one switch may be selected from the group consisting of tetracycline (Tet)/DOX inducible systems, light inducible systems, ABA inducible systems, cumate repressor/operator systems, 4OHT/estrogen inducible systems, ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.

In one aspect of the invention the inducer energy source is electromagnetic energy. The electromagnetic energy may be a component of visible light having a wavelength in the range of 450 nm-700 nm. In a preferred embodiment the component of visible light may have a wavelength in the range of 450 nm-500 nm and may be blue light. The blue light may have an intensity of at least 0.2 mW/cm 2 , or more preferably at least 4 mW/cm 2 . In another embodiment, the component of visible light may have a wavelength in the range of 620-700 nm and is red light.

The invention comprehends systems wherein the at least one functional domain may be selected from the group consisting of: transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domains, transcription-regulatory protein (or transcription complex recruiting) domain, cellular uptake activity associated domain, nucleic acid binding domain, antibody presentation domain, histone modifying enzymes, recruiter of histone modifying enzymes; inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease.

The invention also provides for use of the system for perturbing a genomic or epigenomic locus of interest. Also provided are uses of the system for the preparation of a pharmaceutical compound.

In a further aspect, the invention provides a method of controlling a non-naturally occurring or engineered TALE or CRISPR-Cas system, comprising providing said TALE or CRISPR-Cas system comprising at least one switch wherein the activity of said TALE or CRISPR-Cas system is controlled by contact with at least one inducer energy source as to the switch.

In an embodiment of the invention, the invention provides methods wherein the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system may be activated, enhanced, terminated or repressed. The contact with the at least one inducer energy source may result in a first effect and a second effect. The first effect may be one or more of nuclear import, nuclear export, recruitment of a secondary component (such as an effector molecule), conformational change (of protein, DNA or RNA), cleavage, release of cargo (such as a caged molecule or a co-factor), association or dissociation. The second effect may be one or more of activation, enhancement, termination or repression of the control as to the at least one switch or the activity of said TALE or CRISPR-Cas system. In one embodiment the first effect and the second effect may occur in a cascade.

In another aspect of the methods of the invention the TALE or CRISPR-Cas system may further comprise at least one nuclear localization signal (NLS), nuclear export signal (NES), functional domain, flexible linker, mutation, deletion, alteration or truncation. The one or more of the NLS, the NES or the functional domain may be conditionally activated or inactivated. In another embodiment, the mutation may be one or more of a mutation in a transcription factor homology region, a mutation in a DNA binding domain (such as mutating basic residues of a basic helix loop helix), a mutation in an endogenous NLS or a mutation in an endogenous NES. The invention comprehends that the inducer energy source may be heat, ultrasound, electromagnetic energy or chemical. In a preferred embodiment of the invention, the inducer energy source may be an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid or a steroid derivative. In a more preferred embodiment, the inducer energy source maybe abscisic acid (ABA), doxycyeline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. The invention provides that the at least one switch may be selected from the group consisting of antibiotic based inducible systems, electromagnetic energy based inducible systems, small molecule based inducible systems, nuclear receptor based inducible systems and hormone based inducible systems. In a more preferred embodiment the at least one switch may be selected from the group consisting of tetracycline (Tet)/DOX inducible systems, light inducible systems, ABA inducible systems, cumate repressor/operator systems, 4OHT/estrogen inducible systems, ecdysone-based inducible systems and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.

In one aspect of the methods of the invention the inducer energy source is electromagnetic energy. The electromagnetic energy may be a component of visible light having a wavelength in the range of 450 nm-700 nm. In a preferred embodiment the component of visible light may have a wavelength in the range of 450 nm-500 nm and may be blue light. The blue light may have an intensity of at least 0.2 mW/cm 2 , or more preferably at least 4 mW/cm 2 . In another embodiment, the component of visible light may have a wavelength in the range of 620-700 nm and is red light.

The invention comprehends methods wherein the at least one functional domain may be selected from the group consisting of: transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domains, transcription-regulatory protein (or transcription complex recruiting) domain, cellular uptake activity associated domain, nucleic acid binding domain, antibody presentation domain, histone modifying enzymes, recruiter of histone modifying enzymes; inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease.

Further aspects of the invention provides for systems or methods as described herein wherein the TALE system comprises a DNA binding polypeptide comprising:

(i) a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target a locus of interest or at least one or more effector domains linked to an energy sensitive protein or fragment thereof, wherein the energy sensitive protein or fragment thereof undergoes a conformational change upon induction by an inducer energy source allowing it to bind an interacting partner, and/or (ii) a DNA binding domain comprising at least one or more TALE monomers or half-monomers specifically ordered to target the locus of interest or at least one or more effector domains linked to the interacting partner, wherein the energy sensitive protein or fragment thereof binds to the interacting partner upon induction by the inducer energy source.

The systems and methods of the invention provide for the DNA binding polypeptide comprising a (a) a N-terminal capping region (b) a DNA binding domain comprising at least 5 to 40 Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the locus of interest, and (c) a C-terminal capping region wherein (a), (b) and (c) may be arranged in a predetermined N-terminus to C-terminus orientation, wherein the genomic locus comprises a target DNA sequence 5′-T 0 N 1 N 2 . . . N z N z+1 -3′, where T 0 and N=A, G, T or C, wherein the target DNA sequence binds to the DNA binding domain, and the DNA binding domain may comprise (X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 )z, wherein X 1-11 a chain of 11 contiguous amino acids, wherein X 12 X 13 is a repeat variable diresidue (RVD), wherein X 14-33 or 34 or 35 is a chain of 21, 22 or 23 contiguous amino acids, wherein z may be at least 5 to 40, wherein the polypeptide may be encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to DNA of the locus of interest.

In a further embodiment, the system or method of the invention provides the N-terminal capping region or fragment thereof comprises 147 contiguous amino acids of a wild type N-terminal capping region, or the C-terminal capping region or fragment thereof comprises 68 contiguous amino acids of a wild type C-terminal capping region, or the N-terminal capping region or fragment thereof comprises 136 contiguous amino acids of a wild type N-terminal capping region and the C-terminal capping region or fragment thereof comprises 183 contiguous amino acids of a wild type C-terminal capping region. In another embodiment, the at least one RVD may be selected from the group consisting of (a) HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognition of guanine (G); (b) NI, KI, RI, HI, SI for recognition of adenine (A); (c) NG, HG, KG, RG for recognition of thymine (T); (d) RD, SD, HD, ND, KD, YG for recognition of cytosine (C); (e) NV, HN for recognition of A or G; and (f) H*, HA, KA, N*, NA, NC, NS, RA, S*for recognition of A or T or G or C, wherein (*) means that the amino acid at X 13 is absent.

In yet another embodiment the at least one RVD may be selected from the group consisting of (a) HH, KH, NH, NK, NQ, RH, RN, SS for recognition of guanine (G); (b) SI for recognition of adenine (A); (c) HG, KG, RG for recognition of thymine (T); (d) RD, SD for recognition of cytosine (C); (e) NV, HN for recognition of A or G and (f) H*, HA, KA, N*, NA, NC, NS, RA, S*for recognition of A or T or G or C, wherein (*) means that the amino acid at X 13 is absent. In a preferred embodiment, the RVD for the recognition of G is RN, NH, RH or KH; or the RVD for the recognition of A is SI; or the RVD for the recognition of T is KG or RG; and the RVD for the recognition of C is SD or RD. In yet another embodiment, at least one of the following is present [LTLD] (SEQ ID NO: 1) or [LTLA] (SEQ ID NO: 2) or [LTQV] (SEQ ID NO: 3) at X 1-4 , or [EQHG] (SEQ ID NO: 4) or [RDHG] (SEQ ID NO: 5) at positions X 30-33 or X 31-34 or X 32-35.

In an aspect of the invention the TALE system is packaged into a AAV or a lentivirus vector.

Further aspects of the invention provides for systems or methods as described herein wherein the CRISPR system may comprise a vector system comprising: a) a first regulatory element operably linked to a CRISPR-Cas system guide RNA that targets a locus of interest, b) a second regulatory inducible element operably linked to a Cas protein, wherein components (a) and (b) may be located on same or different vectors of the system, wherein the guide RNA targets DNA of the locus of interest, wherein the Cas protein and the guide RNA do not naturally occur together. In a preferred embodiment of the invention, the Cas protein is a Cas9 enzyme. The invention also provides for the vector being a AAV or a lentivirus.

The invention particularly relates to inducible methods of altering expression of a genomic locus of interest and to compositions that inducibly alter expression of a genomic locus of interest wherein the genomic locus may be contacted with a non-naturally occurring or engineered composition comprising a deoxyribonucleic acid (DNA) binding polypeptide.

This polypeptide may include a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest or at least one or more effector domains linked to an energy sensitive protein or fragment thereof. The energy sensitive protein or fragment thereof may undergo a conformational change upon induction by an energy source allowing it to bind an interacting partner. The polypeptide may also include a DNA binding domain comprising at least one or more variant TALE monomers or half-monomers specifically ordered to target the genomic locus of interest or at least one or more effector domains linked to the interacting partner, wherein the energy sensitive protein or fragment thereof may bind to the interacting partner upon induction by the energy source. The method may also include applying the energy source and determining that the expression of the genomic locus is altered. In preferred embodiments of the invention the genomic locus may be in a cell.

The invention also relates to inducible methods of repressing expression of a genomic locus of interest and to compositions that inducibly repress expression of a genomic locus of interest wherein the genomic locus may be contacted with a non-naturally occurring or engineered composition comprising a DNA binding polypeptide.

The polypeptide may include a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest or at least one or more repressor domains linked to an energy sensitive protein or fragment thereof. The energy sensitive protein or fragment thereof may undergo a conformational change upon induction by an energy source allowing it to bind an interacting partner. The polypeptide may also include a DNA binding domain comprising at least one or more variant TALE monomers or half-monomers specifically ordered to target the genomic locus of interest or at least one or more effector domains linked to the interacting partner, wherein the energy sensitive protein or fragment thereof may bind to the interacting partner upon induction by the energy source. The method may also include applying the energy source and determining that the expression of the genomic locus is repressed. In preferred embodiments of the invention the genomic locus may be in a cell.

The invention also relates to inducible methods of activating expression of a genomic locus of interest and to compositions that inducibly activate expression of a genomic locus of interest wherein the genomic locus may be contacted with a non-naturally occurring or engineered composition comprising a DNA binding polypeptide.

The polypeptide may include a DNA binding domain comprising at least five or more TALE monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest or at least one or more activator domains linked to an energy sensitive protein or fragment thereof. The energy sensitive protein or fragment thereof may undergo a conformational change upon induction by an energy source allowing it to bind an interacting partner. The polypeptide may also include a DNA binding domain comprising at least one or more variant TALE monomers or half-monomers specifically ordered to target the genomic locus of interest or at least one or more effector domains linked to the interacting partner, wherein the energy sensitive protein or fragment thereof may bind to the interacting partner upon induction by the energy source. The method may also include applying the energy source and determining that the expression of the genomic locus is activated. In preferred embodiments of the invention the genomic locus may be in a cell.

In another preferred embodiment of the invention, the inducible effector may be a Light Inducible Transcriptional Effector (LITE). The modularity of the LITE system allows for any number of effector domains to be employed for transcriptional modulation.

In yet another preferred embodiment of the invention, the inducible effector may be a chemical.

The present invention also contemplates an inducible multiplex genome engineering using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems.

The present invention also encompasses nucleic acid encoding the polypeptides of the present invention. The nucleic acid may comprise a promoter, advantageously human Synapsin I promoter (hSyn). In a particularly advantageous embodiment, the nucleic acid may be packaged into an adeno associated viral vector (AAV).

The invention further also relates to methods of treatment or therapy that encompass the methods and compositions described herein.

Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean“includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

FIG. 1 shows a schematic indicating the need for spatial and temporal precision.

FIG. 2 shows transcription activator like effectors (TALEs). TALEs consist of 34 aa repeats at the core of their sequence. Each repeat corresponds to a base in the target DNA that is bound by the TALE. Repeats differ only by 2 variable amino acids at positions 12 and 13. The code of this correspondence has been elucidated (Boch, J et al., Science, 2009 and Moscou, M et al., Science, 2009) and is shown in this figure. Applicants have developed a method for the synthesis of designer TALEs incorporating this code and capable of binding a sequence of choice within the genome (Zhang, F et al., Nature Biotechnology, 2011). FIG. 2 discloses SEQ ID NOS 212-213, respectively, in order of appearance.

FIG. 3 shows a design of a LITE: TALE/Cryptochrome transcriptional activation. Each LITE is a two-component system which may comprise a TALE fused to CRY2 and the cryptochrome binding partner CIB1 fused to VP64, a transcription activor. In the inactive state, the TALE localizes its fused CRY2 domain to the promoter region of the gene of interest. At this point, CIB1 is unable to bind CRY2, leaving the CIB1-VP64 unbound in the nuclear space. Upon stimulation with 488 nm (blue) light, CRY2 undergoes a conformational change, revealing its CIB1 binding site (Liu, H et al., Science, 2008). Rapid binding of CIB1 results in recruitment of the fused VP64 domain, which induces transcription of the target gene.

FIG. 4 shows effects of cryptochrome dimer truncations on LITE activity. Truncations known to alter the activity of CRY2 and CIB1 (Kennedy M et al., Nature Methods 2010) were compared against the full length proteins. A LITE targeted to the promoter of Neurog2 was tested in Neuro-2a cells for each combination of domains. Following stimulation with 488 nm light, transcript levels of Neurog2 were quantified using qPCR for stimulated and unstimulated samples.

FIG. 5 shows a light-intensity dependent response of KLF4 LITE.

FIG. 6 shows activation kinetics of Neurog2 LITE and inactivation kinetics of Neurog2 LITE.

FIG. 7A shows the base-preference of various RVDs as determined using the Applicants' RVD screening system.

FIG. 7B shows the base-preference of additional RVDs as determined using the Applicants' RVD screening system.

FIGS. 8A-D show in (a) Natural structure of TALEs derived from Xanthomonas sp. Each DNA-binding module consists of 34 amino acids, where the RVDs in the 12th and 13th amino acid positions of each repeat specify the DNA base being targeted according to the cipher NG=T, HD=C, NI=A, and NN=G or A. The DNA-binding modules are flanked by nonrepetitive N and C termini, which carry the translocation, nuclear localization (NLS) and transcription activation (AD) domains. A cryptic signal within the N terminus specifies a thymine as the first base of the target site. (b) The TALE toolbox allows rapid and inexpensive construction of custom TALE-TFs and TALENs. The kit consists of 12 plasmids in total: four monomer plasmids to be used as templates for PCR amplification, four TALE-TF and four TALEN cloning backbones corresponding to four different bases targeted by the 0.5 repeat. CMV, cytomegalovirus promoter; N term, nonrepetitive N terminus from the Hax3 TALE; C term, nonrepetitive C terminus from the Hax3 TALE; BsaI, type IIs restriction sites used for the insertion of custom TALE DNA-binding domains; ccdB+CmR, negative selection cassette containing the ccdB negative selection gene and chloramphenicol resistance gene; NLS, nuclear localization signal; VP64, synthetic transcriptional activator derived from VP16 protein of herpes simplex virus; 2A, 2A self-cleavage linker; EGFP, enhanced green fluorescent protein; polyA signal, polyadenylation signal; FokI, catalytic domain from the FokI endonuclease. (c) TALEs may be used to generate custom TALE-TFs and modulate the transcription of endogenous genes from the genome. The TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors needed to initiate transcription. (d) TALENs may be used to generate site-specific double-strand breaks to facilitate genome editing through nonhomologous repair or homology directed repair. Two TALENs target a pair of binding sites flanking a 16-bp spacer. The left and right TALENs recognize the top and bottom strands of the target sites, respectively. Each TALE DNA-binding domain is fused to the catalytic domain of FokI endonuclease; when FokI dimerizes, it cuts the DNA in the region between the left and right TALEN-binding sites. FIG. 8A discloses SEQ ID NOS 212-213, respectively, in order of appearance.

FIG. 9A-F shows a table listing monomer sequences (SEQ ID NOS 214-444, respectively, in order of appearance) (excluding the RVDs at positions 12 and 13) and the frequency with which monomers having a particular sequence occur.

FIG. 10 shows the comparison of the effect of non-RVD amino acid on TALE activity. FIG. 10 discloses SEQ ID NOS 215, 214, 221, 218, 244, 445, 214, 219, 334, 446, 251, and 447, respectively, in order of appearance.

FIG. 11 shows an activator screen comparing levels of activation between VP64, p65 and VP16.

FIGS. 12A-D show the development of a TALE transcriptional repressor architecture. (a) Design of SOX2 TALE for TALE repressor screening. A TALE targeting a 14bp sequence within the SOX2 locus of the human genome was synthesized. (b) List of all repressors screened and their host origin (left). Eight different candidate repressor domains were fused to the C-term of the SOX2 TALE. (c) The fold decrease of endogenous SOX2 mRNA is measured using qRTPCR by dividing the SOX2 mRNA levels in mock transfected cells by SOX2 mRNA levels in cells transfected with each candidate TALE repressor. (d) Transcriptional repression of endogenous CACNA1C. TALEs using NN, NK, and NH as the G-targeting RVD were constructed to target a 18 bp target site within the human CACNA1C locus. Each TALE is fused to the SID repression domain. NLS, nuclear localization signal; KRAB, Krüppel-associated box; SID, mSin interaction domain. All results are collected from three independent experiments in HEK 293FT cells. Error bars indicate s.e.m.; n=3. *p<0.05, Student's t test. FIGS. 12A and 12D disclose SEQ ID NOS 448 and 449, respectively.

FIGS. 13A-C shows the optimization of TALE transcriptional repressor architecture using SID and SID4X. (a) Design of p11 TALE for testing of TALE repressor architecture. A TALE targeting a 20 bp sequence (p11 TALE binding site) within the p11 (s100a10) locus of the mouse ( Mus musculus ) genome was synthesized. (b) Transcriptional repression of endogenous mouse p11 mRNA. TALEs targeting the mouse p11 locus harboring two different truncations of the wild type TALE architecture were fused to different repressor domains as indicated on the x-axis. The value in the bracket indicate the number of amino acids at the N-and C-termini of the TALE DNA binding domain flanking the DNA binding repeats, followed by the repressor domain used in the construct. The endogenous p11 mRNA levels were measured using qRT-PCR and normalized to the level in the negative control cells transfected with a GFP-encoding construct. (c) Fold of transcriptional repression of endogenous mouse p11. The fold decrease of endogenous p11 mRNA is measured using qRT-PCR through dividing the p11 mRNA levels in cells transfected with a negative control GFP construct by p11 mRNA levels in cells transfected with each candidate TALE repressors. The labeling of the constructs along the x-axis is the same as previous panel. NLS, nuclear localization signal; SID, mSin interaction domain; SID4X, an optimized four-time tandem repeats of SID domain linked by short peptide linkers. All results are collected from three independent experiments in Neuro2A cells. Error bars indicate s.e.m.; n=3. ***p<0.001, Student's t test. FIG. 13A discloses SEQ ID NO: 450.

FIG. 14A-D shows a comparison of two different types of TALE architecture.

FIGS. 15A-C show a chemically inducible TALE ABA inducible system. ABI (ABA insensitive 1) and PYL (PYL protein: pyrabactin resistance (PYR)/PYR1-like (PYL)) are domains from two proteins listed below that will dimerize upon binding of plant hormone Abscisic Acid (ABA). This plant hormone is a small molecule chemical that Applicants used in Applicants' inducible TALE system. In this system, the TALE DNA-binding polypeptide is fused to the ABI domain, whereas the VP64 activation domain or SID repressor domain or any effector domains are linked to the PYL domain. Thus, upon the induction by the presence of ABA molecule, the two interacting domains, ABI and PYL, will dimerize and allow the TALE to be linked to the effector domains to perform its activity in regulating target gene expression.

FIGS. 16A-B show a chemically inducible TALE 4OHT inducible system.

FIG. 17 depicts an effect of cryptochrome2 heterodimer orientation on LITE functionality.

FIG. 18 depicts mGlur2 LITE activity in mouse cortical neuron culture.

FIG. 19 depicts transduction of primary mouse neurons with LITE AAV vectors.

FIG. 20 depicts expression of LITE component in vivo.

FIG. 21 depicts an improved design of the construct where the specific NES peptide sequence used is LDLASLIL (SEQ ID NO: 6).

FIG. 22 depicts Sox2 mRNA levels in the absence and presence of 4OH tamoxifen.

FIGS. 23A-E depict a Type II CRISPR locus from Streptococcus pyogenes SF370 can be reconstituted in mammalian cells to facilitate targeted DSBs of DNA. (A) Engineering of SpCas9 and SpRNase III with NLSs enables import into the mammalian nucleus. (B) Mammalian expression of SpCas9 and SpRNase III are driven by the EF1la promoter, whereas tracrRNA and pre-crRNA array (DR-Spacer-DR) are driven by the U6 promoter. A protospacer (blue highlight) from the human EMX1 locus with PAM is used as template for the spacer in the pre-crRNA array. (C) Schematic representation of base pairing between target locus and EMX1-targeting crRNA. Red arrow indicates putative cleavage site. (D) SURVEYOR assay for SpCas9-mediated indels. (E) An example chromatogram showing a micro-deletion, as well as representative sequences of mutated alleles identified from 187 clonal amplicons. Red dashes, deleted bases; red bases, insertions or mutations. Scale bar=10 μm. FIG. 23B discloses SEQ ID NO: 451, FIG. 23C discloses SEQ ID NOS 452-453, and FIG. 23E discloses SEQ ID NOS 454-461, all respectively, in order of appearance.

FIGS. 24A-C depict a SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. (A) Schematic of the human EMX1 locus showing the location of five protospacers, indicated by blue lines with corresponding PAM in magenta. (B) Schematic of the pre-crRNA:tracrRNA complex (top) showing hybridization between the direct repeat (gray) region of the pre-crRNA and tracrRNA. Schematic of a chimeric RNA design (M. Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816 (Aug. 17, 2012)) (bottom). tracrRNA sequence is shown in red and the 20 bp spacer sequence in blue. (C) SURVEYOR assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus. Each protospacer is targeted using either processed pre-crRNA:tracrRNA complex (crRNA) or chimeric RNA (chiRNA). FIG. 24A discloses SEQ ID NO: 462 and FIG. 24B discloses SEQ ID NOS 463-465, respectively, in order of appearance.

FIGS. 25A-D depict an evaluation of the SpCas9 specificity and comparison of efficiency with TALENs. (A) EMX1-targeting chimeric crRNAs with single point mutations were generated to evaluate the effects of spacer-protospacer mismatches. (B) SURVEYOR assay comparing the cleavage efficiency of different mutant chimeric RNAs. (C) Schematic showing the design of TALENs targeting EMX1 . (D) SURVEYOR gel comparing the efficiency of TALEN and SpCas9 (N=3). FIG. 25A discloses SEQ ID NOS 466-478, respectively, in order of appearance, and FIG. 25C discloses SEQ ID NO: 466.

FIGS. 26A-G depict applications of Cas9 for homologous recombination and multiplex genome engineering. (A) Mutation of the RuvC I domain converts Cas9 into a nicking enzyme (SpCas9n) (B) Co-expression of EMX1-targeting chimeric RNA with SpCas9 leads to indels, whereas SpCas9n does not (N=3). (C) Schematic representation of the recombination strategy. A repair template is designed to insert restriction sites into EMX1 locus. Primers used to amplify the modified region are shown as red arrows. (D) Restriction fragments length polymorphism gel analysis. Arrows indicate fragments generated by HindIII digestion. (E) Example chromatogram showing successful recombination. (F) SpCas9 can facilitate multiplex genome modification using a crRNA array containing two spacers targeting EMX1 and PVALB. Schematic showing the design of the crRNA array (top). Both spacers mediate efficient protospacer cleavage (bottom). (G) SpCas9 can be used to achieve precise genomic deletion. Two spacers targeting EMX1 (top) mediated a 118bp genomic deletion (bottom). FIG. 26E discloses SEQ ID NO: 479, FIG. 26F discloses SEQ ID NOS 480-481, and FIG. 26G discloses SEQ ID NOS 482-486, respectively, in order of appearance.

FIG. 27 depicts a schematic of the type II CRISPR-mediated DNA double-strand break. The type II CRISPR locus from Streptococcus pyogenes SF370 contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, 30 bp each) (15-18, 30, 31). Each spacer is typically derived from foreign genetic material (protospacer), and directs the specificity of CRISPR-mediated nucleic acid cleavage. In the target nucleic acid, each protospacer is associated with a protospacer adjacent motif (PAM) whose recognition is specific to individual CRISPR systems (22, 23). The Type II CRISPR system carries out targeted DNA double-strand break (DSB) in sequential steps (M. Jinek et al., Science 337, 816 (Aug. 17, 2012); Gasiunas, R. et al. Proc Natl Acad Sci U S A 109, E2579 (Sep. 25, 2012); J. E. Garneau et al., Nature 468, 67 (Nov. 4, 2010); R. Sapranauskas et al., Nucleic Acids Res 39, 9275 (November 2011); A. H. Magadan et al. PLoS One 7, e40913 (2012)). First, the pre-crRNA array and tracrRNA are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA and associates with Cas9 as a duplex, which mediates the processing of the pre-crRNA into mature crRNAs containing individual,truncated spacer sequences. Third, the mature crRNA:tracrRNA duplex directs Cas9 to the DNA target consisting of the protospacer and the requisite PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer.

FIGS. 28A-C depict a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting. (A) Schematic showing the design and sequences of two tracrRNA transcripts tested (short and long). Each transcript is driven by a U6 promoter. Transcription start site is marked as +1 and transcription terminator is as indicated. Blue line indicates the region whose reverse-complement sequence is used to generate northern blot probes for tracrRNA detection. (B) SURVEYOR assay comparing the efficiency of hSpCas9-mediated cleavage of the EMX1 locus. Two biological replicas are shown for each tracrRNA transcript. (C) Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying long or short tracrRNA, as well as SpCas9 and DR-EMX1(1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III respectively. U6 indicate loading control blotted with a probe targeting human U6 snRNA. Transfection of the short tracrRNA expression construct led to abundant levels of the processed form of tracrRNA (˜75 bp) (E. Deltcheva et al., Nature 471, 602 (Mar. 31, 2011)). Very low amounts of long tracrRNA are detected on the Northern blot. As a result of these experiments, Applicants chose to use short tracrRNA for application in mammalian cells. FIG. 28A discloses SEQ ID NOS 487-488, respectively, in order of appearance.

FIG. 29 depicts a SURVEYOR assay for detection of double strand break-induced micro insertions and deletions (D. Y. Guschin et al. Methods Mol Biol 649, 247 (2010)). Schematic of the SURVEYOR assay used to determine Cas9-mediated cleavage efficiency. First, genomic PCR (gPCR) is used to amplify the Cas9 target region from a heterogeneous population of modified and unmodified cells, and the gPCR products are reannealed slowly to generate heteroduplexes. The reannealed heteroduplexes are cleaved by SURVEYOR nuclease, whereas homoduplexes are left intact. Cas9-mediated cleavage efficiency (% indel) is calculated based on the fraction of cleaved DNA.

FIG. 30A-B depict a Northern blot analysis of crRNA processing in mammalian cells. (A) Schematic showing the expression vector for a single spacer flanked by two direct repeats (DR-EMX1(1)-DR). The 30 bp spacer targeting the human EMX1 locus protospacer 1 (Table 1) is shown in blue and direct repeats are in shown in gray. Orange line indicates the region whose reversecomplement sequence is used to generate northern blot probes for EMX1 (1) crRNA detection. (B) Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying DR-EMX1 (1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III respectively. DR-EMX1(1)-DR was processed into mature crRNAs only in the presence of SpCas9 and short tracrRNA, and was not dependent on the presence of SpRNase III. The mature crRNA detected from transfected 293FT total RNA is ˜33 bp and is shorter than the 39-42 bp mature crRNA from S. pyogenes (E. Deltcheva et al., Nature 471, 602 (Mar 31, 2011)), suggesting that the processed mature crRNA in human 293FT cells is likely different from the bacterial mature crRNA in S. pyogenes . FIG. 30A discloses SEQ ID NO: 489.

FIG. 31A-B depict a bicistronic expression vectors for pre-crRNA array or chimeric crRNA with Cas9. (A) Schematic showing the design of an expression vector for the pre-crRNA array. Spacers can be inserted between two BbsI sites using annealed oligonucleotides. Sequence design for the oligonucleotides are shown below with the appropriate ligation adapters indicated. (B) Schematic of the expression vector for chimeric crRNA. The guide sequence can be inserted between two BbsI sites using annealed oligonucleotides. The vector already contains the partial direct repeat (gray) and partial tracrRNA (red) sequences. WPRE, Woodchuck hepatitis virus posttranscriptional regulatory element. FIG. 31A discloses SEQ ID NOS 490-492, and FIG. 31B discloses SEQ ID NOS 493-495, all respectively, in order of appearance.

FIGS. 32A-B depict a selection of protospacers in the human PVALB and mouse Th loci. Schematic of the human PVALB (A) and mouse Th (B) loci and the location of the three protospacers within the last exon of the PVALB and Th genes, respectively. The 30 bp protospacers are indicated by black lines and the adjacent PAM sequences are indicated by the magenta bar. Protospacers on the sense and anti-sense strands are indicated above and below the DNA sequences respectively. FIGS. 32A-B disclose SEQ ID NOS 496 and 497, respectively.

FIGS. 33A-C depict occurrences of PAM sequences in the human genome. Histograms of distances between adjacent Streptococcus pyogenes

SF370 locus 1 PAM (NGG) (A) and Streptococcus <figure-callout id="1" label="thermophiles LMD9 locus" filena

CLAIMS

Claims ( 46 )

What is claimed is:

1 . An engineered composition comprising:

a Cas9 protein; and a guide comprising: (a) an engineered guide sequence capable of hybridizing o a target sequence adjacent to a protospacer adjacent motif, (b) a tracr mate sequence capable of hybridizing to a tracr sequence, and (c) the tracr sequence.

2 . An engineered composition comprising one or more vectors comprising one or more nucleotide sequences encoding a Cas9 protein, and one or more nucleotide sequences encoding a guide, the guide compri sing:

an engineered guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif, (b) a tracr mate sequence capable of hybridizing to a tracr sequence, and (c) the tracr sequence.

3 . The composition according to claim 1 or 2 , wherein the Cas9 protein is translated from mRNA.

4 . The composition according to claim 1 or 2 , wherein the Cas9 protein is delivered in the form of a protein.

5 . The composition according to claim 1 or 2 , wherein (a), (b), and (c) are arranged in a 5′ to 3′ orientation.

6 . The composition according to claim 1 or 2 , wherein (b) and (c) are fused or covalently linked to one another with intervening nucleotides.

7 . The composition according to claim 1 or 2 , wherein the Cas9 protein is derived from Streptococcus or Staphylococcus.

8 . The composition according to claim 7 , wherein the Cas9 protein is Streptococcus pyogenes Cas9.

9 . The composition according to claim 1 or 2 , further comprising a template.

10 . The composition according to claim 1 or 2 , wherein the Cas9 protein is linked or fused to one or more NLSs.

11 . The composition according to claim 2 , wherein the nucleotide sequence encoding the Cas9 protein is codon optimized for expression in a eukaryotic cell.

12 . The composition according to claim 6 , wherein the tracr sequence comprises at least 40 nucleotides in length.

13 . The composition according to claim 1 or 2 , wherein the composition is configured as a delivery composition suitable for delivery by an electroporation method.

14 . The composition according to claim 1 or 2 , wherein two or more different guide sequences are provided for multiplexed targeting.

15 . The composition according to claim 1 or 2 , wherein the Cas9 protein is linked or fused to at least one functional domain.

16 . The composition according to claim 15 , wherein the functional domain is a transcriptional activation domain or a transcriptional repression domain.

17 . The composition according to claim 1 or 2 , wherein the engineered composition comprises or encodes two or more guides each capable of hybridizing to a different target sequence in the eukaryotic cell.

18 . The composition according to claim 1 or 2 , wherein the engineered composition further comprises a recombination template.

19 . The composition of claim 18 , wherein the recombination template is inserted by homology-directed repair (HDR).

20 . A method of modifying one or more target sequences comprising contacting a cell or population of cells with the composition of claim 1 or 2 , wherein the guide sequence directs sequence-specific binding to a target sequence in the cell or population of cells, whereby the target sequence is modified.

21 . The method of claim 20 , wherein the modification of the one or more target sequences results in:

a. a cell or population of cells comprising altered expression levels of one or more gene products; b. a cell or population of cells that produces and/or secrete an endogenous or non-endogenous biological product or chemical compound

22 . An engineered cell comprising one or more modified genes modified according to the method of claim 20 .

23 . The engineered cell of claim 22 , wherein the cell produces and/or secretes an endogenous or non-endogenous biological product or chemical compound.

24 . The engineered cell of claim 23 , wherein the biological product is a protein or an RNA.

25 . A cell line comprising the engineered cell of claim 22 and progeny thereof.

26 . A plant or animal model comprising the engineered cell of claim 22 and progeny thereof.

27 . A composition comprising the engineered cell of claim 22 .

28 . The composition of claim 27 formulated for use as a therapeutic.

29 . A biological product or chemical compound produced by the engineered cell of claim 22 .

30 . An engineered cell or progeny thereof, the cell being engineered by use of an engineered CRISPR-Cas complex.

31 . The cell or progeny thereof of claim 30 that is isolated.

32 . The cell or progeny thereof of claim 30 that is further used as a therapeutic.

33 . The cell or progeny thereof of claim 30 from which a product is isolated.

34 . A product produced by the cell or progeny thereof of claim 30 .

35 . The product of claim 34 , wherein the product is a protein or an RNA.

36 . The product of claim 35 , wherein the protein comprises a mutation.

37 . A pharmaceutical composition for treatment of a disease or disorder, comprising the cell or progeny thereof of claim 30 .

38 . The pharmaceutical composition according to claim 37 , wherein the treatment results in genetic changes in one or more cells.

39 . The pharmaceutical composition according to claim 37 , wherein the treatment results in correction of one or more defective genotypes.

40 . The pharmaceutical composition according to claim 37 , wherein the treatment results in improved phenotype.

41 . The cell or progeny thereof of claim 30 , wherein the cell comprises a mutation in a protein expressed from a gene comprising the target sequence.

42 . The cell or progeny thereof of claim 41 , wherein the cell comprises deletion of a genomic region comprising the target sequence.

43 . The cell or progeny thereof of claim 41 , wherein the cell comprises integration of an exogenous sequence by homology-directed repair.

44 . The cell or progeny thereof of claim 41 , wherein the cell comprises decreased transcription of a gene associated with the target sequence.

45 . The cell or progeny thereof of claim 41 , wherein the cell comprises increased transcription of a gene associated with the target sequence.

46 . The product of claim 34 , wherein the product is a mutated protein or product provided by a template.

US16/535,042

2012-07-25

2019-08-07

Inducible dna binding proteins and genome perturbation tools and applications thereof

Pending

US20190390204A1

( en )

Priority Applications (1)

Application Number

Priority Date

Filing Date

Title

US16/535,042

US20190390204A1

( en )

2012-07-25

2019-08-07

Inducible dna binding proteins and genome perturbation tools and applications thereof

Applications Claiming Priority (9)

Application Number

Priority Date

Filing Date

Title

US201261675778P

2012-07-25

2012-07-25

US201261721283P

2012-11-01

2012-11-01

US201261736465P

2012-12-12

2012-12-12

US201361794458P

2013-03-15

2013-03-15

US201361835973P

2013-06-17

2013-06-17

PCT/US2013/051418

WO2014018423A2

( en )

2012-07-25

2013-07-21

Inducible dna binding proteins and genome perturbation tools and applications thereof

US14/604,641

US20150291966A1

( en )

2012-07-25

2015-01-23

Inducible dna binding proteins and genome perturbation tools and applications thereof

US15/388,248

US20170166903A1

( en )

2012-07-25

2016-12-22

Inducible dna binding proteins and genome perturbation tools and applications thereof

US16/535,042

US20190390204A1

( en )

2012-07-25

2019-08-07

Inducible dna binding proteins and genome perturbation tools and applications thereof

Related Parent Applications (1)

Application Number

Title

Priority Date

Filing Date

US15/388,248

Continuation

US20170166903A1

( en )

2012-07-25

2016-12-22

Inducible dna binding proteins and genome perturbation tools and applications thereof

Publications (1)

Publication Number

Publication Date

US20190390204A1

true

US20190390204A1 ( en )

2019-12-26

Family

ID=48914461

Family Applications (4)

Application Number

Title

Priority Date

Filing Date

US14/604,641

Abandoned

US20150291966A1

( en )

2012-07-25

2015-01-23

Inducible dna binding proteins and genome perturbation tools and applications thereof

US15/388,248

Abandoned

US20170166903A1

( en )

2012-07-25

2016-12-22

Inducible dna binding proteins and genome perturbation tools and applications thereof

US16/297,560

Abandoned

US20190203212A1

( en )

2012-07-25

2019-03-08

Inducible dna binding proteins and genome perturbation tools and applications thereof

US16/535,042

Pending

US20190390204A1

( en )

2012-07-25

2019-08-07

Inducible dna binding proteins and genome perturbation tools and applications thereof

Family Applications Before (3)

Application Number

Title

Priority Date

Filing Date

US14/604,641

Abandoned

US20150291966A1

( en )

2012-07-25

2015-01-23

Inducible dna binding proteins and genome perturbation tools and applications thereof

US15/388,248

Abandoned

US20170166903A1

( en )

2012-07-25

2016-12-22

Inducible dna binding proteins and genome perturbation tools and applications thereof

US16/297,560

Abandoned

US20190203212A1

( en )

2012-07-25

2019-03-08

Inducible dna binding proteins and genome perturbation tools and applications thereof

Country Status (12)

Country

Link

US

( 4 )

US20150291966A1

( en )

EP

( 3 )

EP3808844A1

( en )

JP

( 2 )

JP2015527889A

( en )

KR

( 2 )

KR20230065381A

( en )

CN

( 2 )

CN116622704A

( en )

AU

( 3 )

AU2013293270B2

( en )

CA

( 1 )

CA2879997A1

( en )

DK

( 1 )

DK3494997T3

( en )

ES

( 1 )

ES2757623T3

( en )

PL

( 1 )

PL3494997T3

( en )

PT

( 1 )

PT3494997T

( en )

WO

( 1 )

WO2014018423A2

( en )

Cited By (7)

* Cited by examiner, † Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

CN110857440A

( en )

*

2018-08-23

2020-03-03

武汉纽福斯生物科技有限公司

Recombinant human II type mitochondrial dynamic protein sample GTP enzyme gene sequence and application thereof

US11814645B2

( en )

2012-05-25

2023-11-14

The Regents Of The University Of California

Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

US11859172B2

( en )

2020-01-02

2024-01-02

The Trustees Of Columbia University In The City Of New York

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

WO2024136952A1

( en )

*

2022-12-20

2024-06-27

Florida Atlantic University Board Of Trustees

Phosphagen system modulators and uses thereof

US12251429B2

( en )

2015-06-01

2025-03-18

Temple University—Of the Commonwealth System of Higher Education

Methods and compositions for RNA-guided treatment of HIV infection

WO2025226836A1

( en )

2024-04-24

2025-10-30

Sarepta Therapeutics, Inc.

Treatment of genetic neurological conditions with genomic editing

WO2025250808A1

( en )

2024-05-29

2025-12-04

The Brigham And Women’S Hospital, Inc.

Anti-crispr delivery compositions and methods

Families Citing this family (560)

* Cited by examiner, † Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

AR085533A1

( en )

2011-03-23

2013-10-09

Pioneer Hi Bred Int

PRODUCTION METHODS OF A COMPLEX TRANSGENIC RISK LOCUS

US20160144003A1

( en )

*

2011-05-19

2016-05-26

The Scripps Research Institute

Compositions and methods for treating charcot-marie-tooth diseases and related neuronal diseases

CA2841710C

( en )

2011-07-15

2021-03-16

The General Hospital Corporation

Methods of transcription activator like effector assembly

WO2013066438A2

( en )

2011-07-22

2013-05-10

President And Fellows Of Harvard College

Evaluation and improvement of nuclease cleavage specificity

US10465042B2

( en )

2011-12-02

2019-11-05

Yale University

Poly(amine-co-ester) nanoparticles and methods of use thereof

GB201122458D0

( en )

2011-12-30

2012-02-08

Univ Wageningen

Modified cascade ribonucleoproteins and uses thereof

US10322949B2

( en )

2012-03-15

2019-06-18

Flodesign Sonics, Inc.

Transducer and reflector configurations for an acoustophoretic device

US9950282B2

( en )

2012-03-15

2018-04-24

Flodesign Sonics, Inc.

Electronic configuration and control for acoustic standing wave generation

US9745548B2

( en )

2012-03-15

2017-08-29

Flodesign Sonics, Inc.

Acoustic perfusion devices

US10967298B2

( en )

2012-03-15

2021-04-06

Flodesign Sonics, Inc.

Driver and control for variable impedence load

US10704021B2

( en )

2012-03-15

2020-07-07

Flodesign Sonics, Inc.

Acoustic perfusion devices

US9458450B2

( en )

2012-03-15

2016-10-04

Flodesign Sonics, Inc.

Acoustophoretic separation technology using multi-dimensional standing waves

US9752113B2

( en )

2012-03-15

2017-09-05

Flodesign Sonics, Inc.

Acoustic perfusion devices

US10689609B2

( en )

2012-03-15

2020-06-23

Flodesign Sonics, Inc.

Acoustic bioreactor processes

US10737953B2

( en )

2012-04-20

2020-08-11

Flodesign Sonics, Inc.

Acoustophoretic method for use in bioreactors

ES2683071T3

( en )

2012-04-25

2018-09-24

Regeneron Pharmaceuticals, Inc.

Nuclease-mediated addressing with large addressing vectors

EP2841572B1

( en )

2012-04-27

2019-06-19

Duke University

Genetic correction of mutated genes

US9890364B2

( en )

2012-05-29

2018-02-13

The General Hospital Corporation

TAL-Tet1 fusion proteins and methods of use thereof

CN116622704A

( en )

2012-07-25

2023-08-22

布罗德研究所有限公司

Inducible DNA-binding proteins and genome interference tools and their applications

EP3789405A1

( en )

2012-10-12

2021-03-10

The General Hospital Corporation

Transcription activator-like effector (tale) - lysine-specific demethylase 1 (lsd1) fusion proteins

EP4397759A3

( en )

*

2012-10-23

2024-10-09

Toolgen Incorporated

Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof

DK3363902T3

( en )

2012-12-06

2020-01-02

Sigma Aldrich Co Llc

CRISPR-BASED RE-MODIFICATION AND REGULATION

EP2954042B1

( en )

*

2013-02-07

2017-12-06

The General Hospital Corporation

Tale transcriptional activators

ES2749624T3

( en )

2013-03-14

2020-03-23

Caribou Biosciences Inc

Nucleic Acid Targeted Nucleic Acid Compositions and Methods

US10760064B2

( en )

2013-03-15

2020-09-01

The General Hospital Corporation

RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci

CN105408483A

( en )

2013-03-15

2016-03-16

通用医疗公司

RNA-guided targeting of genetic and epigenetic regulatory proteins to specific genomic loci

US9828582B2

( en )

2013-03-19

2017-11-28

Duke University

Compositions and methods for the induction and tuning of gene expression

DK3456831T3

( en )

2013-04-16

2021-09-06

Regeneron Pharma

TARGETED MODIFICATION OF RAT GENOMES

WO2014186435A2

( en )

2013-05-14

2014-11-20

University Of Georgia Research Foundation, Inc.

Compositions and methods for reducing neointima formation

US9873907B2

( en )

2013-05-29

2018-01-23

Agilent Technologies, Inc.

Method for fragmenting genomic DNA using CAS9

KR20250012194A

( en )

*

2013-06-17

2025-01-23

더 브로드 인스티튜트, 인코퍼레이티드

Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components

US10421957B2

( en )

2013-07-29

2019-09-24

Agilent Technologies, Inc.

DNA assembly using an RNA-programmable nickase

US9163284B2

( en )

2013-08-09

2015-10-20

President And Fellows Of Harvard College

Methods for identifying a target site of a Cas9 nuclease

CN106133138A

( en )

2013-08-22

2016-11-16

先锋国际良种公司

Use genomic modification and the using method thereof guiding polynucleotide/CAS endonuclease systems

US9359599B2

( en )

2013-08-22

2016-06-07

President And Fellows Of Harvard College

Engineered transcription activator-like effector (TALE) domains and uses thereof

US9526784B2

( en )

2013-09-06

2016-12-27

President And Fellows Of Harvard College

Delivery system for functional nucleases

US9322037B2

( en )

2013-09-06

2016-04-26

President And Fellows Of Harvard College

Cas9-FokI fusion proteins and uses thereof

US9340799B2

( en )

2013-09-06

2016-05-17

President And Fellows Of Harvard College

MRNA-sensing switchable gRNAs

IL312865B2

( en )

2013-09-11

2025-06-01

Eagle Biologics Inc

Liquid protein formulations containing viscosity-reducing agents

US9745569B2

( en )

2013-09-13

2017-08-29

Flodesign Sonics, Inc.

System for generating high concentration factors for low cell density suspensions

WO2015065964A1

( en )

2013-10-28

2015-05-07

The Broad Institute Inc.

Functional genomics using crispr-cas systems, compositions, methods, screens and applications thereof

CN111218447B

( en )

2013-11-07

2024-10-11

爱迪塔斯医药有限公司

CRISPR-related methods and compositions using governing gRNA

MX388127B

( en )

2013-12-11

2025-03-19

Regeneron Pharma

METHODS AND COMPOSITIONS FOR THE TARGETED MODIFICATION OF A GENOME.

WO2015089473A1

( en )

2013-12-12

2015-06-18

The Broad Institute Inc.

Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation

KR20160097338A

( en )

2013-12-12

2016-08-17

더 브로드 인스티튜트, 인코퍼레이티드

Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders

WO2015089364A1

( en )

2013-12-12

2015-06-18

The Broad Institute Inc.

Crystal structure of a crispr-cas system, and uses thereof

CN111206032B

( en )

2013-12-12

2024-07-19

布罗德研究所有限公司

Delivery, use and therapeutic applications of CRISPR-CAS systems and compositions for genome editing

WO2015089419A2

( en )

2013-12-12

2015-06-18

The Broad Institute Inc.

Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using particle delivery components

JP6793547B2

( en )

2013-12-12

2020-12-02

ザ・ブロード・インスティテュート・インコーポレイテッド

Optimization Function Systems, methods and compositions for sequence manipulation with the CRISPR-Cas system

CN105899658B

( en )

2013-12-12

2020-02-18

布罗德研究所有限公司

Delivery, Use and Therapeutic Applications of CRISPR-CAS Systems and Compositions for HBV and Viral Diseases and Disorders

US20150166984A1

( en )

2013-12-12

2015-06-18

President And Fellows Of Harvard College

Methods for correcting alpha-antitrypsin point mutations

CN105939767B

( en )

2014-01-08

2018-04-06

弗洛设计声能学公司

Sound electrophoretic apparatus with alliteration electrophoresis chamber

WO2015113063A1

( en )

2014-01-27

2015-07-30

Georgia Tech Research Corporation

Methods and systems for identifying crispr/cas off-target sites

US10287590B2

( en )

2014-02-12

2019-05-14

Dna2.0, Inc.

Methods for generating libraries with co-varying regions of polynuleotides for genome modification

JP6416939B2

( en )

2014-02-13

2018-10-31

タカラ バイオ ユーエスエー，インコーポレイティド

Method for depleting target molecules from an initial collection of nucleic acids, and compositions and kits for performing the same

WO2015126884A1

( en )

*

2014-02-18

2015-08-27

Stc.Unm

Compositions and methods for controlling cellular function

EP3110971B1

( en )

2014-02-27

2019-05-08

The Broad Institute Inc.

T cell balance gene expression and methods of use thereof

ES2898460T3

( en )

2014-02-27

2022-03-07

Monsanto Technology Llc

Compositions and methods for site-directed genomic modification

US11028388B2

( en )

2014-03-05

2021-06-08

Editas Medicine, Inc.

CRISPR/Cas-related methods and compositions for treating Usher syndrome and retinitis pigmentosa

EP3613854A1

( en )

*

2014-03-05

2020-02-26

National University Corporation Kobe University

Genomic sequence modification method for specifically converting nucleic acid bases of targeted dna sequence, and molecular complex for use in same

US11141493B2

( en )

2014-03-10

2021-10-12

Editas Medicine, Inc.

Compositions and methods for treating CEP290-associated disease

US9938521B2

( en )

2014-03-10

2018-04-10

Editas Medicine, Inc.

CRISPR/CAS-related methods and compositions for treating leber&#39;s congenital amaurosis 10 (LCA10)

US11339437B2

( en )

2014-03-10

2022-05-24

Editas Medicine, Inc.

Compositions and methods for treating CEP290-associated disease

EP3981876A1

( en )

2014-03-26

2022-04-13

Editas Medicine, Inc.

Crispr/cas-related methods and compositions for treating sickle cell disease

EP3540061A1

( en )

2014-04-02

2019-09-18

Editas Medicine, Inc.

Crispr/cas-related methods and compositions for treating primary open angle glaucoma

WO2015153940A1

( en )

*

2014-04-03

2015-10-08

Massachusetts Institute Of Technology

Methods and compositions for the production of guide rna

EP3129485B2

( en )

*

2014-04-09

2022-12-21

Editas Medicine, Inc.

Crispr/cas-related methods and compositions for treating cystic fibrosis

CN103952424B

( en )

2014-04-23

2017-01-11

尹熙俊

Method for producing double-muscular trait somatic cell cloned pig with MSTN (myostatin) bilateral gene knockout

CN104630267B

( en )

*

2014-07-17

2017-10-31

清华大学

Utilize the kit of TALE Transcription inhibitions built modular synthetic gene circuit in mammalian cell

US11918695B2

( en )

2014-05-09

2024-03-05

Yale University

Topical formulation of hyperbranched polymer-coated particles

EP3721875B1

( en )

2014-05-09

2023-11-08

Yale University

Particles coated with hyperbranched polyglycerol and methods for their preparation

US9970001B2

( en )

2014-06-05

2018-05-15

Sangamo Therapeutics, Inc.

Methods and compositions for nuclease design

SG11201610357UA

( en )

2014-06-13

2017-01-27

Illumina Cambridge Ltd

Methods and compositions for preparing sequencing libraries

WO2016003814A1

( en )

2014-06-30

2016-01-07

Illumina, Inc.

Methods and compositions using one-sided transposition

US9744483B2

( en )

2014-07-02

2017-08-29

Flodesign Sonics, Inc.

Large scale acoustic separation device

EP3166391A1

( en )

2014-07-11

2017-05-17

E. I. du Pont de Nemours and Company

Compositions and methods for producing plants resistant to glyphosate herbicide

EP3169776B1

( en )

*

2014-07-14

2025-09-03

The Regents of The University of California

Crispr/cas transcriptional modulation

AU2015298571B2

( en )

2014-07-30

2020-09-03

President And Fellows Of Harvard College

Cas9 proteins including ligand-dependent inteins

CN107429241B

( en )

2014-08-14

2025-10-24

百奥赛图(北京)医药科技股份有限公司

DNA knock-in system

EP3180426B1

( en )

2014-08-17

2019-12-25

The Broad Institute, Inc.

Genome editing using cas9 nickases

ES2730378T3

( en )

2014-08-27

2019-11-11

Caribou Biosciences Inc

Procedures to increase the efficiency of the modification mediated by Cas9

BR112017003757A2

( en )

2014-09-12

2017-12-26

Du Pont

? corn plants, parts of corn plants or corn seeds?

WO2016049024A2

( en )

2014-09-24

2016-03-31

The Broad Institute Inc.

Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling competition of multiple cancer mutations in vivo

WO2016049163A2

( en )

2014-09-24

2016-03-31

The Broad Institute Inc.

Use and production of chd8+/- transgenic animals with behavioral phenotypes characteristic of autism spectrum disorder

WO2016049251A1

( en )

2014-09-24

2016-03-31

The Broad Institute Inc.

Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling mutations in leukocytes

WO2016049258A2

( en )

2014-09-25

2016-03-31

The Broad Institute Inc.

Functional screening with optimized functional crispr-cas systems

US10370673B2

( en )

2014-09-26

2019-08-06

Purecircle Sdn Bhd

Single nucleotide polymorphism (SNP) markers for stevia

SG11201702614SA

( en )

2014-10-01

2017-04-27

Eagle Biolog Inc

Polysaccharide and nucleic acid formulations containing viscosity-lowering agents

EP3204496A1

( en )

2014-10-10

2017-08-16

Editas Medicine, Inc.

Compositions and methods for promoting homology directed repair

US20170247762A1

( en )

2014-10-27

2017-08-31

The Board Institute Inc.

Compositions, methods and use of synthetic lethal screening

WO2016073990A2

( en )

2014-11-07

2016-05-12

Editas Medicine, Inc.

Methods for improving crispr/cas-mediated genome-editing

EP3221457B1

( en )

2014-11-21

2019-03-20

Regeneron Pharmaceuticals, Inc.

Methods and compositions for targeted genetic modification using paired guide rnas

EP3626832B1

( en )

2014-11-25

2024-06-12

The Brigham and Women&#39;s Hospital, Inc.

Related documents

• Protein id is O97980#638788
• A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture#1174909
• 1950–2000: five decades of curiosity-driven discovery in alternative DNA structures#202427
• Protein id is O96008#638779
• Self-assembling functional programmable protein array for studying protein-protein interactions in malaria parasites#236920