ABSTRACT
Abstract
The present invention provides RNA-guided gene editing systems and methods of use thereof.
Description
RELATED APPLICATIONS
This application is a national stage entry under 35 U.S.C. section 371 of PCT application PCT/IB2016/052243, filed Apr. 20, 2016, and claims priority to U.S. Ser. No. 62/150,353, filed Apr. 21, 2015. The entire contents of each of these applications are incorporated herein by reference.
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 Apr. 18, 2016, is named PAT056693-WO-PCT_SL.txt and is 46,997 bytes in size.
BACKGROUND
Recently, gene editing systems such as zinc finger nucleases, CRISPR/Cas systems, transcription activator-like effector nucleases (TALENs) and meganucleases have emerged as tools for the regulation of genes.
Monomeric Cas9 nuclease-based systems are directed to cleave specific DNA sequences by an associated Ë100-nt single RNA comprising 17-20 nucleotides that target the Cas9 nuclease to a site of interest (targeting RNA) by hybridizing to the target DNA site. While the simplicity of designing the targeting RNA to recognize predetermined sequences of DNA makes the CRISPR/Cas-based gene editing systems powerful, the large size of the Cas9 enzyme makes it nearly impossible to incorporate the system into a single vector for gene delivery in humans, and the sequence requirements of the Cas9 enzyme itself (e.g., PAM sequence) limit the sites to which it can be targeted.
In contrast, Zinc finger nucelase and TALEN-based gene editing system use much smaller enzyme components, such as the FokI nuclease, and the nucleases do not require specific binding sites. However, these systems are hampered by the use of protein-based DNA-targeting domains, which are difficult to engineer to bind specific DNA target sequences.
There is thus a need for a gene editing system which can be guided to a target DNA binding site using an easy-to-engineer component such as a targeting RNA but which utilizes a compact gene editing enzyme with no required sequence specificity.
SUMMARY
In a first aspect, the invention features a non-naturally occurring gene editing system including:
a) nucleic acid including a first targeting RNA capable of hybridizing with a target DNA sequence;
b) nucleic acid including a first guide RNA capable of binding to a first guide RNA-binding domain, and
c) a polypeptide including the first guide RNA-binding domain and a first cleavage domain,
wherein the polypeptide of c) includes fewer than approximately 1200 amino acids.
In a second aspect, the invention further features d) nucleic acid including a second targeting RNA capable of hybridizing with a second target DNA sequence, e) nucleic acid including a second guide RNA capable of binding to the first guide RNA-binding domain or a second guide-RNA-binding domain, and, optionally, f) a polypeptide including the second guide RNA-binding domain and a second cleavage domain, wherein the polypeptide of f) includes fewer than approximately 1200 amino acids.
In aspects featuring a polypeptide of c), the polypeptide of c) can include, for example, fewer than approximately 1100 amino acids, fewer than approximately 1000 amino acids, fewer than approximately 900 amino acids, fewer than approximately 800 amino acids, fewer than approximately 700 amino acids, fewer than approximately 600 amino acids, fewer than approximately 500 amino acids, fewer than approximately 400 amino acids, fewer than approximately 300 amino acids, or fewer than approximately 200 amino acids.
In some aspects, the invention features a non-naturally occurring gene editing system, wherein the nucleic acid of a) and the nucleic acid of b) are disposed on separate nucleic acid molecules. In aspects where the nucleic acid of a) and the nucleic acid of b) are disposed on separate nucleic acid molecules, the nucleic acid of a) may further include a hybridization domain A and the nucleic acid of b) may further include a hybridization domain Aâ², wherein the hybridization domain A and hybridization domain Aâ² are capable of specific hybridization. In one aspect, the hybridization domain A and the hybridization domain Aâ² each include, for example, 10-50 complimentary nucleic acid residues, e.g., 20-40 complimentary nucleic acid residues, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 complimentary nucleic acid residues.
In some aspects, the non-naturally-occurring gene editing systems described above include, for example, a nucleic acid of a) and a nucleic acid of b) that are disposed on the same molecule. In some aspects the nucleic acid may further include, for example, additional nucleic acids disposed between the nucleic acid of a) and the nucleic acid of b).
In some aspects, the invention features a non-naturally occurring gene editing system, wherein the first guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof. In some aspects where the first guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 3.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain is a fibronectin.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain is an antibody or antigen-binding fragment or analog thereof. In some aspects, the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof. In embodiments where the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 11.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain includes a sequence of an IgG1 Fc domain, or guide RNA-binding fragment or analog thereof. In embodiments, the first guide RNA-binding domain includes SEQ ID NO: 40, or a guide RNA-binding fragment or analog thereof. In embodiments in which the first guide RNA-binding domain includes SEQ ID NO: 40 or a guide RNA-binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 41, or fragment or analog thereof.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain includes a polyhistidine sequence, for example, a sequence of a histidine tag, or guide RNA-binding fragment or analog thereof. In embodiments, the first guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a guide RNA-binding fragment or analog of any of said sequences. In embodiments in which the first guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 or a guide RNA-binding fragment or analog of any of said sequences, the first guide RNA includes, for example, SEQ ID NO: 8, or fragment or analog thereof.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain includes a streptavidin sequence, or guide RNA-binding fragment or analog thereof. In embodiments, the first guide RNA-binding domain includes SEQ ID NO: 9, or a guide RNA-binding fragment or analog thereof. In embodiments in which the first guide RNA-binding domain includes SEQ ID NO: 9 or a guide RNA-binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 39, or fragment or analog thereof.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain is a fluorescent protein or functional fragment thereof, for example, is selected from proteins identified in Tables 1-4, or an RNA-binding fragment or analog of any proteins identified in Tables 1-4. In some embodiments, the first guide RNA-binding domain is green fluorescent protein (e.g., UniProt code P42212), or an RNA-binding fragment or analog thereof. In embodiments where the first guide RNA-binding domain is a fluorescent protein or functional fragment thereof, the first guide RNA can include, for example, SEQ ID NO: 10.
In some aspects, the invention features a non-naturally occurring gene editing system, wherein the first cleavage domain includes a functional fragment of a nuclease capable of inducing a double-strand break in DNA, for example, a functional fragment of a GIY-YIG homing endonuclease, e.g., a functional fragment of I-TevI, e.g., SEQ ID NO: 13 or a functional fragment of SEQ ID NO: 13. In some embodiments, the first cleavage domain includes a polypeptide derived from a Type IIS restriction enzyme. In some aspects, the first cleavage domain includes a polypeptide capable of inducing a single strand break in DNA, e.g., includes a functional fragment of a nuclease selected from the group including of FokI and PvuII, e.g., SEQ ID NO: 12, SEQ ID NO: 49, or a functional fragment of SEQ ID NO: 12 or SEQ ID NO: 49.
In aspects featuring a polypeptide of f), the polypeptide of f) may include, for example, fewer than approximately 1100 amino acids, fewer than approximately 1000 amino acids, fewer than approximately 900 amino acids, fewer than approximately 800 amino acids, fewer than approximately 700 amino acids, fewer than approximately 600 amino acids, fewer than approximately 500 amino acids, fewer than approximately 400 amino acids, fewer than approximately 300 amino acids, or fewer than approximately 200 amino acids.
In aspects featuring the nucleic acid of d) and the nucleic acid of e), these may be disposed, for example on separate nucleic acid molecules. In such embodiments, the nucleic acid of d) may further include, for example, a hybridization domain B and the nucleic acid of e) may further include, for example, a hybridization domain Bâ², wherein the hybridization domain B and hybridization domain Bâ² are capable of specific hybridization. In some embodiments, the hybridization domain B and the hybridization domain Bâ² each include, for example, 10-50 complimentary nucleic acid residues, e.g., 20-40, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleic acid residues.
In some aspects featuring the nucleic acid of d) and the nucleic acid of e), the nucleic acid of d) and the nucleic acid of e) may be disposed on the same molecule.
In some aspects of the invention featuring a second guide RNA-binding domain, the second guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof. In embodiments where the second guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 3.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain is a fibronectin.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain is an antibody or antigen-binding fragment or analog thereof. In some aspects, the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof. In embodiments where the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 11.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain includes a sequence of an IgG1 Fc domain, or guide RNA-binding fragment or analog thereof. In embodiments, the second guide RNA-binding domain includes SEQ ID NO: 40, or a guide RNA-binding fragment or analog thereof. In embodiments in which the second guide RNA-binding domain includes SEQ ID NO: 40 or a guide RNA-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 41, or fragment or analog thereof.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain includes a polyhistidine sequence, for example, a sequence of a histidine tag, or guide RNA-binding fragment or analog thereof. In embodiments, the second guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a guide RNA-binding fragment or analog thereof. In embodiments in which the second guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a guide RNA-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 8, or fragment or analog thereof.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain includes a streptavidin sequence, or guide RNA-binding fragment or analog thereof. In embodiments, the second guide RNA-binding domain includes SEQ ID NO: 9, or a guide RNA-binding fragment or analog thereof. In embodiments in which the second guide RNA-binding domain includes SEQ ID NO: 9 or a guide RNA-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 39, or fragment or analog thereof.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain is a fluorescent protein or functional fragment thereof, for example, is selected from proteins identified in Tables 1-4, or an RNA-binding fragment or analog of any proteins identified in Tables 1-4. In some embodiments, the second guide RNA-binding domain is green fluorescent protein (e.g., UniProt code P42212), or an RNA-binding fragment or analog thereof. In embodiments where the second guide RNA-binding domain is a fluorescent protein or functional fragment thereof, the second guide RNA can include, for example, SEQ ID NO: 10.
In some aspects of the invention featuring a first guide RNA and a second guide RNA, the first guide RNA and the second guide RNA include different sequences. In some aspects the first guide RNA and the second guide RNA include the same sequence.
In some aspects, the non-naturally occurring gene editing systems that include a first guide RNA and a second guide RNA, the first guide RNA and second guide RNA may each independently bind to the first guide RNA-binding domain, e.g., a first guide RNA-binding domain described herein.
In some aspects featuring a first guide RNA and a second guide RNA, the first guide RNA and second guide RNA may each include, for example SEQ ID NO: 3. In such aspects, the first guide RNA-binding domain includes, e.g., lysozyme or a guide RNA binding fragment or analog thereof, e.g., SEQ ID NO: 2, SEQ ID NO: 50 or a guide RNA binding fragment or analog thereof.
In some aspects featuring a first guide RNA and a second guide RNA, the first and second guide RNA may each include, for example SEQ ID NO: 11. In such aspects, the first guide RNA-binding domain may include, for example, an IgE antibody or an antigen binding fragment or analog thereof.
In some aspects of the invention featuring a first guide RNA and a second guide RNA-binding domain, the first and second guide RNA may each include, for example SEQ ID NO: 41, or fragment or analog thereof. In such aspects, the first guide RNA-binding domain may include, for example, a sequence of an IgG1 Fc domain, or guide RNA-binding fragment or analog thereof, e.g., SEQ ID NO:
RELATED APPLICATIONS
This application is a national stage entry under 35 U.S.C. section 371 of PCT application PCT/IB2016/052243, filed Apr. 20, 2016, and claims priority to U.S. Ser. No. 62/150,353, filed Apr. 21, 2015. The entire contents of each of these applications are incorporated herein by reference.
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 Apr. 18, 2016, is named PAT056693-WO-PCT_SL.txt and is 46,997 bytes in size.
BACKGROUND
Recently, gene editing systems such as zinc finger nucleases, CRISPR/Cas systems, transcription activator-like effector nucleases (TALENs) and meganucleases have emerged as tools for the regulation of genes.
Monomeric Cas9 nuclease-based systems are directed to cleave specific DNA sequences by an associated Ë100-nt single RNA comprising 17-20 nucleotides that target the Cas9 nuclease to a site of interest (targeting RNA) by hybridizing to the target DNA site. While the simplicity of designing the targeting RNA to recognize predetermined sequences of DNA makes the CRISPR/Cas-based gene editing systems powerful, the large size of the Cas9 enzyme makes it nearly impossible to incorporate the system into a single vector for gene delivery in humans, and the sequence requirements of the Cas9 enzyme itself (e.g., PAM sequence) limit the sites to which it can be targeted.
In contrast, Zinc finger nucelase and TALEN-based gene editing system use much smaller enzyme components, such as the FokI nuclease, and the nucleases do not require specific binding sites. However, these systems are hampered by the use of protein-based DNA-targeting domains, which are difficult to engineer to bind specific DNA target sequences.
There is thus a need for a gene editing system which can be guided to a target DNA binding site using an easy-to-engineer component such as a targeting RNA but which utilizes a compact gene editing enzyme with no required sequence specificity.
SUMMARY
In a first aspect, the invention features a non-naturally occurring gene editing system including:
a) nucleic acid including a first targeting RNA capable of hybridizing with a target DNA sequence;
b) nucleic acid including a first guide RNA capable of binding to a first guide RNA-binding domain, and
c) a polypeptide including the first guide RNA-binding domain and a first cleavage domain,
wherein the polypeptide of c) includes fewer than approximately 1200 amino acids.
In a second aspect, the invention further features d) nucleic acid including a second targeting RNA capable of hybridizing with a second target DNA sequence, e) nucleic acid including a second guide RNA capable of binding to the first guide RNA-binding domain or a second guide-RNA-binding domain, and, optionally, f) a polypeptide including the second guide RNA-binding domain and a second cleavage domain, wherein the polypeptide of f) includes fewer than approximately 1200 amino acids.
In aspects featuring a polypeptide of c), the polypeptide of c) can include, for example, fewer than approximately 1100 amino acids, fewer than approximately 1000 amino acids, fewer than approximately 900 amino acids, fewer than approximately 800 amino acids, fewer than approximately 700 amino acids, fewer than approximately 600 amino acids, fewer than approximately 500 amino acids, fewer than approximately 400 amino acids, fewer than approximately 300 amino acids, or fewer than approximately 200 amino acids.
In some aspects, the invention features a non-naturally occurring gene editing system, wherein the nucleic acid of a) and the nucleic acid of b) are disposed on separate nucleic acid molecules. In aspects where the nucleic acid of a) and the nucleic acid of b) are disposed on separate nucleic acid molecules, the nucleic acid of a) may further include a hybridization domain A and the nucleic acid of b) may further include a hybridization domain Aâ², wherein the hybridization domain A and hybridization domain Aâ² are capable of specific hybridization. In one aspect, the hybridization domain A and the hybridization domain Aâ² each include, for example, 10-50 complimentary nucleic acid residues, e.g., 20-40 complimentary nucleic acid residues, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 complimentary nucleic acid residues.
In some aspects, the non-naturally-occurring gene editing systems described above include, for example, a nucleic acid of a) and a nucleic acid of b) that are disposed on the same molecule. In some aspects the nucleic acid may further include, for example, additional nucleic acids disposed between the nucleic acid of a) and the nucleic acid of b).
In some aspects, the invention features a non-naturally occurring gene editing system, wherein the first guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof. In some aspects where the first guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 3.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain is a fibronectin.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain is an antibody or antigen-binding fragment or analog thereof. In some aspects, the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof. In embodiments where the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 11.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain includes a sequence of an IgG1 Fc domain, or guide RNA-binding fragment or analog thereof. In embodiments, the first guide RNA-binding domain includes SEQ ID NO: 40, or a guide RNA-binding fragment or analog thereof. In embodiments in which the first guide RNA-binding domain includes SEQ ID NO: 40 or a guide RNA-binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 41, or fragment or analog thereof.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain includes a polyhistidine sequence, for example, a sequence of a histidine tag, or guide RNA-binding fragment or analog thereof. In embodiments, the first guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a guide RNA-binding fragment or analog of any of said sequences. In embodiments in which the first guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 or a guide RNA-binding fragment or analog of any of said sequences, the first guide RNA includes, for example, SEQ ID NO: 8, or fragment or analog thereof.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain includes a streptavidin sequence, or guide RNA-binding fragment or analog thereof. In embodiments, the first guide RNA-binding domain includes SEQ ID NO: 9, or a guide RNA-binding fragment or analog thereof. In embodiments in which the first guide RNA-binding domain includes SEQ ID NO: 9 or a guide RNA-binding fragment or analog thereof, the first guide RNA includes, for example, SEQ ID NO: 39, or fragment or analog thereof.
In some aspects, the non-naturally occurring gene editing systems of the present invention include those in which the first guide RNA-binding domain is a fluorescent protein or functional fragment thereof, for example, is selected from proteins identified in Tables 1-4, or an RNA-binding fragment or analog of any proteins identified in Tables 1-4. In some embodiments, the first guide RNA-binding domain is green fluorescent protein (e.g., UniProt code P42212), or an RNA-binding fragment or analog thereof. In embodiments where the first guide RNA-binding domain is a fluorescent protein or functional fragment thereof, the first guide RNA can include, for example, SEQ ID NO: 10.
In some aspects, the invention features a non-naturally occurring gene editing system, wherein the first cleavage domain includes a functional fragment of a nuclease capable of inducing a double-strand break in DNA, for example, a functional fragment of a GIY-YIG homing endonuclease, e.g., a functional fragment of I-TevI, e.g., SEQ ID NO: 13 or a functional fragment of SEQ ID NO: 13. In some embodiments, the first cleavage domain includes a polypeptide derived from a Type IIS restriction enzyme. In some aspects, the first cleavage domain includes a polypeptide capable of inducing a single strand break in DNA, e.g., includes a functional fragment of a nuclease selected from the group including of FokI and PvuII, e.g., SEQ ID NO: 12, SEQ ID NO: 49, or a functional fragment of SEQ ID NO: 12 or SEQ ID NO: 49.
In aspects featuring a polypeptide of f), the polypeptide of f) may include, for example, fewer than approximately 1100 amino acids, fewer than approximately 1000 amino acids, fewer than approximately 900 amino acids, fewer than approximately 800 amino acids, fewer than approximately 700 amino acids, fewer than approximately 600 amino acids, fewer than approximately 500 amino acids, fewer than approximately 400 amino acids, fewer than approximately 300 amino acids, or fewer than approximately 200 amino acids.
In aspects featuring the nucleic acid of d) and the nucleic acid of e), these may be disposed, for example on separate nucleic acid molecules. In such embodiments, the nucleic acid of d) may further include, for example, a hybridization domain B and the nucleic acid of e) may further include, for example, a hybridization domain Bâ², wherein the hybridization domain B and hybridization domain Bâ² are capable of specific hybridization. In some embodiments, the hybridization domain B and the hybridization domain Bâ² each include, for example, 10-50 complimentary nucleic acid residues, e.g., 20-40, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleic acid residues.
In some aspects featuring the nucleic acid of d) and the nucleic acid of e), the nucleic acid of d) and the nucleic acid of e) may be disposed on the same molecule.
In some aspects of the invention featuring a second guide RNA-binding domain, the second guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof. In embodiments where the second guide RNA-binding domain is, for example, lysozyme, e.g., SEQ ID NO: 2, SEQ ID NO: 50, or an RNA binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 3.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain is a fibronectin.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain is an antibody or antigen-binding fragment or analog thereof. In some aspects, the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof. In embodiments where the antibody or antigen-binding fragment or analog thereof is, for example, an IgE-derived antibody or antigen-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 11.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain includes a sequence of an IgG1 Fc domain, or guide RNA-binding fragment or analog thereof. In embodiments, the second guide RNA-binding domain includes SEQ ID NO: 40, or a guide RNA-binding fragment or analog thereof. In embodiments in which the second guide RNA-binding domain includes SEQ ID NO: 40 or a guide RNA-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 41, or fragment or analog thereof.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain includes a polyhistidine sequence, for example, a sequence of a histidine tag, or guide RNA-binding fragment or analog thereof. In embodiments, the second guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a guide RNA-binding fragment or analog thereof. In embodiments in which the second guide RNA-binding domain includes SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a guide RNA-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 8, or fragment or analog thereof.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain includes a streptavidin sequence, or guide RNA-binding fragment or analog thereof. In embodiments, the second guide RNA-binding domain includes SEQ ID NO: 9, or a guide RNA-binding fragment or analog thereof. In embodiments in which the second guide RNA-binding domain includes SEQ ID NO: 9 or a guide RNA-binding fragment or analog thereof, the second guide RNA includes, for example, SEQ ID NO: 39, or fragment or analog thereof.
In some aspects of the invention featuring a second guide RNA-binding domain, the non-naturally occurring gene editing systems of the present invention include those in which the second guide RNA-binding domain is a fluorescent protein or functional fragment thereof, for example, is selected from proteins identified in Tables 1-4, or an RNA-binding fragment or analog of any proteins identified in Tables 1-4. In some embodiments, the second guide RNA-binding domain is green fluorescent protein (e.g., UniProt code P42212), or an RNA-binding fragment or analog thereof. In embodiments where the second guide RNA-binding domain is a fluorescent protein or functional fragment thereof, the second guide RNA can include, for example, SEQ ID NO: 10.
In some aspects of the invention featuring a first guide RNA and a second guide RNA, the first guide RNA and the second guide RNA include different sequences. In some aspects the first guide RNA and the second guide RNA include the same sequence.
In some aspects, the non-naturally occurring gene editing systems that include a first guide RNA and a second guide RNA, the first guide RNA and second guide RNA may each independently bind to the first guide RNA-binding domain, e.g., a first guide RNA-binding domain described herein.
In some aspects featuring a first guide RNA and a second guide RNA, the first guide RNA and second guide RNA may each include, for example SEQ ID NO: 3. In such aspects, the first guide RNA-binding domain includes, e.g., lysozyme or a guide RNA binding fragment or analog thereof, e.g., SEQ ID NO: 2, SEQ ID NO: 50 or a guide RNA binding fragment or analog thereof.
In some aspects featuring a first guide RNA and a second guide RNA, the first and second guide RNA may each include, for example SEQ ID NO: 11. In such aspects, the first guide RNA-binding domain may include, for example, an IgE antibody or an antigen binding fragment or analog thereof.
In some aspects of the invention featuring a first guide RNA and a second guide RNA-binding domain, the first and second guide RNA may each include, for example SEQ ID NO: 41, or fragment or analog thereof. In such aspects, the first guide RNA-binding domain may include, for example, a sequence of an IgG1 Fc domain, or guide RNA-binding fragment or analog thereof, e.g., SEQ ID NO: 40, or a guide RNA-binding fragment or analog thereof.
In some aspects of the invention featuring a first guide RNA and a second guide RNA-binding domain, the first and second guide RNA may each include, for example, SEQ ID NO: 8, or fragment or analog thereof. In such aspects, the first guide RNA-binding domain may include, for example, a sequence including a polyhistidine sequence, or guide RNA-binding fragment or analog thereof, e.g., SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or a guide RNA-binding fragment or analog thereof.
In some aspects of the invention featuring a first guide RNA and a second guide RNA-binding domain, the first and second guide RNA may each include, for example, SEQ ID NO: 39, or fragment or analog thereof. In such aspects, the first guide RNA-binding domain may include, for example, a streptavidin sequence, or guide RNA-binding fragment or analog thereof, e.g., SEQ ID NO: 9, or a guide RNA-binding fragment or analog thereof.
In some aspects featuring a first guide RNA and a second guide RNA, the first and second guide RNA each include SEQ ID NO: 10. In such aspects, the first guide RNA-binding domain includes, for example, a fluorescent protein or an active fragment thereof, e.g., is selected from the proteins identified in Tables 1-4, and an RNA-binding fragment or analog of any of the preceding.
In some aspects featuring a first guide RNA and a second guide RNA, the invention features a non-naturally occurring gene editing system, wherein the first guide RNA binds specifically to the first guide RNA-binding domain and the second guide RNA binds specifically to the second guide RNA-binding domain. For example, the first guide RNA does not bind the second guide RNA-binding domain and the second guide RNA does not bind the first guide RNA-binding domain. In some aspects, the first guide RNA-binding domain and the second guide RNA-binding domain are independently selected from the group including: lysozyme, a fibronectin, an antibody or antigen-binding fragment or analog thereof, and a fluorescent protein or functional fragment or analog thereof, with the proviso that the first guide RNA-binding domain and the second guide RNA-binding domain are not identical.
In another aspect, the invention features a non-naturally occurring gene editing system including: a) nucleic acid including a first targeting RNA capable of hybridizing with a target DNA sequence, b) nucleic acid including a first guide RNA capable of binding directly to a first cleavage domain, and c) a polypeptide including the first cleavage domain, wherein the polypeptide of c) includes fewer than approximately 1200 amino acids, for example, fewer than approximately 1100 amino acids, fewer than approximately 1000 amino acids, fewer than approximately 900 amino acids, fewer than approximately 800 amino acids, fewer than approximately 700 amino acids, fewer than approximately 600 amino acids, fewer than approximately 500 amino acids, fewer than approximately 400 amino acids, fewer than approximately 300 amino acids, or fewer than approximately 200 amino acids.
In some embodiments that include a first guide RNA capable of binding directly to a first cleavage domain, the nucleic acid of a) and the nucleic acid of b) are disposed on the same molecule. In some aspects, the invention features a non-naturally occurring gene editing system, wherein the nucleic acid of a) and the nucleic acid of b) are disposed on separate nucleic acid molecules. In aspects where the nucleic acid of a) and the nucleic acid of b) are disposed on separate nucleic acid molecules, the nucleic acid of a) may further include a hybridization domain A and the nucleic acid of b) may further include a hybridization domain Aâ², wherein the hybridization domain A and hybridization domain Aâ² are capable of specific hybridization. In one aspect, the hybridization domain A and the hybridization domain Aâ² each include, for example, 10-50 complimentary nucleic acid residues, e.g., 20-40, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleic acid residues.
In some aspects of the invention that include a first guide RNA capable of binding directly to a first cleavage domain, the invention may further include d) nucleic acid including a second targeting RNA capable of hybridizing with a target DNA sequence, e) nucleic acid including a second guide RNA capable of binding directly to the first or a second cleavage domain, and, optionally, f) a polypeptide including the second cleavage domain, wherein the polypeptide of f) includes fewer than approximately 1200 amino acids, for example, fewer than approximately 1100 amino acids, fewer than approximately 1000 amino acids, fewer than approximately 900 amino acids, fewer than approximately 800 amino acids, fewer than approximately 700 amino acids, fewer than approximately 600 amino acids, fewer than approximately 500 amino acids, fewer than approximately 400 amino acids, fewer than approximately 300 amino acids, or fewer than approximately 200 amino acids.
In some aspects of the invention that include a first guide RNA capable of binding directly to a first cleavage domain and featuring the nucleic acid of d) and the nucleic acid of e), the nucleic acid of d) and the nucleic acid of e) may be disposed, for example, on the same nucleic acid molecule. In other embodiments, the nucleic acid of d) and the nucleic acid of e), these may be disposed, for example, on separate nucleic acid molecules. In such embodiments, the nucleic acid of d) may further include, for example, a hybridization domain B and the nucleic acid of e) may further include, for example, a hybridization domain Bâ², wherein the hybridization domain B and hybridization domain Bâ² are capable of specific hybridization. In some embodiments, the hybridization domain B and the hybridization domain Bâ² each include, for example, 10-50 complimentary nucleic acid residues, e.g., 20-40, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleic acid residues.
In one aspect, one or more of the polypeptide-based components the non-naturally occurring gene editing systems of the present invention further include a nuclear localization sequence (NLS).
In another aspect, the present invention features a chimeric polypeptide including a RNA-binding domain and a cleavage domain that are not naturally associated, wherein the chimeric polypeptide includes fewer than approximately 1200 amino acids, for example, fewer than approximately 1100 amino acids, fewer than approximately 1000 amino acids, fewer than approximately 900 amino acids, fewer than approximately 800 amino acids, fewer than approximately 700 amino acids, fewer than approximately 600 amino acids, fewer than approximately 500 amino acids, fewer than approximately 400 amino acids, fewer than approximately 300 amino acids, or fewer than approximately 200 amino acids.
In some embodiments, the chimeric polypeptide features an RNA binding domain that includes lysozyme or an RNA-binding fragment or analog thereof, e.g., is SEQ ID NO: 2, SEQ ID NO: 50, or an RNA-binding fragment or analog thereof.
In some embodiments, the chimeric polypeptide features an RNA-binding domain that includes a fibronectin.
In some embodiments, the chimeric polypeptide features an RNA-binding domain that includes an antibody or guide RNA-binding fragment or analog thereof, e.g., an IgE antibody or RNA-binding fragment thereof.
In some embodiments, the chimeric polypeptide features an RNA-binding domain that includes an IgG1 Fc or guide RNA-binding fragment or analog thereof, e.g., SEQ ID NO: 40, or guide RNA-binding fragment or analog thereof.
In some embodiments, the chimeric polypeptide features an RNA-binding domain that includes streptavidin or guide RNA-binding fragment or analog thereof, e.g., SEQ ID NO: 9, or guide RNA-binding fragment or analog thereof.
In some embodiments, the chimeric polypeptide features an RNA-binding domain that includes polyhistidine sequence, e.g., a histidine tag, or guide RNA-binding fragment or analog thereof, e.g., SEQ ID NO: 7, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or guide RNA-binding fragment or analog thereof.
In some embodiments, the chimeric polypeptide features an RNA-binding domain that includes fluorescent protein or functional fragment or analog thereof. In some embodiments the florescent protein or functional fragment or analog thereof is selected from the group including of the proteins identified in Tables 1-4, and an RNA-binding fragment or analog of any of the proteins identified in Tables 1-4.
In embodiments of the foregoing chimeric polypeptides, the cleavage domain includes a functional fragment of a nuclease capable of inducing a double-strand break in DNA. For example, the cleavage domain can include a a GIY-YIG homing endonuclease or a functional fragment of a GIY-YIG homing endonuclease, for example, I-TevI or a functional fragment of I-TevI, e.g., SEQ ID NO: 13.
In embodiments of the foregoing chimeric polypeptides, the first cleavage domain includes a functional fragment of a nuclease capable of inducing a single-strand break in DNA. For example, the first cleavage domain includes Fok I or PvuII, or a functional fragment of FokI or PvuII, e.g., SEQ ID NO: 12 or SEQ ID NO: 49.
In another aspect of the invention, any of the foregoing chimeric polypeptides further include a NLS, e.g., an NLS selected from the group including SEQ ID NOS: 22-37.
In one aspect, the invention features a nucleic acid, e.g., an isolated nucleic acid, including sequence encoding any of the foregoing gene editing systems or any of the foregoing chimeric polypeptides.
In one aspect, the invention features a vector including any of the foregoing nucleic acids.
In some aspects, the vector is selected from the group including of a viral vector, a plasmid, a minicircle, and a nanoplasmid. In one embodiment the vector is a viral vector. For example, the viral vector is selected from the group including of a lentivirus vector, adenovirus vector, adenoassociated vector and a retrovirus vector.
In embodiments, the vector includes fewer than 10,000 nucleic acid residues, for example, fewer than 9000 nucleic acid residues, fewer than 8000 nucleic acid residues, fewer than 7000 nucleic acid residues, fewer than 6000 nucleic acid residues, fewer than 5000 nucleic acid residues, fewer than 4000 nucleic acid residues, or fewer than 3000 nucleic acid residues.
In an aspect, the invention features a cell that includes any of the foregoing gene editing systems, any of the foregoing chimeric polypeptides, any of the foregoing nucleic acids, or any of the foregoing vectors. In embodiments the cell is a human cell, e.g., a human stem or progenitor cell, e.g., a hematopoietic stem cell (HSC). In embodiments the cell is a T cell, e.g., a human T cell. In embodiments the cell is a NK cell, e.g., a human NK cell. In embodiments the cell is a cancer cell, e.g., a human cancer cell.
In one aspect the invention features a cell derived from any of the forgoing cells, e.g., a daughter or progeny cell of any of the foregoing cells.
In one aspect, the invention features a method of making a cell, e.g., any of the foregoing cells, including introducing into the cell a) a gene editing system of the present invention, e.g., as described herein; b) a chimeric polypeptide of the present invention, e.g., as described herein; c) a nucleic acid of the present invention, e.g., as described herein; or d) a vector of the present invention, e.g., as described herein. In one aspect, the method is performed in vitro. In one aspect, the method is performed ex vivo. In one aspect the method is performed in vivo.
In one aspect the invention features a method of modulating expression of a gene in a cell including introducing into the cell a) a gene editing system of the present invention, e.g., as described herein; b) a chimeric polypeptide of the present invention, e.g., as described herein; c) a nucleic acid of the present invention, e.g., as described herein; or d) a vector of the present invention, e.g., as described herein, such that expression of a gene in a cell is modulated. In one aspect, the method is performed in vitro. In one aspect, the method is performed ex vivo. In one aspect the method is performed in vivo. In one aspect the method of modulation results in repression of a gene in a cell, e.g., reduced expression in a cell. In one aspect the method of modulation results in activation of a gene in a cell, e.g., increased expression in a cell.
In one aspect, the invention features a method of modifying an endogenous nucleic acid sequence, e.g., a gene, in a cell, including administering to the cell a) a gene editing system of the present invention, e.g., as described herein; b) a chimeric polypeptide of the present invention, e.g., as described herein; c) a nucleic acid of the present invention, e.g., as described herein; or d) a vector of the present invention, e.g., as described herein, such that an endogenous nucleic acid sequence, e.g., a gene, in a cell is modified. In some aspects the method of modifying includes deletion of one or more endogenous nucleic acid residues. In some aspects the method of modifying includes the replacement of one or more endogenous nucleic acid residues with nucleic acids from a donor nucleic acid molecule. In one aspect, the method is performed in vitro. In one aspect, the method is performed ex vivo. In one aspect the method is performed in vivo.
In one aspect, the invention features a cell, wherein expression of one or more endogenous genes has been modulated by the method of any of the foregoing methods of modulation of expression of a gene in a cell.
In one aspect, the invention features a cell, wherein one or more endogenous nucleic acid sequences, e.g., genes, have been modified by the method of any of the foregoing methods of modifying an endogenous nucleic acid sequence, e.g., a gene, in a cell.
In some embodiments, the gene that is modified or modulated includes an HLA gene. In some embodiments, the gene that is modified or modulated includes a beta 2-microglobulin (B2M) gene. In some embodiments, the gene that is modified or modulated includes a TCR gene, e.g., TCRα or TCRβ. In some embodiments, the gene that is modified or modulated includes an inhibitory molecule selected from the group including of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In embodiments combinations, e.g., two or more, genes are modified and/or modulated in the same cell, resulting in a cell in which combinations, e.g., two or more, genes have been modified and/or modulated. In one embodiment, the invention features a cell in which both HLA and a TCR gene, e.g., TCRα or TCRβ, have been modified and/or modulated
In one aspect, the invention features a cell derived from the cell of any of the foregoing, e.g. a daughter cell or progeny cell.
In one aspect, the invention features a method of treating a subject, e.g., a mammal, having a disease associated with aberrant gene expression, e.g., a disease described herein, including administering to the subject an effective amount of a) a gene editing system of the present invention, e.g., any of the foregoing; b) a chimeric polypeptide of the present invention, e.g., any of the foregoing; c) a nucleic acid of the present invention, e.g., any of the foregoing; d) a vector of the present invention, e.g., any of the foregoing; or e) a cell of the present invention, e.g., any of the foregoing.
In one aspect, the invention features a) a gene editing system of the present invention, e.g., any of the foregoing; b) a chimeric polypeptide of the present invention, e.g., any of the foregoing; c) a nucleic acid of the present invention, e.g., any of the foregoing; d) a vector of the present invention, e.g., any of the foregoing; or e) a cell of the present invention, e.g., any of the foregoing, for use as a medicament.
In one aspect, the invention features a) a gene editing system of the present invention, e.g., any of the foregoing; b) a chimeric polypeptide of the present invention, e.g., any of the foregoing; c) a nucleic acid of the present invention, e.g., any of the foregoing; d) a vector of the present invention, e.g., any of the foregoing; or e) a cell of the present invention, e.g., any of the foregoing, for use in treating a disease associated with aberrant gene expression.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphical representation of a monomeric gene editing system of the present invention. In this example, the guide RNA-binding domain is lysozyme and the cleavage domain is derived from the ITev-I nuclease. In this figure, âgRNAâ represents a chimeric fusion between a targeting RNA and a guide RNA (e.g., SEQ ID NO: 3 when the guide RNA-binding domain is lysozyme or a guide RNA-binding fragment thereof).
FIG. 2 is a graphical representation of a dimeric gene editing system of the present invention. In this example, the guide RNA-binding domain is lysozyme and the cleavage domain is a FokI domain. In this figure, âgRNAâ represents a chimeric fusion between a targeting RNA and a guide RNA (e.g., SEQ ID NO: 3 when the guide RNA-binding domain is lysozyme or a guide RNA-binding fragment thereof).
FIG. 3 is a graphical representation of a dimeric gene editing system of the present invention. In this example, the guide RNA binds directly to the Fok-I nuclease domain. In this figure, âgRNAâ represents a chimeric fusion between a targeting RNA and a guide RNA.
DESCRIPTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
âAâ and âanâ as used herein, refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, âan elementâ means one element or more than one element.
The term âapproximatelyâ as used herein, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some aspects ±10%, or in some aspects ±5%, or in some aspects ±1%, or in some aspects ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
The term âamino acidâ as used herein, refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
The term âconservatively modified variantâ as used herein, applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are âsilent variations,â which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
For polypeptide sequences, âconservatively modified variantsâ include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some aspects, the term âconservative sequence modificationsâ are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
The term âoptimizedâ as used herein refers to a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the âparentalâ sequence.
The terms âpercent identicalâ or âpercent identity,â as used herein in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Two sequences are âsubstantially identicalâ if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A âcomparison windowâ, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, 2003).
Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=â4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=â4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
The term âamino acidâ as used herein, refers to naturally occurring, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
The term âconservatively modified variantâ as used herein, applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are âsilent variations,â which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
For polypeptide sequences, âconservatively modified variantsâ include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some aspects, the term âconservative sequence modificationsâ are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
The term âoptimizedâ as used herein refers to a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the âparentalâ sequence.
The terms âpercent identicalâ or âpercent identity,â as used herein in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Two sequences are âsubstantially identicalâ if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference
CLAIMS
Claims ( 15 )
We claim:
1. A non-naturally occurring gene editing system comprising:
a) nucleic acid comprising a first targeting RNA capable of hybridizing with a target DNA sequence;
b) nucleic acid comprising a first guide RNA capable of binding to a first guide RNA-binding domain; and
c) a polypeptide comprising the first guide RNA-binding domain and a first cleavage domain,
wherein the first guide RNA-binding domain is lysozyme.
2. The non-naturally occurring gene editing system of claim 1 , wherein:
(i) the nucleic acid of a) and the nucleic acid of b) are disposed on separate nucleic acid molecules, and optionally, wherein the nucleic acid of a) further comprises a hybridization domain A and the nucleic acid of b) further comprises a hybridization domain Aâ², and wherein said hybridization domain A and hybridization domain Aâ² are capable of specific hybridization; or
(ii) the nucleic acid of a) and the nucleic acid of b) are disposed on the same molecule.
3. The non-naturally occurring gene editing system of claim 1 , wherein the first cleavage domain comprises:
(a) a functional fragment of a nuclease capable of inducing a double-strand break in DNA; or
(b) a functional fragment of a nuclease capable of inducing a single-strand break in DNA.
4. The non-naturally occurring gene editing system of claim 3 , wherein the first cleavage domain comprises:
(a) a functional fragment of a GIY-YIG homing endonuclease;
(b) a functional fragment of I-TevI, e.g., a functional fragment of SEQ ID NO: 13;
(c) a functional fragment of FokI; or
(d) a functional fragment of PvuII.
5. The non-naturally occurring gene editing system of claim 1 , further comprising d) nucleic acid comprising a second targeting RNA capable of hybridizing with a second target DNA sequence, e) nucleic acid comprising a second guide RNA capable of binding to the first guide RNA-binding domain or a second guide-RNA-binding domain, and, optionally, f) a polypeptide comprising the second guide RNA-binding domain and a second cleavage domain, wherein the polypeptide of f) comprises:
(i) fewer than approximately 1200 amino acids;
(ii) fewer than approximately 1100 amino acids;
(iii) fewer than approximately 1000 amino acids;
(iv) fewer than approximately 900 amino acids;
(v) fewer than approximately 800 amino acids;
(vi) fewer than approximately 700 amino acids;
(vii) fewer than approximately 600 amino acids;
(viii) fewer than approximately 500 amino acids;
(ix) fewer than approximately 400 amino acids;
(x) fewer than approximately 300 amino acids; or
(xi) fewer than approximately 200 amino acids.
6. The non-naturally occurring gene editing system of claim 5 , wherein the first guide RNA and the second guide RNA comprise:
(a) different sequences; or
(b) the same sequence.
7. The non-naturally-occurring gene editing system of claim 1 , wherein one or more of the recited polypeptide components further comprises a nuclear localization sequence (NLS).
8. A nucleic acid comprising sequence encoding the gene editing system of claim 1 .
9. A vector comprising the nucleic acid of claim 8 .
10. The vector of claim 9 , wherein the vector is selected from the group consisting of a viral vector, a plasmid, a minicircle, a lentivirus vector, an adenovirus vector, an adenoassociated vector, a retrovirus vector and a nanoplasmid.
11. The vector of claim 9 , comprising:
(i) fewer than 10,000 nucleic acid residues;
(ii) fewer than 9,000 nucleic acid residues;
(iii) fewer than 8,000 nucleic acid residues;
(iv) fewer than 7,000 nucleic acid residues;
(v) fewer than 6,000 nucleic acid residues;
(vi) fewer than 5,000 nucleic acid residues;
(vii) fewer than 4,000 nucleic acid residues; or
(viii) fewer than 3,000 nucleic acid residues.
12. A cell comprising a gene editing system of claim 1 .
13. A method of (i) modulating expression of a gene in a cell, or (ii) modifying an endogenous nucleic acid sequence in a cell, comprising administering to the cell a gene editing system of claim 1 , such that (i) expression of a gene in a cell is modulated or (ii) an endogenous nucleic acid sequence in a cell is modified.
14. The method of claim 13 , wherein the administering to the cell is performed:
(i) in vivo;
(ii) in vitro; or
(iii) ex vivo.
15. A method of treating a subject having a disease associated with aberrant gene expression, comprising administering to the subject an effective amount of a gene editing system of claim 1 .
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