Patent Application
20190359973
Mounting evidence indicates that growth of pathologically identical cancers in each individual patient is fueled by different sets of driving mutations. The need to identify these drivers stems from the recognized necessity for tailoring therapy and scheduling future surveillance. A major advancement in patient diagnosis is the use of next-generation sequencing to identify the cancer-causing mutations. However, functional characterization of patient mutations and their sensitivity to different targeted therapy drugs is needed.
One possible way to address this issue is to monitor the activity levels of signaling pathways by means of a transfected cell-based assay. As a functional platform, this system reveals activated pathways regardless of the type of mutation, i.e., whether it is a known mutation or a Variant of Unknown Significance (VUS). A central step in this process is the synthesis of the patient mutations into plasmids, ready to be expressed in live cells that are then tested in the assay. Currently, there is a lack of in-vitro tools to generate mutations in an automated robust manner. Instead, a PCR-based site-directed mutagenesis is used, but this method requires multiple PCR steps and is therefore time consuming.
As an alternative to the lengthy serial procedure associated with PCR based methods, a technique that allows precise and rapid mutagenesis of numerous mutations at the same time is needed. One of the most progressive and promising gene editing methods is CRISPR/Cas9. This system is constructed by either using a plasmid base system from which the guide RNAs and Cas9 are expressed or using a ribonucleoprotein (RNP) complex in which the guide RNAs are coupled with purified Cas9 protein prior to inclusion in the reaction mixture.
There is an unmet need in the art for a rapid and robust in vitro technique that allows simultaneous generation of mutations without requiring sequencing. The present invention satisfies this need.
As described herein, the present invention relates to compositions and methods for in vitro mutagenesis.
One aspect of the invention includes a method of performing in vitro mutagenesis of a targeted sequence. In one embodiment, the method comprises incubating a mixture comprising an isolated ribonucleotide particle (RNP), a first plasmid, an oligonucleotide, and a cell-free extract. In one embodiment, the RNP comprises a tracrRNA, a crRNA and a Cas9 enzyme. In one embodiment, the tracrRNA is annealed with the crRNA. In one embodiment, the first plasmid comprises a nucleotide sequence of the targeted sequence, and the oligonucleotide comprises a nucleotide sequence that is complementary to the targeted sequence but contains at least one mismatched nucleotide, thus generating a second plasmid. In one embodiment, the second plasmid is administered to a plurality of cells. In one embodiment, at least one cell is selected from the plurality of cells wherein in vitro mutagenesis has occurred in the targeted sequence.
Another aspect of the invention includes a method of performing in vitro mutagenesis of a plurality of targeted sequences. In one embodiment, the method comprises incubating a mixture comprising a plurality of ribonucleotide particles (RNPs), a plurality of first plasmids, a plurality of oligonucleotides, and a cell-free extract. In one embodiment, the plurality of RNPs comprise a plurality of tracrRNAs, a plurality of crRNAs and a Cas9 enzyme, wherein the plurality of tracrRNAs are annealed with the plurality of crRNAs. In one embodiment, the first plurality of plasmids comprises a plurality of nucleotide sequences of the plurality of targeted sequences. In one embodiment, the plurality of oligonucleotides comprise a plurality of nucleotide sequences that are complementary to the plurality of targeted sequences but contain at least one mismatched nucleotide per targeted sequence, thus generating a plurality of second plasmids. In one embodiment, the plurality of second plasmids are administered to a plurality of cells. In one embodiment, the plurality of cells wherein in vitro mutagenesis has occurred in the targeted sequences is selected.
In another aspect, the invention includes an in vitro mutagenesis kit for a targeted sequence comprising an isolated ribonucleotide particle (RNP), an oligonucleotide, a plasmid, a buffer, a cell-free extract and instructional material for use thereof In one embodiment, the RNP comprises a tracrRNA, a crRNA and a Cas9. In one embodiment, the plasmid comprises a nucleotide sequence of a targeted sequence. In one embodiment, the oligonucleotide comprises a nucleotide sequence that is complementary to the targeted sequence but contains at least one mismatched nucleotide as to the targeted sequence therein.
Another aspect of the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a first mixture comprising an isolated ribonucleotide particle (RNP) and a plasmid. In one embodiment, the RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the RNP generates a double stranded break in the plasmid. In one embodiment, a second mixture comprising the first plasmid containing a double stranded break, a double stranded oligonucleotide, a cell-free extract, and a DNA ligase is incubated.
In one embodiment, the double stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
Yet another aspect of the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a first mixture comprising an isolated RNP and a plasmid. In one embodiment, the RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the RNP generates a double stranded break in the plasmid. In one embodiment, a second mixture is incubated comprising the first plasmid containing a double stranded break, a single stranded oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the single stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
In another aspect, the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a mixture comprising an isolated RNP, a plasmid, a single stranded oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the RNP generates a double stranded break in the plasmid. In one embodiment, the single stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
In yet another aspect, the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a first mixture comprising a first isolated RNP, a second isolated RNP and a plasmid. In one embodiment, the first RNP comprises a crRNA complementary to a first target sequence and a first Cpf1 enzyme. In one embodiment, the second RNP comprises a second crRNA complementary to a second target sequence and a second Cpf1 enzyme. In one embodiment, the first RNP generates a first double stranded break in the plasmid and the second RNP generates a second double stranded break in the plasmid. In one embodiment, second mixture is incubated comprising the first plasmid containing the double stranded breaks, an oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut sites. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the target sequence.
One aspect of the invention includes an in vitro mutagenesis kit for a targeted sequence comprising an isolated ribonucleotide particle (RNP), a double stranded oligonucleotide, a plasmid, a buffer, a cell-free extract, a DNA ligase, and instructional material for use thereof. In one embodiment, the RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the double stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site.
Another aspect of the invention includes an in vitro mutagenesis kit for a targeted sequence comprising an isolated ribonucleotide particle (RNP), a single stranded oligonucleotide, a plasmid, a buffer, a cell-free extract, a DNA ligase, and instructional material for use thereof. In one embodiment, the RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the single stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site.
In another aspect, the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a mixture comprising an isolated ribonucleotide particle (RNP), a first plasmid, an oligonucleotide, and a cell-free extract. In one embodiment, the RNP comprises a tracrRNA, a crRNA and a Cas endonuclease. In one embodiment, the tracrRNA is annealed with the crRNA. In one embodiment, the first plasmid comprises a nucleotide sequence of the targeted sequence. In one embodiment, the oligonucleotide comprises a nucleotide sequence that is complementary to the targeted sequence but contains at least one mismatched nucleotide. In one embodiment, a second plasmid is generated. In one embodiment, the second plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
Another aspect of the invention includes a method of performing in vitro mutagenesis of a plurality of targeted sequences comprising incubating a mixture comprising a plurality of ribonucleotide particles (RNPs), a plurality of first plasmids, a plurality of oligonucleotides, and a cell-free extract. In one embodiment, the plurality of RNPs comprise a plurality of tracrRNAs, a plurality of crRNAs and a Cas endonuclease. In one embodiment, the plurality of tracrRNAs are annealed with the plurality of crRNAs. In one embodiment, the first plurality of plasmids comprise a plurality of nucleotide sequences of the plurality of targeted sequences. In one embodiment, the plurality of oligonucleotides comprise a plurality of nucleotide sequences that are complementary to the plurality of targeted sequences but contain at least one mismatched nucleotide per targeted sequence. In one embodiment, a plurality of second plasmids is generated. In one embodiment, the plurality of second plasmids are administered to a plurality of cells. In one embodiment, the plurality of cells wherein in vitro mutagenesis has occurred in the targeted sequences is selected.
Yet another aspect of the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a first mixture comprising an isolated ribonucleotide particle (RNP) and a plasmid. In one embodiment, the RNP comprises a crRNA and a Cas endonuclease, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the RNP generates a double stranded break in the plasmid. In one embodiment, second mixture is incubated comprising the first plasmid containing a double stranded break, a double stranded oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the double stranded oligonucleotide comprises 5′ overhangs complementary to the RNP cut site. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
Still another aspect of the invention includes a method of performing in vitro mutagenesis of a plurality of targeted sequences comprising incubating a mixture comprising a plurality of ribonucleotide particles (RNPs), a plurality of first plasmids, a plurality of oligonucleotides, and a cell-free extract. In one embodiment, the plurality of RNPs comprise a plurality of tracrRNAs, a plurality of crRNAs and a Cas endonuclease. In one embodiment, the plurality of tracrRNAs are annealed with the plurality of crRNAs. In one embodiment, the first plurality of plasmids comprise a plurality of nucleotide sequences of the plurality of targeted sequences. In one embodiment, the plurality of oligonucleotides comprise a plurality of nucleotide sequences that are complementary to the plurality of targeted sequences but contain at least one mismatched nucleotide per targeted sequence. In one embodiment, a plurality of second plasmids is generated. In one embodiment, the plurality of second plasmids is administered to a plurality of cells. In one embodiment, the plurality of cells wherein in vitro mutagenesis has occurred in the targeted sequences is selected.
Another aspect of the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a first mixture comprising an isolated ribonucleotide particle (RNP) and a plasmid. In one embodiment, the RNP comprises a crRNA and a Cas endonuclease, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the RNP generates a double stranded break in the plasmid. In one embodiment, a second mixture is incubated comprising the first plasmid containing a double stranded break, a double stranded oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the double stranded oligonucleotide comprises 5′ overhangs complementary to the RNP cut site. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
In another aspect, the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a first mixture comprising an isolated RNP and a plasmid. In one embodiment, the RNP comprises a crRNA and a Cas endonuclease, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the RNP generates a double stranded break in the plasmid. In one embodiment, a second mixture is incubated comprising the first plasmid containing a double stranded break, a single stranded oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the single stranded oligonucleotide comprises 5′ overhangs complementary to the RNP cut site. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
In yet another aspect, the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a mixture comprising an isolated RNP, a plasmid, a single stranded oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the RNP comprises a crRNA and a Cas endonuclease, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the RNP generates a double stranded break in the plasmid. In one embodiment, the single stranded oligonucleotide comprises 5′ overhangs complementary to the RNP cut site. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
In still another aspect, the invention includes a method of performing in vitro mutagenesis of a targeted sequence comprising incubating a first mixture comprising a first isolated RNP, a second isolated RNP and a plasmid. In one embodiment, the first RNP comprises a crRNA complementary to a first target sequence and a first Cas endonuclease and the second RNP comprises a second crRNA complementary to a second target sequence, and second Cas endonuclease. In one embodiment, the first RNP generates a first double stranded break in the plasmid and the second RNP generates a second double stranded break in the plasmid. In one embodiment, a second mixture is incubated comprising the first plasmid containing the double stranded breaks, an oligonucleotide, a cell-free extract, and a DNA ligase. In one embodiment, the oligonucleotide comprises 5′ overhangs complementary to the RNP cut sites. In one embodiment, a re-circularized plasmid is generated. In one embodiment, the re-circularized plasmid is administered to a plurality of cells. In one embodiment, selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the target sequence.
Another aspect of the invention includes an in vitro mutagenesis kit for a targeted sequence comprising an isolated ribonucleotide particle (RNP), an oligonucleotide, a plasmid, a buffer, a cell-free extract and instructional material for use thereof In one embodiment, the RNP comprises a tracrRNA, a crRNA and a Cas endonuclease. In one embodiment, the plasmid comprises a nucleotide sequence of a targeted sequence. In one embodiment, the oligonucleotide comprises a nucleotide sequence that is complementary to the targeted sequence but contains at least one mismatched nucleotide as to the targeted sequence therein.
Yet another aspect of the invention includes an in vitro mutagenesis kit for a targeted sequence comprising an isolated ribonucleotide particle (RNP), an oligonucleotide, a plasmid, a buffer, a cell-free extract, a DNA ligase, and instructional material for use thereof. In one embodiment, the RNP comprises a crRNA and a Cas endonuclease, wherein the crRNA is complementary to the targeted sequence. In one embodiment, the oligonucleotide comprises 5′ overhangs complementary to the RNP cut site.
In various embodiments of the above aspects or any other aspect of the invention delineated herein, the in vitro mutagenesis comprises at least one mutation in the nucleotide sequence of the targeted sequence selected from the group consisting of a single base nucleotide modification, a deletion, and an insertion. In another embodiment, the cell-free extract is derived from at least one cell selected from the group consisting of HEK, HUH-7, DLD1, HCT116, and S. cerevisiae.
In one embodiment, the mixture is incubated at about 37° C. for about 120 minutes. In another embodiment, each oligonucleotide is independently between about 25 and about 200 bases in length. In yet another embodiment, each oligonucleotide is independently about 72 bases in length. In still another embodiment, each oligonucleotide independently further comprises a chemically modified terminal linkage. In one embodiment, the chemically modified terminal linkage comprises a 2′-O-methyl modification.
In certain embodiments, the oligonucleotide further comprises a NotI restriction site. In one embodiment, the selecting comprises digesting the cell with a NotI enzyme.
In certain embodiments, the kit or method of the invention further comprises a second RNP comprising a second crRNA complementary to a second targeted sequence.
In certain embodiments, the Cas endonuclease is selected from the group consisting of Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, T7, spCas9, Cpf1, Cpf2, CasY, CasX, and saCas9.
For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.
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. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“About” 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 ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein the term “amount” refers to the abundance or quantity of a constituent in a mixture.
As used herein, the term “bp” refers to base pair.
The term “complementary” refers to the degree of anti-parallel alignment between two nucleic acid strands. Complete complementarity requires that each nucleotide be across from its opposite. No complementarity requires that each nucleotide is not across from its opposite. The degree of complementarity determines the stability of the sequences to be together or anneal/hybridize. Furthermore various DNA repair functions as well as regulatory functions are based on base pair complementarity.
The term “CRISPR/Cas” or “clustered regularly interspaced short palindromic repeats” or “CRISPR” refers to DNA loci containing short repetitions of base sequences followed by short segments of spacer DNA from previous exposures to a virus or plasmid. Bacteria and archaea have evolved adaptive immune defenses termed CRISPR/CRISPR-associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. In bacteria, the CRISPR. system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage.
“Cas endonuclease” refers to a CRISPR-associated endonuclease enzyme. A non-limiting example of a Cas endonuclease is Cas9. Other exemplary Cas endonucleases include but are not limited to Cpf1, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, T7, spCas9, Cpf2, CasY, CasX, and/or saCas9.
The “CRISPR/Cas9” system or “CRISPR/Cas9-mediated gene editing” refers to a type II CRISPR/Cas system that has been modified for genome editing/engineering. It is typically comprised of a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). “Guide RNA (gRNA)” is used interchangeably herein with “short guide RNA (sgRNA)”. The gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ˜20 nucleotide “spacer” or “targeting” sequence that defines the genomic target to be modified. The genomic target of Cas9 can changed by changing the targeting sequence present in the gRNA.
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.
As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
“Homologous” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules, two RNA molecules, a DNA and an RNA molecule, a DNA and a sgRNA molecule, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
“Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.
A “mutation” as used therein is a change in a DNA sequence resulting in an alteration from a given reference sequence (which may be, for example, an earlier collected DNA sample from the same subject). The mutation can comprise deletion and/or insertion and/or duplication and/or substitution of at least one deoxyribonucleic acid base such as a purine (adenine and/or thymine) and/or a pyrimidine (guanine and/or cytosine). Mutations may or may not produce discernible changes in the observable characteristics (phenotype) of an organism (subject).
By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 100 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T”.
As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
The term “polynucleotide” includes DNA, cDNA, RNA, DNA/RNA hybrid, anti-sense RNA, siRNA, miRNA, snoRNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatized, synthetic, or semisynthetic nucleotide bases. Also, included within the scope of the invention are alterations of a wild type or synthetic gene, including but not limited to deletion, insertion, substitution of one or more nucleotides, or fusion to other polynucleotide sequences.
Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5′-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5′-direction.
A “primer” is an oligonucleotide, usually of about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length, that is capable of hybridizing in a sequence specific fashion to the target sequence and being extended during the PCR.
The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
A “sample” or “biological sample” as used herein means a biological material from a subject, including but is not limited to organ, tissue, exosome, blood, plasma, saliva, urine and other body fluid. A sample can be any source of material obtained from a subject.
The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.
A “targeted gene”, “targeted sequence”, or “target sequence” as used interchangeably herein refers to a nucleic acid sequence that is specifically targeted for mutagenesis. The nucleic acid sequence that is targeted can be in a coding (gene) or non-coding region of a genome.
The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The invention relates to a novel method for performing in vitro site-directed mutagenesis using gene editing technologies. In certain embodiments, the invention includes an in vitro site-directed mutagenesis kit comprising a ribonucleotide particle (RNP), an oligonucleotide, a buffer, a cell-free extract and instructional material for use thereof.
In certain embodiments, the invention includes a method of performing in vitro mutagenesis. In other embodiments, the method comprises assembling a ribonucleotide particle (RNP). In yet other embodiments, the method comprises incubating a mixture comprising the RNP, a first plasmid, an oligonucleotide, a buffer, and a cell-free extract, thus forming a second plasmid. In yet other embodiments, the method comprises isolating the second plasmid. In yet other embodiments, the method comprises administering the isolated second plasmid to a plurality of cells. In yet other embodiments, the method comprises selecting from the plurality of cells at least one cell wherein in vitro mutagenesis of the target gene has occurred. In certain embodiments, the RNP is assembled by annealing tracrRNA with crRNA then combining with Cas9. The plasmid contains a gene target. The gene target can be any gene in a cell. In one embodiment, the target gene is EGFP.
In certain embodiments, the invention includes a method of performing in vitro mutagenesis. In other embodiments, the method comprises incubating a first mixture comprising an isolated ribonucleotide particle (RNP) and a plasmid, wherein the RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted gene, and wherein the RNP generates a double stranded break in the plasmid. In yet other embodiments, the method comprises incubating a second mixture comprising the first plasmid containing a double stranded break, a double stranded oligonucleotide, a cell-free extract, and a DNA ligase, wherein the double stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site, and wherein a re-circularized plasmid comprising a NotI restriction site is generated. In yet other embodiments, the method comprises administering the re-circularized plasmid to a plurality of cells, and selecting from the plurality of cells at least one cell wherein in vitro mutagenesis has occurred in the targeted gene.
In certain embodiments, the invention includes a method of performing in vitro mutagenesis of a targeted sequence. In other embodiments, the method comprises incubating a first mixture comprising an isolated RNP and a plasmid, wherein the RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted sequence, and wherein the RNP generates a double stranded break in the plasmid. In yet other embodiments, the method comprises incubating a second mixture comprising the first plasmid containing a double stranded break, a single stranded oligonucleotide, a cell-free extract, and a DNA ligase, wherein the single stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site, and wherein a re-circularized plasmid comprising a NotI restriction site is generated. In yet other embodiments, the method comprises administering the re-circularized plasmid to a plurality of cells. In yet other embodiments, the method comprises selecting from the plurality of cells at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
In certain embodiments, the invention includes a method of performing in vitro mutagenesis of a targeted gene. In other embodiments, the method comprises incubating a first mixture comprising a first isolated RNP, a second isolated RNP, and a plasmid, wherein the first RNP comprises a crRNA complementary to a first target sequence and a first Cpf1 enzyme and the second RNP comprises a second crRNA complementary to a second target sequence, and wherein the first RNP generates a first double stranded break in the plasmid and the second RNP generates a second double stranded break in the plasmid. In yet other embodiments, the method comprises incubating a second mixture comprising the first plasmid containing the double stranded breaks, an oligonucleotide, a cell-free extract, and a DNA ligase, wherein the oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut sites, and wherein a re-circularized plasmid comprising a NotI restriction site is generated. In yet other embodiments, the method comprises administering the re-circularized plasmid to a plurality of cells. In yet other embodiments, the method comprises selecting from the plurality of cells at least one cell wherein in vitro mutagenesis has occurred in the target sequence.
In certain embodiments, the invention includes a method of performing in vitro mutagenesis of a targeted sequence, comprising incubating a mixture comprising an isolated RNP, a plasmid, a single stranded oligonucleotide, a cell-free extract, and a DNA ligase. The RNP comprises a crRNA and a Cpf1 enzyme. The crRNA is complementary to the targeted sequence, and the RNP generates a double stranded break in the plasmid. The single stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site. A re-circularized plasmid is generated and administered to a plurality of cells. Selected from the plurality of cells is at least one cell wherein in vitro mutagenesis has occurred in the targeted sequence.
In certain embodiments, the double stranded oligonucleotide comprises a NotI restriction site.
The cells can be selected using any standard mean known to one of ordinary skill in the art. In one embodiment, the selecting can comprise digesting the cell with a NotI enzyme to confirm that in vitro mutagenesis has occurred. The cell-free extract can be derived from any cells or cell line known in the art, including but not limited to HEK, HUH-7, DLD1, HCT116, and S. cerevisiae.
Demonstrated herein is a new system in which an RNP particle contains crRNA and/or tracRNA as separate entities, similar to what is found in natural systems and bacteria. Coupled with the Cas9 or Cpf1 protein, this system increases efficacy and precision reducing the concern of offsite mutagenesis. A series of capabilities for mutation synthesis are possible, ranging from single base point mutations, single base deletions or insertions, small insertions or deletions, and small duplications within the coding region of the target genes.
The fundamental steps for this new assay were established and validated for a wide-range of DNA sequence mutations. This focused primarily on increasing the efficacy and efficiency of creating point mutations in specific target genes. In addition, a more universal RNP-type particle is used that expands the versatility of the assay and enables precise mutagenesis at multiple sites simultaneously within the coding region of the gene.
Single-stranded oligonucleotides are well studied and useful synthetic DNA molecules for gene editing because a large number of chemical modifications and variations can be incorporated into their composition. Some of these modifications enable a higher binding affinity to a duplex DNA target, ensuring that the critical reaction intermediate, the D-loop, is stable enough to direct genetic exchange. Several classes of chemical modifications can be used in this study so that the desired genetic alteration in the target gene will be created at a higher efficiency. In certain embodiments, single base nucleotide modifications, deletions, or insertions, are executed by an oligonucleotide of about 72 bases in length bearing chemically modified terminal linkages to prevent against nuclease digestion in the cell free extract. This workhorse oligonucleotide is designed so that it binds in perfect homologous register and complementarity with the target gene sequence except for a single mismatch which is created at the nucleotide in the target gene designated for change. The mismatched base pair is most efficiently corrected when it is created at the central base in the oligonucleotide during the alignment of complementary strands. For target gene alterations where double nucleotide substitutions or small insertions or deletions are desired, two types of chemical modifications in the targeting oligonucleotide are utilized. Both are designed to increase target affinity so that the section of the targeted gene can be deleted or small insertions can be placed within the gene sequence. Chemical modifications that increase binding affinity and stable DNA pairing between an incoming single stranded oligo donor (ssODN) and the duplex as it incorporates into the helix are synthesized alone or in combination into the targeting oligonucleotide.
The 2′-O-(methyl, fluoro, and so forth) group of modifications offers a significant increase in binding affinity with both RNA and DNA targets and have also been shown to increase resistance to nuclease digestion in both cell free conditions and after microinjection into cells. Typically, a series of 2′-O-methyl modifications (ranging from 3-5) are incorporated in the left and/or right arms of the workhorse vector (72-mer), as well as within the basis in the center of the molecule surrounding the target site. Another 2′ modification that improves binding affinity for DNA is the addition of a fluoro- group to designated bases in the oligonucleotide. Once again, a series of fluoro-group modified bases are placed at 3′ and 5′ arms as well as in the central region of the targeting vector. Linked Nucleic Acid (LNA) has become a prominent modification for increasing binding affinity. LNA is a bicyclic nucleic acid that tethers the 2′-O to the 4′ C to create a methylene bridge effectively locking the structure into a 3′-sugar conformation. Since the length of the oligonucleotide can be extended from about 72 to about 200 bases, where the desired end product is a 20 base insertion, lateral sections of the oligonucleotide can bear a series of these modifications to improve binding target stability. The same is true in the case where the objective is to delete 20 bases. In certain embodiments, the targeting oligonucleotide is 52 bases in length and bears lateral sections of chemical moieties that improve binding avidity. In certain embodiments, the oligonucleotide is between 25 and 200 bases in length, and any and all numbers therebetween. In this way genetic modifications beyond the single base substitution can be created with relatively high efficiency and identified through the dual targeting approach outlined herein.
The oligonucleotides may be engineered to be between about 10 nucleotides to about 200 nucleotides, or about 50 nucleotides to about 125 nucleotides, or about 60 nucleotides to about 100 nucleotides, or about 70 nucleotides to about 90 nucleotides in length. The oligonucleotide can be about 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200 nucleotides, or any number of nucleotides therebetween. In one embodiment, the oligonucleotide is greater than 60 nucleotides in length. In another embodiment, the oligonucleotide is greater than 70 nucleotides in length. In yet another embodiment, the oligonucleotide is about 72 nucleotides in length. In still another embodiment, the oligonucleotide is between about 25 and about 200 bases in length.
There are a number of sources for the cell extracts that provide the enzymatic activity for editing genes located in expression vectors or episomal targets. While the tendency might be to isolate and purify nuclear extracts from mammalian cells solely, whole cell extracts containing cytoplasmic activities enable a higher level of gene editing, and thus are used in the experiments described herein. Mammalian cell free extracts can be obtained from any type of cell, including but not limited to HEK, HUH-7, DLD1,HCT116, and S. cerevisiae cells. The latter two originate from colon cancer cells while the former originates from liver. Each cell line has demonstrated a rich source for cell extracts that can catalyze gene editing activity. These cell lines are known to be deficient in one or more mismatch repair protein activities which, while somewhat counterintuitive, actually enables higher levels of gene editing. When a mismatch is created by an incoming oligonucleotide with the target gene, to enable nucleotide exchange, insertion or deletion, the natural tendency of a wild type mismatch repair pathway is to recognize and destabilize that pairing. Extensive genetic and biochemical studies were carried out to show that nucleotide exchange driven by ssODNs at the target site is enhanced in such mutant cell backgrounds (Dekker et al. Nucleic Acids Res. 31(6) e27). Preparation and utilization of cell free extracts from yeast, primarily S. cerevisiae, to enable genetic modification of expression vectors provides an innovative approach to modifying episomal targets. The variety of genetic backgrounds in the remarkably genetically tractable S. cerevisiae, provides a rich source of enzymatic activity that could be more efficient in executing single base repairs, small segment deletions, small segment insertions, or gene fragment duplications. Thus, depending on the objective, the appropriate strain can be utilized as a source for the cell free extract to achieve success in the most validated and expeditious fashion.
In certain embodiments, chemical modification of the oligonucleotide provides the most efficient strategy to produce a wide range of appropriate genetic modifications. However, should a particular series of gene alterations become problematic, cell extracts supplemented with additional genetic tools can be used. A wide variety of CRISPR/Cas9 expression constructs that are highly expressed in mammalian cell lines are available. Non-limiting examples of these constructs include plasmid p42230, which contains a human codon optimized SpCas9 and an insertion site for chimeric guide RNA, and other constructs with a pX330 backbone vector (Addgene). A particular CRISPR/Cas9 construct can be transfected into mammalian cells and expressed prior to preparing a cell free extract, thereby enriching the enzymatic activity of gene editing. Without wishing to be bound by specific theory, supplementation of the cell free extract may help direct the oligonucleotide to a specific site and indirectly enhance the frequency of editing of the target gene. In certain embodiments, if simple deletion or insertion of a DNA segment of the target gene is the experimental objective, it may be preferable to utilize the cell free extract supplemented with a CRISPR/Cas9 function initially to enable the generation of that genetic alteration. An innovative series of experiments have been performed in which a long single-stranded DNA molecule was inserted into a mammalian gene, in frame, to tag the disabled gene (Miura et al. Sci Rep. 2015 Aug. 5; 5:12799). Such an approach can also be used in vitro using cell free extracts.
In certain aspects, the invention provides in vitro mutagenesis kits for a targeted sequence. In one embodiment, the kit comprises an isolated ribonucleotide particle (RNP), a double stranded oligonucleotide, a plasmid, a buffer, a cell-free extract, a DNA ligase, and instructional material for use thereof. The RNP comprises a crRNA and a Cpf1 enzyme, wherein the crRNA is complementary to the targeted sequence, and the double stranded oligonucleotide comprises 5′ overhangs complementary to the Cpf1 cut site. In certain embodiments, the double stranded oligonucleotide comprises a NotI restriction site.
In another embodiment, the in vitro mutagenesis kit comprises an isolated ribonucleotide particle (RNP), a single stranded oligonucleotide, a plasmid, a buffer, a cell-free extract, a DNA ligase, and instructional material for use thereof. The RNP comprises a crRNA and a Cpf1 enzyme. The crRNA is complementary to the targeted gene and the single stranded oligonucleotide comprises a NotI restriction site and 5′ overhangs complementary to the Cpf1 cut site.
The kits can further comprise a second RNP comprising a second crRNA complementary to a second targeted sequence.
The CRISPR/Cas system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRNA) and a conserved tri-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR/CAS system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA for use in cell lines (such as 2931 cells), primary cells, and CAR T cells. The CRISPR/CAS system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes.
One example of a CRISPR/Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Publication No.: 2014/0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.
CRISPR/Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In another embodiment, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof. In certain embodiments, Cas9 further includes spCas9, Cpf1, Cpf2, CasY, CasX, and/or saCas9.
In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.
The guide nucleic acid sequence is specific for a gene and targets that gene for Cas endonuclease-induced double strand breaks. The sequence of the guide nucleic acid sequence may be within a loci of the gene. In certain embodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.
The guide nucleic acid sequence may be specific for any gene, such as a gene that would reduce immunogenicity or reduce sensitivity to an immunosuppressive microenvironment. The guide nucleic acid sequence includes a RNA sequence, a DNA sequence, a combination thereof (a RNA-DNA combination sequence), or a sequence with synthetic nucleotides. The guide nucleic acid sequence can be a single molecule or a double molecule. In certain embodiments, the guide nucleic acid sequence comprises a single guide RNA.
In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. Typically, in the context of a CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional. In certain embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In other embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).
In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.
Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Another delivery mode for the CRISPR/Cas9 comprises a combination of RNA and purified Cas9 protein in the form of a Cas9-guide RNA ribonucleoprotein (RNP) complex (Lin et al., 2014, ELife 3:e04766). Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu et al., 1994, Gene Therapy 1:13-26).
In certain embodiments, the CRISPR/Cas is derived from a type II CRISPR/Cas system. In other embodiments, the CRISPR/Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.
In general, CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with the guiding RNA. CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. The CRISPR/Cas proteins can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein. In certain embodiments, the CRISPR/Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the CRISPR/Cas can be derived from modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-stranded break in DNA (Jinek et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a “nickase”), but not cleave the double-stranded DNA. In any of the above-described embodiments, any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
In one non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is replete with suitable vectors that are useful in the present invention. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the present invention may also be used for nucleic acid standard gene delivery protocols. Methods for gene delivery are known in the art, such as in U.S. Pat. Nos. 5,399,346, 5,580,859 & 5,589,466, incorporated by reference herein in their entireties.
Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, reaction temperatures and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.
The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the exemplary embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
The materials and methods employed in these experiments are now described.
Preparation of Cell-Free Extract: Cell-free extract was prepared from 200 million HCT116-19 cells following the outlined technique in Cole-Strauss et al., 1999. Nucl. Acids Res. 27 (5), 1323-1330. The resulting extract was aliquoted immediately and stored at −80° C. HEK293 cells (ATCC, American Type Cell Culture) were cultured and 8×106 cells were harvested and immediately washed in cold hypotonic buffer (20 mM HEPES, 5 mM KCl, 1.5 mM MgCl2 1 mM DTT, and 250 mM sucrose). Cells were centrifuged, washed and re-suspended in cold hypotonic buffer without sucrose, followed by incubation on ice for 15 minutes before being lysed by 25 strokes of a Dounce homogenizer. Cytoplasmic fraction of enriched cell lysate was incubated on ice for 60 minutes and centrifuged for 15 minutes at 12,000 g, 4° C. The supernatant was then aliquoted out and immediately frozen at −80° C. Cell-free extract concentrations were determined using a Bradford assay.
RNP Construction: Cas9 and tracrRNA:crRNA components were assembled as an RNP complex to accommodate eight reactions at 10 pmoles per reaction.
In vitro Reactions: In certain embodiments, reactions were prepared with components shown in Table 1 and incubated at 37° C. for 120 minutes. In certain embodiments, in vitro DNA cleavage reaction mixtures contained 250 ng of pHSG299 (Takara Bio Company, Shiga, Japan) or pKRAS6163 plasmid DNA and 10 pmols of RNP mixed in a reaction buffer (100 mM NaCl, 20 mM Tris-HCl, 10 mM MgCl2, 100 ug/ml BSA) at a final volume of 20 ul. Each reaction was incubated for 15 minutes at 37° C. after which DNA was isolated from reaction mixtures and purified using silica spin columns. Secondary in vitro re-circularization reactions contained DNA isolated from the initial cleavage reaction and 175 ug of cell-free extract supplemented with ligase mixed in a reaction buffer (20 mM TRIS, 15 mM MgCl2, 0.4 mM DTT, and 1.0 mM ATP) brought to a final volume of 35 ul. Each reaction was incubated for 15 minutes at 37° C. For reactions including duplexed insertion or replacement fragments, 100 pmol were added into the final reaction mixture. DNA from the final reaction mixture was then purified using silica spin columns (Qiagen, Hilden, Germany).
Isolation of Plasmid DNA: After incubation, each reaction mixture was diluted with PB buffer at a 1:5 ratio. Plasmid DNA was isolated using silica membrane spin columns following Qiagen's QIAprep Miniprep protocol and eluted in 104 of water. Miniprep plasmid yields ranged from 620 ng-1.8 μg, with control mixtures yielding higher returns on plasmid than full-component mixtures.
Transformation of Isolated Plasmids into Competent Cells: Eight transformations were performed using all 10 μL of the varied full plasmid DNA concentrations isolated from each of the eight reaction mixtures. Transformations were performed following the heat shock technique protocol outlined by the Invitrogen OneShot TOP10 Chemically Competent E. coli protocol.
Plating of Transformed Cells and Kanamycin Selection: Transformed cells were plated onto kanamycin plates after 1:5 and 1:100 dilutions and incubated overnight to allow kanamycin-resistant colony growth. After incubation, 30 colonies were selected over 8 of the 1:100 diluted plates, favoring the last three full-component reactions, sealed and sent out for sequencing.
Transformation, selection, DNA isolation and analysis: Plasmid DNA recovered from in vitro reactions was transformed into DH5α (Invitrogen, Carlsbad, Calif.) or TOP10 One Shot (Thermo Fisher Scientific Wilmington, Del.) competent E. coli via the heat shock methodology. Competent cells were incubated on ice for 30 minutes, heat shocked for 20 seconds at 42° C., placed on ice for 2 minutes and incubated at 37° C. in 1 ml of SOC media for 1 hour at 225 rpm. Undiluted competent cells were plated on media containing kanamycin and incubated overnight at 37° C. Single kanamycin resistant colonies were selected, and plasmid DNA was isolated via QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). Modifications to the plasmid DNA selected from bacterial colonies was evaluated after DNA sequencing (GeneWiz, South Plainfield, N.J.). Sequence analysis and disruption patterns were evaluated using SnapGene software for alignment of sample sequences to a wild-type sequence of the relevant plasmids.
The results of the experiments are now described.
For RNP assembly, tracrRNA and (cr)isprRNA were annealed separately followed by addition of the purified Cas9 protein (
DNA cleavage activity of the purified RNP complex is shown in
An experiment was designed to show the specificity of RNP-directed cleavage (
The initialization and validation strategy of the genetic readout system for the present invention is depicted in
Two genetic readouts are used in the present study. The first is the conversion of kanamycin sensitivity to kanamycin resistance through correction of a G residue to a T residue in the plasmid pKan (
The cell free extract is capable of catalyzing a single base repair at low levels as evidenced by a variety of combinations tested (Table 2). Addition of gene editing components individually, including the RNP or the oligonucleotide, does not catalyze site specific mutagenesis and conversion of the pKan plasmid. Simultaneous addition of the RNP, single-stranded oligonucleotide and the cell free extract catalyzed a highly significant increase in the conversion of the pKan sensitive to pKan resistant plasmid. Four experiments were carried out in triplicate to generate the data are presented in Table 3; the average range of colony numbers is presented.
A single base alteration or mutagenesis in converting peGFP− to peGFP+ required all reaction components including the RNP, single-stranded oligonucleotide and the cell free extract (Table 3). In the absence of the single-stranded DNA or RNP, the cell free extract catalyzed no mutagenic events within the eGFP gene (without selection). Sequences in
Representative DNA sequencing results from plasmid DNA modified in vitro and isolated from clones are shown in
The Cas9 protein contains two nucleolytic domains buried within the binding domains of the intact protein. Genetic engineering carried out on Cas9 has now generated two variations of the wild type protein; inactive Cas9, often referred to as dead Cas9 (dCas9) and Nickase, an enzyme in which one of the two nuclease domains has been altered to inactivity. The enzyme retains the capacity to cleave one of the two strands of the double helix. Both of these Cas9 variants can be tested in a methodical fashion within the in vitro mutagenesis assay in order to improve the frequency and versatility of the overall reaction. The rationale is that the introduction of a single break or the unwinding of the DNA helix at the site targeted for mutagenesis by the oligonucleotide can improve the specificity and efficiency of the overall reaction.
The optimization process began by examining the interaction of the dCas9 and Nickase on superhelical DNA. As illustrated in
The present invention provides a novel method to modify target genes and utilizes a cell-free extract system, a RNP, an oligonucleotide and a plasmid expression vector. Expression vectors contain the gene of interest (target gene) and, in certain embodiments, a mutated Kanamycin gene which serves as the correction marker for selection of genetically altered plasmids. Gene alteration, directed by specific oligonucleotides, is measured by utilizing a genetic readout in E. coli. In this embodiment, a mutant gene conferring resistance to Kanamycin inserted into the expression construct is co-targeted for correction by a standard oligonucleotide, 72 bases in length bearing specific phosphorothioate linkages. Gene editing occurs in a cell-free extract, generated from mammalian or yeast cells, and expression of the repaired Kanamycin gene is measured by the appearance of kanamycin resistance bacterial colonies on agar plates. For Kanamycin resistance, the objective is to convert a G/C base pair, (mutant) at position 4021 in the gene, to a C/G base pair (functional) concurrently with the genetic alteration in the target gene. The isolated plasmids are then electroporated into E. coli lacking a functional RecA protein. Bacterial strains deficient in homologous recombination and mismatch repair are used for this readout since these bacteria do not catalyze targeted nucleotide substitution on their own. This strategy minimizes background levels and enables a more efficient identification of plasmids bearing the specific targeted gene alteration. Isolation and DNA sequencing of the plasmids from E. coli confirms genetic alteration through the initial selection of Kanamycin resistance which signals that gene editing activity has taken place on that plasmid.
The same basic reaction workflow described herein was utilized for nucleotide insertion: DNA cleavage catalyzed by an RNP particle, addition of donor DNA, in this case a pre-annealed double strand fragment, followed by the addition of a mammalian cell free extract generated from HEK293 cells supplemented with exogenously added DNA ligase. The objective here was to insert a 15 base double-stranded fragment at the site of RNP cleavage as opposed to the intent in previous examples of creating a double strand break to execute deletion of DNA sequences contained within the plasmid. This demonstration expands the applicability of the gene editing in vitro mutagenesis assay into the realm of gene or DNA insertion, an important barrier to be crossed for the in vitro reaction.
The details of the reaction itself are as follows (
The re-circularized DNA generated from the second reaction was then isolated and purified using a silica membrane column and transformed into DH5α competent E. coli. Bacteria were plated onto agar plates containing Kanamycin antibiotics and incubated overnight at 37° C. Single colonies were picked from overnight plates and cultured in broth containing Kanamycin antibiotics in a shake incubator at 37° C. for 16 hours. Supercoiled DNA from overnight cultures was isolated and included in a NotI digestion assay and linearized DNA containing the NotI insert was visualized on an agarose gel. DNA showing NotI enzyme cleavage was then subject to PCR amplification and sequenced to confirm the NotI site insertion.
Restriction enzyme cleavage, RFLP, and DNA sequence analyses of purified plasmid DNA that has been subjected to NotI restriction digestion were performed. Results illustrated the linearization of the circular plasmid DNA. The NotI restriction site does not appear within the plasmid and thus linearization could only have occurred if the fragment had been successfully inserted through the process of in vitro gene editing (
A fully integrated cell-free system for studying the process of gene editing was constructed herein.
A variety of Cas9 and Cpf1 sites were targeted along the lacZ gene region as directed by the appropriate guide RNA sequences displayed in
A mammalian cell-free extract was previously utilized to investigate the mechanism and regulation of gene editing directed by ssODNs (Engstrom et al. BioEssays 31, 159-168 (2009)). Much of the data that led to the elucidation and re-construction of that gene editing pathway originated from studies carried out in vitro (Cole-Strauss. et al. Nucleic Acids Res. 27, 1323-30 (1999)). The same experimental workflow was employed in developing the cell-free, CRISPR-directed gene editing system.
As described herein, a CRISPR-based gene editing system was established that could be used to elucidate the molecular pathways and regulatory circuitry surrounding genome modification in human cells. Toward this end, the DNA cleavage activity on pHSG299 was taken advantage of in an in vitro reaction involving Cas9 RNP, pHSG299 and the mammalian cell-free extract. The main objective was to create a specific deletion at the break site followed by recirculation and genetic readout in bacteria.
Next, this reaction was repeated and the nuclease Cpf1 substituted in place of Cas9.
The experimental system was expanded by attempting to carry out DNA insertion via homology directed repair (HDR) at the designated Cpf1 cleavage site. To do so, two short, complimentary DNA molecules were synthesized that, upon annealing, created a double-stranded DNA fragment containing a cleavage site for the restriction enzyme NotI, with no inherent site in pHSG299. The NotI restriction site and experimental system is displayed in
Plasmid DNA samples exhibiting NotI restriction were amplified by PCR and sequenced to verify DNA insertion.
The possibility that HDR and fragment insertion could be directed by single-stranded DNA molecules, instead of by the annealed double strand DNA fragment, was examined.
DNA sequencing was carried out across the region of interest for NotI positive plasmid samples recovered from both of the in vitro single-stranded DNA molecule insertion reactions. A representative panel for each is presented in
In this experiment, two Cpf1 nucleases, cutting at different sites, were used to excise a fragment from the parent plasmid and replace it with a fragment of 186 base pairs, created by the annealing of two complementary oligonucleotides with complementary overhangs as indicated in
The site-specific DNA cleavage activity of an RNP complex, containing the Cpf1 nuclease, was combined with a mammalian cell-free extract that can catalyze DNA resection, DNA insertion, and gene segment replacement within a plasmid using donor DNA fragments (
This in vitro system demonstrated great versatility in enabling the insertion of a donor DNA fragment at a single Cpf1 RNP cleavage site or the replacement of a gene segment with a donor DNA fragment through the action of two Cpf1 RNP complexes at distinct cleavage sites. In addition, this system also produced plasmid molecules containing site-specific deletions through the resection of the DNA ends created by the action of the RNP complex. Thus, a multiplicity of gene editing reactions were occurring simultaneously, competitively recapitulating how gene editing tools act when introduced into cells. These three pathways are depicted schematically in
As described herein, a CRISPR-based gene editing system that could be used to elucidate the molecular pathways and regulatory circuitry surrounding genome modification in human cells was established. Cpf1 was utilized to initiate the cleavage of supercoiled DNA followed by the addition of the cell-free extract as described herein.
The possibility that this system could reflect the reaction being carried out in mammalian cells wherein homology-directed repair, catalyzed by a single-stranded DNA fragment, is taking place was explored. Single-stranded DNA templates of the preferred substrates for homology-directed repair and successful incorporation of these fragments has been seen reproducibly, whether through imprecise or precise alignment. By designing a system that could recapitulate these reactions in a controllable environment, the function of some of the controlling factors of homology-directed repair in mammalian cells could be identified and elucidated.
Herein it was demonstrated that both site-specific deletion, perhaps reflecting a non-homologous end joining reaction, and site-specific insertion of the single-stranded DNA fragment, perhaps reflecting homology-directed repair, was possible in this in vitro system. The capability of the system to catalytically support the precise replacement of a segment of a gene was tested. One long-term objective of gene editing is to successfully replace a functional copy of the disabled gene or a dysfunctional exon. The same reaction strategy was carried out as was done for assessment of insertion (see
The success of DNA replacement of a section of the lacZ gene with a high degree of precision prompted testing of a unique application of in vitro gene editing. Perfect gene segment replacement was observed at a notable frequency, particularly when using donor DNA fragments with lengths between 81-186 bases. It was decided to target the KRAS gene and reengineer well-known oncogenic mutations that are found within the coding region. Two prominent mutations appeared within the first 13 codons; at position 35, a G to A transversion changes the amino acid produced from these codons from glycine to aspartic acid. (
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/444,629, filed Jan. 10, 2017, U.S. Provisional Patent Application No. 62/514,494, filed Jun. 2, 2017, and U.S. Provisional Patent Application No. 62/533,170, filed Jul. 17, 2017, all of which are incorporated by reference in their entireties herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/13009 | 1/9/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62444629 | Jan 2017 | US | |
| 62514494 | Jun 2017 | US | |
| 62533170 | Jul 2017 | US |
