Patent Application
20210310022
The invention relates to methods and compositions for the rapid generation of high-order combinations of genetic elements comprising a CRISPR guide sequence and scaffold sequence, and the identification of said genetic elements. The invention also relates to compositions of inhibitors that target epigenetic genes to inhibit cell proliferation and related methods.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system was initially discovered in bacterial and archaeal species as a defense mechanism against foreign genetic material (e.g., plasmids and bacteriophages). The naturally occurring CRISPR/Cas systems rely on expression of three components: a guide RNA sequence that is complementary to a target sequence, scaffold RNA that aids in recruiting the third component, an endonuclease, to the site. Though in many bacterial and archaeal species CRISPR/Cas systems are used to degrade foreign genetic material, the system has been adapted for use in a wide variety of prokaryotic and eukaryotic organisms and have been used for many methods including gene knockout, mutagenesis, and expression activation or repression (Hsu, et al. Cell (2014) 157(6):1262-1278). In genetically engineered CRISPR/Cas systems, the requirement for three independent components can be circumvented by expression of a small guide RNA (sgRNA) that contains both the CRISPR guide RNA sequence for binding a target sequence and the scaffold RNA that together mimics the structure formed by the individual guide RNA sequence and scaffold sequence and is sufficient to recruit the endonuclease to the appropriate target site (Jinek, et al. Science (2012) 337(6096):816-821).
Generation of vectors and genetic elements for the expression of multiple CRISPR systems comprising more than one sgRNA (guide sequence and scaffold sequence) is very laborious, and the complexity of libraries of CRISPR systems built using traditional cloning methods is very limited. The methods described herein allow for the generation of vectors comprising multiple sgRNAs each comprising a CRISPR guide sequences and a scaffold sequence, and concatenated barcode elements that can be detected and used as indicators of the identity of the CRISPR guide sequence(s). The methods also provide simple and rapid generation of highly complex libraries of vectors.
Aspects of the present invention provide genetic constructs comprising a first DNA element comprising a CRISPR guide sequence and a scaffold sequence; a first compatible end element and a second compatible end element flanking the first DNA element, wherein the first and second compatible end elements are capable of annealing to each other; a barcode element; a third compatible end element and a fourth compatible end element flanking the barcode element, wherein the third and fourth compatible end elements are capable of annealing to each other but are not capable of annealing to the first or second compatible end elements; and a separation site located between the fourth compatible end element and the first compatible end element, wherein the DNA element, first compatible end element, and second compatible end element are on one side of the separation site, and the barcode element, the third compatible end element, and the fourth compatible end element are on the other side of the separation site.
In some embodiments, the genetic construct further comprises a promoter element upstream of the first DNA element.
Aspects provide vectors comprising any of the genetic constructs provided herein.
Other aspects provide genetic constructs comprising a plurality of DNA elements, wherein each DNA element of the plurality of DNA element comprises a CRISPR guide sequence and a scaffold sequence; a first compatible end element and a second compatible end element flanking the plurality of DNA elements, wherein the first and second compatible end elements are capable of annealing to each other; a plurality of barcode elements; a third compatible end element and a fourth compatible end element flanking the plurality of barcode elements, wherein the third and fourth compatible end elements are capable of annealing to each other but are not capable of annealing to the first or second compatible end elements; and a separation site located between the plurality of DNA elements and the plurality of barcode elements.
Other aspects provide vectors comprising any of the genetic constructs provided herein and a promoter sequence located upstream of each of the CRISPR guide sequences.
Yet other aspects provide methods for generating a combinatorial vector, comprising (a) providing a vector containing a first genetic construct comprising a CRISPR guide sequence; a second compatible end element and a first recognition site for a first restriction enzyme flanking the CRISPR guide sequence; a barcode element; and a third compatible end element and a second recognition site for a second restriction enzyme flanking the barcode element; (b) cleaving the first genetic construct at the first recognition site, resulting in a fifth compatible end element, and cleaving the vector at the second recognition site, resulting in a sixth compatible end element; (c) providing a scaffold element comprising a scaffold sequence; a separation site comprising a first compatible end element and a fourth compatible end element; and a seventh compatible end element and an eighth compatible end element flanking the scaffold element, wherein the seventh compatible end element is capable of annealing to the fifth compatible end element and the eighth compatible end element is capable of annealing to the sixth compatible end element; and (d) annealing the scaffold element to the cleaved first genetic construct, wherein the annealing occurs at compatible end elements within the vector and the scaffold element that are capable of annealing to each other, and wherein after the annealing, the scaffold element is integrated between the CRISPR guide sequence and the barcode element, and wherein the separation site is located between the scaffold sequence and the barcode element, creating a combinatorial vector.
In some embodiments, the method further comprises (a) providing any of the combinatorial vector as described herein; (b) cleaving the vector at the separation site within the scaffold element, resulting in a first compatible end element and a fourth compatible end element; (c) providing a second genetic construct comprising a CRISPR guide sequence; a scaffold sequence; a barcode element; and a second compatible end element and a third compatible end element flanking the second genetic construct, wherein the second compatible end element of the second genetic construct is capable of annealing with the first compatible end element of the vector and the third compatible end element of the second genetic construct is capable of annealing to the fourth compatible end element of the vector; and (d) annealing the second genetic construct to the cleaved vector, wherein the annealing occurs at compatible end elements within the second genetic construct and the vector that are capable of annealing to each other, and wherein after annealing, the second genetic construct is integrated into the vector, creating a combinatorial vector comprising concatenated barcode elements and concatenated CRISPR guide and scaffold sequences.
In some embodiments, the combinatorial vector further comprises one or more promoter upstream of the CRISPR guide sequence. In some embodiments, the method is iterative. In some embodiments, the first recognition site and the second recognition sites have the same recognition site sequence, and the first restriction enzyme and the second restriction enzyme are the same restriction enzymes.
Other aspects of the invention provide genetic constructs comprising at least two CRISPR guide sequences; a barcode element; and a restriction recognition site located between each CRISPR guide sequence and between the barcode element and the CRISPR guide sequence nearest to the barcode element.
Other aspects provide genetic constructs comprising a plurality of DNA elements, each comprising a CRISPR guide sequence and a scaffold sequence; a barcode element; and a promoter sequence located upstream of each of the DNA elements of the plurality of DNA elements. In some embodiments, the barcode element is located at the 5′ end of the genetic construct. In some embodiments, the barcode element is located at the 3′ end of the genetic construct.
Other aspects provide vectors comprising any of the genetic constructs described herein.
Yet other aspects provide methods for generating a combinatorial vector, comprising (a) providing a vector comprising: a plurality of CRISPR guide sequences; a barcode element, wherein the barcode element is located downstream of the plurality of CRISPR guide sequences; optionally a promoter sequence located upstream of at least one of the plurality of CRISPR guide sequences; and a plurality of recognition sites for a plurality of restriction enzymes, wherein each of the plurality of recognition sites is located downstream of one of the plurality of CRISPR guide sequences; (b) cleaving the vector at at least one of the plurality of recognition sites with at least one of the plurality of restriction enzymes, resulting in a first compatible end element and a second compatible end element; (c) providing a first scaffold element comprising: a scaffold sequence, optionally a promoter sequence, and a third compatible end element and fourth compatible end element flanking the first scaffold element, wherein the third compatible end element is capable of annealing to the first compatible end element of the cleaved vector and the fourth compatible end element is capable of annealing to the second compatible end element of the cleaved vector; and (d) annealing the first scaffold element to the cleaved vector, wherein the annealing occurs at compatible end elements within the first scaffold element and the cleaved vector, and wherein after annealing, the first scaffold element is integrated downstream of one of the plurality of CRISPR guide sequences, thereby producing a combinatorial vector. In some embodiments, the method is iterative.
Other aspects provide methods for generating a combinatorial vector comprising (a) providing a vector comprising: a plurality of CRISPR guide sequences, a barcode element, wherein the barcode element is located upstream of the plurality of CRISPR guide sequences; optionally a promoter sequence located upstream of at least one of the plurality of CRISPR guide sequences; and a plurality of recognition sites for a plurality of restriction enzymes, wherein each of the plurality of recognition sites is located upstream of one of the plurality of CRISPR guide sequences; (b) cleaving the vector at least one of the plurality of recognition sites with at least one of the plurality of restriction enzymes, resulting in a first compatible end element and a second compatible end element; (c) providing a first scaffold element comprising optionally a scaffold sequence, a promoter sequence, and a third compatible end element and fourth compatible end element flanking the first scaffold element, wherein the third compatible end element is capable of annealing to the first compatible end element of the cleaved vector and the fourth compatible end element is capable of annealing to the second compatible end element of the cleaved vector; (d) annealing the first scaffold element to the cleaved vector, wherein the annealing occurs at compatible end elements within the first scaffold element and the cleaved vector, and wherein after annealing, the first scaffold element is integrated upstream of one of the plurality of CRISPR guide sequences, thereby producing a combinatorial vector. In some embodiments, the method is iterative.
Aspects of the invention provide compositions comprising two or more inhibitors targeting two or more epigenetic genes selected from the combinations of epigenetic genes set forth in Table 2. In some embodiments, each of the two or more inhibitors reduce or prevent expression of an epigenetic gene or reduce or prevent activity of a protein encoded by the epigenetic gene. In some embodiments, each of the inhibitors is selected from the group consisting of a CRISPR guide sequence and scaffold sequence; an shRNA; and a small molecule. In some embodiments, at least one of the inhibitors is a CRISPR guide sequence and scaffold sequence; and the composition further comprises or encodes a Cas9 endonuclease. In some embodiments, the CRISPR guide sequence or shRNA is expressed from a recombinant expression vector. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM4C or BRD4 and KDM6B. In some embodiments, the inhibitor of BRD4 is JQ1 ((6S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester). In some embodiments, the inhibitor of KDM4C is SD70 (N-(furan-2-yl(8-hydroxyquinolin-7-yl)methyl)isobutyramide). In some embodiments, the inhibitor of KDM6B is GSK-J4 (ethyl 3-((6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propanoate, monohydrochloride).
Other aspects provide methods for reducing proliferation of a cell, comprising contacting the cell with a combination of two or more inhibitors targeting two or more epigenetic genes selected from the combinations of epigenetic genes set forth in Table 2. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is an ovarian cancer cell. In some embodiments, each of the two or more inhibitors reduce or prevent expression of an epigenetic gene or reduce or prevent activity of a protein encoded by the epigenetic gene. In some embodiments, each of the inhibitors is selected from the group consisting of a CRISPR guide sequence and scaffold sequence; an shRNA; and a small molecule. In some embodiments, at least one of the inhibitors is a CRISPR guide sequence and scaffold sequence; and the composition further comprises or encodes a Cas9 endonuclease. In some embodiments, the CRISPR guide sequence or shRNA is expressed from a recombinant expression vector. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM4C or BRD4 and KDM6B. In some embodiments, the inhibitor of BRD4 is JQ1 ((6S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester). In some embodiments, the inhibitor of KDM4C is SD70 (N-(furan-2-yl(8-hydroxyquinolin-7-yl)methyl)isobutyramide). In some embodiments, the inhibitor of KDM6B is GSK-J4 (ethyl 3-((6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propanoate, monohydrochloride).
Other aspects provide methods for treating cancer in a subject comprising administering to the subject a combination of two or more inhibitors targeting two or more epigenetic genes selected from the combinations of epigenetic genes set forth in Table 2, wherein each of the two or more inhibitors are administered in an effective amount. In some embodiments, each of the inhibitors is selected from the group consisting of a CRISPR guide sequence, an shRNA, and a small molecule. In some embodiments, the effective amount of each of the two or more inhibitors administered in the combination is less than the effective amount of the inhibitor when not administered in the combination. In some embodiments, each of the two or more inhibitors reduce or prevent expression of an epigenetic gene or reduce or prevent activity of a protein encoded by the epigenetic gene. In some embodiments, each of the inhibitors is selected from the group consisting of a CRISPR guide sequence and scaffold sequence; an shRNA; and a small molecule. In some embodiments, at least one of the inhibitors is a CRISPR guide sequence and scaffold sequence; and the composition further comprises or encodes a Cas9 endonuclease. In some embodiments, the CRISPR guide sequence or shRNA is expressed from a recombinant expression vector. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM4C or BRD4 and KDM6B. In some embodiments, the inhibitor of BRD4 is JQ1 ((6S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester). In some embodiments, the inhibitor of KDM4C is SD70 (N-(furan-2-yl(8-hydroxyquinolin-7-yl)methyl)isobutyramide). In some embodiments, the inhibitor of KDM6B is GSK-J4 (ethyl 3-((6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propanoate, monohydrochloride).
Other aspects provide methods for identifying a combination of inhibitors of epigenetic genes that reduces proliferation of a cell comprising contacting a first population of cells and a second population of cells with a plurality of combinations of two or more CRISPR guide sequences and scaffold sequences and a Cas9 endonuclease; culturing the first population of cells and the second population of cells such that the second population of cells is cultured for a longer duration compared to the first population of cells; identifying the combinations of two or more CRISPR guide sequences and scaffold sequences in the first population of cells and the combinations of two or more CRISPR guide sequences and scaffold sequences in the second population of cells; comparing the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the first population of cells to the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the second population of cells; and identifying a combination of two or more CRISPR guide sequences and scaffold sequences that is absent from or in reduced abundance in the second population of cells but present in or in increased abundance in the first population of cells as a combination of CRISPR guide sequences and scaffold sequences that reduces cell proliferation.
Yet other aspects provide methods for identifying a combination of genes to be inhibited to reduce proliferation of a cell comprising contacting a first population of cells and a second population of cells with a plurality of combinations of two or more CRISPR guide sequences and scaffold sequences and a Cas9 endonuclease; culturing the first population of cells and the second population of cells such that the second population of cells is cultured for a longer duration compared to the first population of cells; identifying the combinations of two or more CRISPR guide sequences and scaffold sequences in the first population of cells and the combinations of two or more CRISPR guide sequences and scaffold sequences in the second population of cells; comparing the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the first population of cells to the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the second population of cells; and identifying a combination of two or more CRISPR guide sequences and scaffold sequences that is absent from or in reduced abundance in the second population of cells but present in or in increased abundance in the first population of cells as a combination of genes to be inhibited to reduce proliferation.
These and other aspects of the invention, as well as various embodiments thereof, will become more apparent in reference to the drawings and detailed description of the invention.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combination of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.
The accompanying drawings are not intended to be drawn to scale. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
Generation of vectors and genetic elements for the expression of multiple CRISPR systems comprising more than one sgRNA (guide sequence and scaffold sequence) is very laborious, and the complexity of libraries of CRISPR systems built using traditional cloning methods is very limited. The methods described herein result in vectors with concatenated barcodes and CRISPR guide sequences and scaffold sequences. The methods are potentially highly efficient for building large libraries for combinatorial genetic screening and leverage the fact that large numbers of oligonucleotides can be readily printed and that guide sequences with target specificity determining regions can be printed onto these oligonucleotides because the guide sequences and barcode elements are of short lengths.
The Massively Parallel Combinatorial Genetics approach to generating CRISPR constructs and vectors described herein allows the rapid generation of combinatorial sets of genetic constructs comprising components of the CRISPR system (CRISPR guide sequences and scaffold sequences) capable of targeting nucleic acid of a host cell. The methods also enable the pooled screening of multiple combination orders (e.g., pairwise, tri-wise, and n-wise combination can be pooled and screened together simultaneously), identifying minimal combinations needed for a given application. Combinatorial sets of genetic constructs, such as those generated using the methods described herein, may be useful for the identification of genes and genetic pathways that interact synergistically to regulate a cellular process or phenotype, such as cancer cell growth. Also described herein are novel combinations of epigenetic genes identified, using combinatorial CRISPR constructs described herein that, when inhibited together, have anti-cancer effects, such as reducing proliferation of cells.
Aspects of the present disclosure relate to genetic constructs, vectors comprising genetic constructs, combinatorial vectors, and methods of generating combinatorial vectors using in the Massively Parallel Combinatorial Genetics approach, which can be found in PCT Publication No. WO2014/00542, herein incorporated by reference in its entirety. As used herein, a “genetic construct” refers to one or more DNA element(s) comprising a CRISPR guide sequence and a scaffold sequence and a barcode element, such that each DNA element is associated with a barcode element. As used herein, association between a specific DNA element and a barcode element means that a specific DNA element and a barcode element are always contained within the same genetic construct. Accordingly, the presence or detection of a specific barcode element within a genetic construct indicates that the associated specific DNA element(s) is also present within the same genetic construct.
In a host cell, the DNA element comprising a CRISPR guide sequence and a scaffold sequence is transcribed and forms a CRISPR small-guide RNA (sgRNA) that functions to recruit an endonuclease to a specific target nucleic acid in a host cell, which may result in site-specific CRISPR activity. As used herein, a “CRISPR guide sequence” refers to a nucleic acid sequence that is complementary to a target nucleic acid sequence in a host cell. The CRISPR guide sequence targets the sgRNA to a target nucleic acid sequence, also referred to as a target site. The CRISPR guide sequence that is complementary to the target nucleic acid may be between 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length. In some embodiments, the CRISPR guide sequence that is complementary to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the CRISPR guide sequence that is complementary to the target nucleic acid is 20 nucleotides in length.
It will be appreciated that a CRISPR guide sequence is complementary to a target nucleic acid in a host cell if the CRISPR guide sequence is capable of hybridizing to the target nucleic acid. In some embodiments, the CRISPR guide sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to a target nucleic acid (see also U.S. Pat. No. 8,697,359, which is incorporated by reference for its teaching of complementarity of a CRISPR guide sequence with a target polynucleotide sequence). It has been demonstrated that mismatches between a CRISPR guide sequence and the target nucleic acid near the 3′ end of the target nucleic acid may abolish nuclease cleavage activity (Upadhyay, et al. Genes Genome Genetics (2013) 3(12):2233-2238). In some embodiments, the CRISPR guide sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3′ end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3′ end of the target nucleic acid).
The CRISPR guide sequence may be obtained from any source known in the art. For example, the CRISPR guide sequence may be any nucleic acid sequence of the indicated length present in the nucleic acid of a host cell (e.g., genomic nucleic acid and/or extra-genomic nucleic acid). In some embodiments, CRISPR guide sequences may be designed and synthesized to target desired nucleic acids, such as nucleic acids encoding transcription factors, signaling proteins, transporters, etc. In some embodiments, the CRISPR guide sequences are designed and synthesized to target epigenetic genes. For example, the CRISPR guide sequences may be designed to target any of the combinations of epigenetic genes presented in Table 2. In some embodiments, the CRISPR guide sequences comprise any of the example CRISPR guide sequences provided in Table 1.
As used herein, a “scaffold sequence,” also referred to as a tracrRNA, refers to a nucleic acid sequence that recruits an endonuclease to a target nucleic acid bound (hybridized) to a complementary CRISPR guide sequence. Any scaffold sequence that comprises at least one stem loop structure and recruits an endonuclease may be used in the genetic elements and vectors described herein. Exemplary scaffold sequences will be evident to one of skill in the art and can be found for example in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Application No. WO2014/093694, and PCT Application No. WO2013/176772.
The terms “target nucleic acid,” “target site,” and “target sequence” may be used interchangeably throughout and refer to any nucleic acid sequence in a host cell that may be targeted by the CRISPR guide sequences described herein. The target nucleic acid is flanked downstream (on the 3′ side) by a protospacer adjacent motif (PAM) that may interact with the endonuclease and be further involved in targeting the endonuclease activity to the target nucleic acid. It is generally thought that the PAM sequence flanking the target nucleic acid depends on the endonuclease and the source from which the endonuclease is derived. For example, for Cas9 endonucleases that are derived from Streptococcus pyogenes, the PAM sequence is NGG. For Cas9 endonucleases derived from Staphylococcus aureus, the PAM sequence is NNGRRT. For Cas9 endonucleases that are derived from Neisseria meningitidis, the PAM sequence is NNNNGATT. For Cas9 endonucleases derived from Streptococcus thermophilus, the PAM sequence is NNAGAA. For Cas9 endonuclease derived from Treponema denticola, the PAM sequence is NAAAAC. For a Cpf1 nuclease, the PAM sequence is TTN.
In some embodiments, the CRISPR guide sequence and the scaffold sequence are expressed as separate transcripts. In such embodiments, the CRISPR guide sequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence and recruit the endonuclease to the target nucleic acid. In other embodiments, the CRISPR guide sequence and the scaffold sequence are expressed as a single transcript, as a chimeric RNA that may be referred to as a single guide RNA (sgRNA). An sgRNA has the dual function of both binding (hybridizing) to the target nucleic acid and recruiting the endonuclease to the target nucleic acid. In such embodiments, the scaffold sequence may further comprise a linker loop sequence.
The barcode elements can be used as identifiers for a genetic construct and may indicate the presence of one or more specific CRISPR guide sequences in a vector or genetic element. Members of a set of barcode elements have a sufficiently unique nucleic acid sequence such that each barcode element is readily distinguishable from the other barcode elements of the set. Barcode elements may be any length of nucleotide but are preferably less than 30 nucleotides in length. In some embodiments, the barcode element is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, or 30 or more nucleotides in length. Detecting barcode elements and determining the nucleic acid sequence of a barcode element or plurality of barcode elements are used to determine the presence of an associated DNA element of a genetic construct. Barcode elements as described herein can be detected by any method known in the art, including sequencing or microarray methods.
Compatible ends can be created in a variety of ways that will be evident to one of skill in the art and can consist of a variety of different sequences. As used herein, “compatible end elements” refer to regions of DNA that are capable of ligating or annealing to each other. Compatible end elements that are capable of ligating or annealing to each other will be apparent to one of skill in the art and refers to end elements that are complementary in nucleotide sequence to one another and therefore, are capable of base-pairing to one another. In several non-limiting embodiments, compatible end elements can be composed of restriction sites with compatible overhangs, Gibson assembly sequences, or functional elements of any other DNA assembly method, including recombinases, meganucleases, TAL Effector/Zinc-finger nucleases, trans-cleaving ribozymes/DNAzymes or integrases.
In some embodiments, Gibson assembly is used to generate compatible overhangs. Gibson assembly refers to an isothermal DNA end-linking technique whereby multiple DNA fragments can be joined in a single reaction. This method is described further in, and incorporated by reference from, Gibson et al. (2009) Nature Methods 6:343-5.
In other embodiments, restriction digestion is used to generate compatible ends, as depicted in
In some embodiments, the first compatible end element is generated by recognition and cleavage with the restriction enzyme BamHI, and the second compatible end element is generated by recognition and cleavage with the restriction enzyme BglII. In some embodiments, the third compatible end element is generated by recognition and cleavage with the restriction enzyme MfeI, and the fourth compatible end element is generated by recognition and cleavage with the restriction enzyme EcoRI.
As used herein, a “separation site” of a genetic construct refers to a region that allows linearization of the construct. It should be appreciated that the separation site is a site within the nucleic acid of a construct at which cleavage linearizes the construct and may allow for insertion of additional genetic elements. In some embodiments, the separation site is a restriction enzyme recognition site. For example, in
Aspects of the invention relate to methods for producing a combinatorial vector comprising genetic constructs described herein. As depicted in step 3 of
It should be appreciated that a variety of different enzyme combinations can be used to cleave the first and second recognition sites. In some embodiments, the two recognition sites located outside of the CRISPR guide sequence and the barcode element are recognized by the same restriction enzyme, which produces compatible ends with the scaffold element. In other embodiments, the two restriction sites located outside of the CRISPR guide sequence and the barcode element are recognized by two different restriction enzymes, each of which produces compatible ends with the scaffold element.
Further aspects of the invention relate to combinatorial constructs, and methods for producing combinatorial constructs. As used herein, a “combinatorial construct” refers to a genetic construct that contains a plurality of DNA elements. As used herein, a plurality of DNA elements refers to more than one DNA element, each of the DNA elements comprising a CRISPR guide sequence and a scaffold sequence. As shown in step 5 of
The (n)-wise guide RNA library of step 5 of
The methods described herein for generating combinatorial constructs can be iterative. For example, the combinatorial vector depicted in
It should be appreciated that combinatorial constructs can contain any number of DNA elements and associated barcode elements. In some embodiments a combinatorial construct contains 2, 3, 4, 5, 6, 7, 8, 9, 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, 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 or more than 100 DNA elements and associated barcode elements.
Another aspect of the present invention relates to genetic constructs and vectors comprising more than one CRISPR guide sequence associated with a single barcode element.
Also within the scope of the present invention are genetic constructs comprising a plurality of DNA elements and one barcode element. In step 3 of
Aspects of the invention relate to methods for producing a combinatorial vector comprising the genetic constructs described herein. In some embodiments, the methods involve providing a vector containing a plurality of CRISPR guide sequences and a barcode element located downstream of the plurality of CRISPR guide sequences. As shown in step 1 of
It should be appreciated that combinatorial vectors can contain any number of DNA elements associated with one barcode element. In some embodiments a combinatorial construct contains 2, 3, 4, 5, 6, 7, 8, 9, 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, 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 or more than 100 DNA elements and one barcode element. The number of DNA elements associated with one barcode element may depend on the length of the genetic construct containing at the CRISPR guide sequences, barcode and recognition sites that is capable of being synthesized.
In any of the constructs or vectors described herein, one or more RNA domains may be inserted into one or more CRISPR guide sequences. In some embodiments, the CRISPR guide sequence is fused to one or more RNA domain. In some embodiments, the RNA is a non-coding RNA or fragment thereof. In such embodiments, the RNA domain may be targeted to a DNA loci. Such constructs or vectors may be used for CRISPR display.
Further aspects of the invention relate to methods for identifying one or more DNA elements within a genetic construct or vector. After a combination event, a unique barcode that is associated with a specific DNA element(s) remains within the same genetic construct as the specific DNA element. Accordingly, identification of a barcode element or plurality of barcode elements allows for the identification of the associated DNA element or plurality of DNA elements within the same genetic construct. In some embodiments, the sequence of a barcode element and/or a DNA element is determined by sequencing or by microarray analysis. It should be appreciated that any means of determining DNA sequence is compatible with identifying one or more barcode elements and corresponding DNA elements. Significantly, in a combinatorial construct, such as is depicted in step 5 of
Further aspects of the invention relate to libraries comprising two or more genetic constructs as described herein that are compatible with methods for Massively Parallel Combinatorial Genetics. As used herein, a library of genetic constructs refers to a collection of two or more genetic constructs. In some embodiments, a library of genetic constructs is generated in which each unique DNA element is on a plasmid. This plasmid library can be pooled to form a vector library. An insert library can be generated, for example, by conducting PCR on the vector library. In a first combination event, all of the vectors can be paired with all of the inserts, generating a full combinatorial set of pairwise combinations. Further reactions between this pairwise library and an insert library can lead to a tri-wise, quad-wise or more than quad-wise library arising from a single vector library. Libraries of combinatorial constructs can used to conduct screens of host cells expressing said libraries of combinatorial constructs. In some embodiments, the libraries of combinatorial constructs contain DNA elements or combinations of DNA elements with CRISPR guide sequences that target epigenetic genes, such as the example CRISPR guide sequences presented in Table 1.
It should be appreciated that since the combinatorial step is conducted in vitro, this technology can be scaled to any host cell or organism that can receive DNA. In some embodiments, the host cell is a bacterial cell. In some embodiments, the organism is bacteria and the constructs are carried on plasmids or phages. In some embodiments, the host cell is a yeast cell. In other embodiments, the organism is yeast and the constructs are carried on plasmids or shuttle vectors. In other embodiments, the host cell is a mammalian cell, such as a human cell. In such embodiments, the genetic constructs described herein can be carried on plasmids or delivered by viruses such as lentiviruses or adenoviruses.
The genetic constructs and vectors described herein relate to the expression of components of a CRISPR system including a CRISPR guide sequence and scaffold sequence. The host cell in which the CRISPR system is expressed may express one or more additional CRISPR components, such as an endonuclease. In some embodiments, the host cell also expresses an endonuclease, such as a Cas endonuclease. In some embodiments, the Cas endonuclease is Cas1, Cas2, or Cas9 endonuclease. In some embodiments, the host cell expresses a Cas9 endonuclease derived from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophilus, or Treponema denticola. In some embodiments, the nucleotide sequence encoding the Cas9 endonuclease may be codon optimized for expression in a host cell or organism. In some embodiments, the endonuclease is a Cas9 homology or ortholog.
In some embodiments, the nucleotide sequence encoding the Cas9 endonuclease is further modified to alter the activity of the protein. In some embodiments, the Cas9 endonuclease is a catalytically inactive Cas9. For example, dCas9 contains mutations of catalytically active residues (D10 and H840) and does not have nuclease activity. Alternatively or in addition, the Cas9 endonuclease may be fused to another protein or portion thereof. In some embodiments, dCas9 is fused to a repressor domain, such as a KRAB domain. In some embodiments, such dCas9 fusion proteins are used with the constructs described herein for multiplexed gene repression (e.g. CRISPR interference (CRISPRi)). In some embodiments, dCas9 is fused to an activator domain, such as VP64 or VPR. In some embodiments, such dCas9 fusion proteins are used with the constructs described herein for multiplexed gene activation (e.g. CRISPR activation (CRISPRa)). In some embodiments, dCas9 is fused to an epigenetic modulating domain, such as a histone demethylase domain or a histone acetyltransferase domain. In some embodiments, dCas9 is fused to a LSD1 or p300, or a portion thereof. In some embodiments, the dCas9 fusion is used for CRISPR-based epigenetic modulation. In some embodiments, dCas9 or Cas9 is fused to a Fok1 nuclease domain. In some embodiments, Cas9 or dCas9 fused to a Fok1 nuclease domain is used for multiplexed gene editing. In some embodiments, Cas9 or dCas9 is fused to a fluorescent protein (e.g., GFP, RFP, mCherry, etc). In some embodiments, Cas9/dCas9 proteins fused to fluorescent proteins are used for multiplexed labeling and/or visualization of genomic loci.
Alternatively or in addition, the endonuclease is a Cpf1 nuclease. In some embodiments, the host cell expresses a Cpf1 nuclease derived from Provetella spp. or Francisella spp. In some embodiments, the nucleotide sequence encoding the Cpf1 nuclease may be codon optimized for expression in a host cell or organism.
The invention encompasses any cell type in which DNA can be introduced, including prokaryotic and eukaryotic cells. In some embodiments the cell is a bacterial cell, such as Escherichia spp., Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Therms spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. The bacterial cell can be a Gram-negative cell such as an Escherichia coli (E. coli) cell, or a Gram-positive cell such as a species of Bacillus.
In other embodiments, the cell is a fungal cell such as a yeast cell, e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Phaffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains. Preferably the yeast strain is a S. cerevisiae strain. Other examples of fungi include Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.
In other embodiments, the cell is an algal cell, a plant cell, an insect cell, a rodent cell or a mammalian cell, including a rodent cell or a human cell (e.g., a human embryonic kidney cell (e.g., HEK293T cell), a human dermal fibroblast, a human cancer cells, such as a OVCAR8 cell or a OVCAR8-ADR cell. In some embodiments, the cell is a human cancer cell, such as a human ovarian cancer cell.
Also provided herein are compositions comprising inhibitors targeting epigenetic genes. As used herein, the term “epigenetic gene” refers to any gene that affects epigenetic regulation of another molecule or process in a cell. In some embodiments, the epigenetic gene encodes a protein that is involved in epigenetic regulation. In some embodiments, the epigenetic gene encodes a nucleic acid, such as an RNA (e.g., a microRNA), that affects epigenetic regulation. In general, epigenetics refers to any alteration to a molecule or process that does not involve mutation of the genomic DNA of the cell (Jaenisch and Bird Nat. Gene. (2003) 33: 245-254). Epigenetic regulation involves DNA-mediated processes in a cell, such as transcription, DNA repair, and replication through mechanisms including DNA methylation, histone modification, nucleosome remodeling, and RNA-mediating targeting (Dawson and Kouzarides Cell (2012) 150(1): 12-27). Non-limiting examples of epigenetic genes include: DNMT1, DNMT3A, DNMT3B, DNMT3L, MBD1, MBD2, CREBBP, EP300, HDAC1, HDAC2, SIRT1, CARM1, EZH1, EZH2, MLL, MLL2, NSD1, PRMT1, PRMT2, PRMT3, PRMTS, PRMT6, PRMT7, SETD2, KDM1A-, KDM1B, KDM2A, KDM2B, KDM3A, KDM3B, KDM4A, KDM4B, KDM4C, KDMSA, KDMSB, KDMSC, KDMSD, KDM6A, KDM6B, PHF2, PHF8, BMI1, BRD1, BRD3, BRD4, ING1, ING2, ING3, ING4, and ING5.
As used herein, the term “inhibitor” refers to any molecule, such as a protein, nucleic acid, or small molecule that reduces or prevents expression of an epigenetic gene or reduces or prevents activity of a protein encoded by the epigenetic gene. In some embodiments, the combination of two or more inhibitors of epigenetic genes reduces expression of an epigenetic gene by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or at least 65% as compared to expression of the epigenetic gene in the absence of the combination of inhibitors. In some embodiments, the combination of two or more inhibitors of epigenetic genes reduces activity of a protein encoded by an epigenetic gene by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or at least 65% as compared to activity of the protein encoded by the epigenetic gene in the absence of the combination of inhibitors.
In some embodiments, the combination of inhibitors of epigenetic genes comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 or more inhibitors of epigenetic genes. In some embodiments, the combination of inhibitors of epigenetic genes comprises two inhibitors of epigenetic genes. In some embodiments, the combination of inhibitors inhibit 2, 3, 4, 5, 6, 7, 8, 9, 10 or more epigenetic genes. In some embodiments, the combination of inhibitors of epigenetic genes comprises two inhibitors that target and inhibit two epigenetic genes.
In some embodiments, at least one inhibitor of the combination of inhibitors is a protein that directly or indirectly reduces or prevents expression of an epigenetic gene or reduces or prevents activity of a protein encoded by the epigenetic gene. For example, the protein may be a repressor that reduces or prevents expression of the epigenetic gene or an allosteric inhibitor of a protein encoded by the epigenetic gene. In some embodiments, two or more inhibitors are proteins targeting two or more epigenetic genes. In some embodiments, the two or more inhibitors are proteins that target any of the combinations of epigenetic genes presented in Table 2.
In some embodiments, at least one inhibitor of the combination of inhibitors is a nucleic acid that reduces or prevents expression of an epigenetic gene or reduces or prevents activity of a protein encoded by the epigenetic gene. In some embodiments, the nucleic acid is a CRISPR guide sequence that, along with a scaffold sequence, recruits an endonuclease to the epigenetic gene. In some embodiments, two or more inhibitors are CRISPR guide sequences targeting two or more epigenetic genes. In some embodiments, the two or more inhibitors are CRISPR guide sequences that target any of the combinations of epigenetic genes presented in Table 2. In some embodiments, the two or more inhibitors are CRISPR guide sequences selected from the example CRISPR guide sequences targeting epigenetic genes provided in Table 1. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM4C. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM6B.
In some embodiments, the nucleic acid is a shRNA that is processed by the RNA interference (RNAi) pathway of the cell to silence expression of the target gene (e.g., reduce mRNA levels and/or protein production). In some embodiments, two or more inhibitors are shRNAs targeting two or more epigenetic genes. In some embodiments, the two or more inhibitors are shRNAs that target any of the combinations of epigenetic genes presented in Table 2. In some embodiments, the two or more inhibitors are shRNAs selected from the example shRNAs targeting epigenetic genes provided in Table 4. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM4C. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM6B.
In some embodiments, at least one inhibitor of the combination of inhibitors is a small molecule that reduces or prevents expression of an epigenetic gene or reduces or prevents activity of a protein encoded by the epigenetic gene. In some embodiments, two or more inhibitors are small molecules targeting two or more epigenetic genes. In some embodiments, the two or more inhibitors are small molecules that target any of the combinations of epigenetic genes presented in Table 2. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM4C. In some embodiments, the combination of epigenetic genes comprises BRD4 and KDM6B.
Any small molecule that reduces or prevents expression of BRD4 or reduces or prevents activity of a protein encoded by BRD4 may be compatible with the compositions and methods described herein. Examples of BRD4 inhibitors include, without limitation, JQ1 ((6S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester), MS417 (methyl [(6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetate), or RVX-208 (2-[4-(2-Hydroxyethoxy)-3,5-dimethylphenyl]-5,7-dimethoxy-4(3H)-quinazolinone). In some embodiments, the BRD4 inhibitor is JQ1. Additional BRD4 inhibitors will be evident to one of skill in the art and can be found, for example, in PCT Publication WO 2014/154760 A1 and Vidler et al. J. Med. Chem. (2013) 56: 8073-8088.
Any small molecule that reduces or prevents expression of KDM4C (also referred to as JMJD2) or reduces or prevents activity of a protein encoded by KDM4C may be compatible with the compositions and methods described herein. In some embodiments, the KDM4C inhibitor is SD70 (N-(furan-2-yl(8-hydroxyquinolin-7-yl)methyl)isobutyramide) or caffeic acid. Additional KDM4C inhibitors will be evident to one of skill in the art and can be found, for example, in Leurs et al. Bioorg. & Med. Chem. Lett. (2012) 22(12): 5811-5813 and Hamada et al. Bioorg. & Med. Chem. Lett. (2009) 19: 2852-2855).
Any small molecule that reduces or prevents expression of KDM6B (also referred to as JMJD3) or reduces or prevents activity of a protein encoded by KDM6B may be compatible with the compositions and methods described herein. Examples of KDM6B inhibitors include, without limitation, GSK-J4 (ethyl 3-((6-(4,5-dihydro-1H-benzo[d]azepin-3(2H)-yl)-2-(pyridin-2-yl)pyrimidin-4-yl)amino)propanoate, monohydrochloride), GSK-J1 (N-[2-(2-Pyridinyl)-6-(1,2,4,5-tetrahydro-3H-3-benzazepin-3-yl)-4-pyrimidinyl]-β-alanine), and IOX1 (8-Hydroxy-5-quinolinecarboxylic acid; 8-Hydroxy-5-quinolinecarboxylic acid). In some embodiments, the KDM6B inhibitor is GSK-J4. Additional KDM4C inhibitors will be evident to one of skill in the art.
The combination of two or more inhibitors may comprise two or more protein inhibitors, two or more CRISPR guide sequences, two or more shRNAs, or two or more small molecule inhibitors. In some embodiments, the two or more inhibitors are different types of inhibitors (e.g., proteins, nucleic acids, small molecules). In some embodiments, the combination comprises a protein inhibitor and one or more additional inhibitors (e.g., CRISPR guide sequences, shRNAs, and/or small molecule inhibitors). In other embodiments, the combination comprises a CRISPR guide sequence and one or more additional inhibitors (e.g., proteins, shRNAs, and/or small molecule inhibitors). In other embodiments, the combination comprises a shRNA and one or more additional inhibitors (e.g., proteins, CRISPR guide sequences, and/or small molecule inhibitors). In other embodiments, the combination comprises a small molecule inhibitor and one or more additional inhibitors (e.g., proteins, shRNAs, and/or shRNAs).
The methods and compositions described herein may be useful for reducing proliferation of a cell, such as a cancer cell or other cell for which reduced proliferation is desired. In some embodiments, contacting a cell with a combination of two or more inhibitors of epigenetic genes (e.g., combinations of inhibitors targeting epigenetic genes presented in Table 2) partially or completely reduces proliferation of the cell. In some embodiments, contacting a cell with a combination of two or more inhibitors of epigenetic genes partially or completely reduces proliferation of the cell as compared to a cell that is not contacted with the combination of inhibitors. In some embodiments, contacting cells with a combination of two or more inhibitors of epigenetic genes reduces proliferation of the cells by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or at least 65% as compared to cells that were not contacted with the combination of inhibitors. In some embodiments, contacting a cell with a combination of two or more inhibitors of epigenetic genes partially or completely reduces proliferation of a cancer cell as compared to a non-cancer cell that is contacted with the combination of inhibitors. Cell proliferation may be assessed and quantified by any method known in the art, for example using cell viability assays, MTT assays, or BrdU cell proliferation assays.
Other aspects of the invention relate to methods and compositions for treating cancer in a subject. Cancer is a disease characterized by uncontrolled or aberrantly controlled cell proliferation and other malignant cellular properties. As used herein, the term “cancer” refers to any type of cancer known in the art, including without limitation, breast cancer, biliary tract cancer, bladder cancer, brain cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms, T-cell acute lymphoblastic leukemia/lymphoma, hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, AIDS-associated leukemias and adult T-cell leukemia/lymphoma, intraepithelial neoplasms, liver cancer, lung cancer, lymphomas, neuroblastomas, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, skin cancer, testicular cancer, thyroid cancer, and renal cancer. The cancer cell may be a cancer cell in vivo (i.e., in an organism), ex vivo (i.e., removed from an organism and maintained in vitro), or in vitro.
The methods involve administering to a subject a combination of two or more inhibitors of epigenetic genes in an effective amount. In some embodiments, the subject is a subject having, suspected of having, or at risk of developing cancer. In some embodiments, the subject is a mammalian subject, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. In some embodiments, the subject is a human subject, such as a patient. The human subject may be a pediatric or adult subject. Whether a subject is deemed “at risk” of having a cancer may be determined by a skilled practitioner.
As used herein “treating” includes ameliorating, curing, preventing it from becoming worse, slowing the rate of progression, or preventing the disorder from re-occurring (i.e., to prevent a relapse). An effective amount of a composition refers to an amount of the composition that results in a therapeutic effect. For example, in methods for treating cancer in a subject, an effective amount of a combination of inhibitors targeting epigenetic genes is any amount that provides an anti-cancer effect, such as reduces or prevents proliferation of a cancer cell or is cytotoxic towards a cancer cell. In some embodiments, the effective amount of an inhibitor targeting an epigenetic gene is reduced when an inhibitor is administered concomitantly or in combination with one or more additional inhibitors targeting epigenetic genes as compared to the effective amount of the inhibitor when administered in the absence of one or more additional inhibitors targeting epigenetic genes. In some embodiments, the inhibitor targeting an epigenetic gene does not reduce or prevent proliferation of a cancer cell when administered in the absence of one or more additional inhibitors targeting epigenetic genes.
Inhibitors targeting epigenetic genes or combinations of inhibitors targeting epigenetic genes (e.g., combinations of epigenetic genes presented in Table 2) may be administered to a subject using any method known in the art. In some embodiments, the inhibitors are administered by a topical, enteral, or parenteral route of administration. In some embodiments, the inhibitors are administered intravenously, intramuscularly, or subcutaneously.
Any of the inhibitors of epigenetic genes described herein may be administered to a subject, or delivered to or contacted with a cell by any methods known in the art. In some embodiments, the inhibitors of epigenetic genes are delivered to the cell by a nanoparticle, cell-permeating peptide, polymer, liposome, or recombinant expression vector. In other embodiments, the inhibitors of epigenetic genes are conjugated to one or more nanoparticle, cell-permeating peptide, and/or polymer. In other embodiments, the inhibitors of epigenetic genes are contained within a liposome.
Also provided are methods for identifying combinations of inhibitors of epigenetic genes that reduce or prevent proliferation of a cell or population of cells. As depicted in
Other methods are provided for identifying combinations of epigenetic genes that when inhibited reduce or prevent proliferation of a cell or population of cells. As depicted in
In some embodiments, one or more of the genes or inhibitors targeting an epigenetic gene associated with the invention is expressed in a recombinant expression vector. As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence or sequences may be inserted by restriction and ligation (e.g., using the CombiGEM method) or by recombination for transport between different genetic environments or for expression in a host cell (e.g., a cancer cell). Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes. In some embodiments, the vector is a lentiviral vector. In some embodiments, two or more genes or inhibitors targeting epigenetic genes are expressed on the same recombinant expression vector. In some embodiments, two or more genes or inhibitors targeting epigenetic genes are expressed on two or more recombinant expression vectors.
A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated or recombination sites at which an insert with compatible ends can be integrated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation or recombination such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein, red fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
As used herein, a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
When the nucleic acid molecule is expressed in a cell, a variety of transcription control sequences (e.g., promoter/enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule. In some embodiments, the promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, the promoter is a human ubiquitin C promoter (UBCp). In some embodiments, the promoter is a viral promoter. In some embodiments, the promoter is a human cytomegalovirus promoter (CMVp). In some embodiments, the promoter is a RNA polymerase III promoter. Examples of RNA polymerase III promoters include, without limitation, H1 promoter, U6 promoter, mouse U6 promoter, swine U6 promoter. In some embodiments, the promoter is a U6 promoter (U6p).
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5′ non-transcribed and 5′ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5′ non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5′ leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA). That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
A nucleic acid molecule associated with the invention can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, viral transduction, particle bombardment, etc. In some embodiments, the viral transduction is achieved using a lentivirus. Expressing the nucleic acid molecule may also be accomplished by integrating the nucleic acid molecule into the genome.
The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, particularly for the teachings referenced herein.
As shown in
In step 4, the pooled library of storage vectors then underwent a single-pot digestion using BbsI to open the vector between the CRISPR guide sequence and the barcode element to allow for the insertion of gRNA scaffold element in between the CRISPR guide sequence and the barcode element. Since the insert of gRNA scaffold element contains a separation site formed by the restriction recognition sites BamHI and EcoRI at the 3′ end of the scaffold element, the library of gRNA storage vectors can undergo iterative cloning steps to generate progressively more complex n-wise barcoded library encompassing multiple gRNA expression cassettes that can mediate combinatorial gene knockout, activation or repression using Cas9 nuclease or effectors, as shown in step 5. Briefly, the barcoded guide RNA library is digested using restriction enzyme complementary to the restriction recognition sites at the separation site (e.g., BamHI and EcoRI). Digestion allows for insertion of additional segments encoding a CRISPR guide sequence, scaffold sequence, separation site, and barcode element. Because the same sets of restriction enzymes can be used for the multiple rounds of cloning to build an (n)-wise library, one of the advantages of this strategy is that the (n+1)-wise library does not increase the set of restriction enzymes required.
To build a (n)-wise library of (m) gRNA members, it takes (n+2) rounds of cloning steps. An additional advantage of this strategy is that the same CRISPR guide libraries can be used to generate higher order complexity libraries.
As shown in
In step 2, the pooled synthesized oligonucleotides were ligated into a destination vector in a single-pot assembly. The destination vector may contain an promoter to drive expression of at least one of the CRISPR guide sequences. As shown in step 3, the vector was sequentially digested at each of the restriction recognition sites following each CRISPR guide sequence with different restriction enzymes, allowing for insertion of a scaffold element, and in some cases a promoter element to drive expression of a downstream CRISPR guide sequence. The method resulted in the generation of a barcoded combinatorial guide RNA library encoding multiple CRISPR guide sequences and scaffold sequences with a single barcode element for combinatorial gene knockout, activation or repression using Cas9 nuclease or effectors.
In step 2, the pooled synthesized oligonucleotides were ligated into a destination vector in a single-pot assembly. The destination vector may contain an promoter to drive expression of at least one of the CRISPR guide sequences. As shown in step 3, the vector was sequentially digested at each of the restriction recognition sites following each CRISPR guide sequence with different restriction enzymes, allowing for insertion of a scaffold element, and in some cases a promoter element to drive expression of a downstream CRISPR guide sequence. The method resulted in the generation of a barcoded combinatorial guide RNA library encoding multiple CRISPR guide sequences and scaffold sequences with a single barcode element for combinatorial gene knockout, activation or repression using Cas9 nuclease or effectors.
Using the strategies depicted in
The CombiGEM-based DNA assembly method was used for the efficient and scalable assembly of barcoded combinatorial gRNA libraries. The libraries were delivered into human cells by lentiviruses in order to create genetically ultra-diverse cell populations harboring unique gRNA combinations that may be tracked via barcode sequencing in pooled assays. This strategy, termed CombiGEM-CRISPR, uses simple one-pot cloning steps to enable the scalable assembly of high-order combinatorial gRNA libraries, thus simplifying and accelerating the workflow towards systematic analysis of combinatorial gene functions.
To create the initial barcoded sgRNA library, an array of oligo pairs encoding a library of barcoded gRNA target sequences was first synthesized, annealed, and pooled in equal ratios for cloning downstream of a U6 promoter in the storage vector (
To evaluate the functionality of our lentiviral combinatorial gRNA expression system, gRNA combinations were constructed targeting sequences encoding green fluorescent protein (GFP) and red fluorescent protein (RFP) (Table 1). The combinatorial gene perturbation phenotypes were determined by using flow cytometry (
Diverse epigenetic modifications tend to act cooperatively to regulate gene expression patterns (Wang, et al. Nat. Genet. (2008) 40:897-903), and combinatorial epigenetic modulation is emerging as a promising strategy for effective cancer therapeutics (Dawson, et al. Cell (2012) 150: 12-27; Juergens, et al. Cancer Discov. (2011) 1: 598-607). Using the CombiGEM-CRISPR methods and compositions described herein, the combinatorial effects of epigenetic gene perturbations on anti-cancer phenotypes were systematically evaluated. A library was constructed containing 153 barcoded sgRNAs targeting a set of 50 epigenetic genes (3 sgRNAs per gene) and 3 control sgRNAs based on the GeCKOv2 library (Shalem, et al. Science (2014) 343:84-87)(Table 1).
Expression of these 50 epigenetic genes was evaluated in OVCAR8-ADR cells using qRT-PCR. A two-wise (153×153 sgRNAs=23,409 total combinations) pooled barcoded gRNA library was generated using the CombiGEM method. Lentiviral pools were produced to deliver the library into OVCAR8-ADR-Cas9 cells. Genomic DNA from the pooled cell populations was isolated for unbiased barcode amplification by polymerase chain reaction (PCR). The representation of individual barcoded combinations in the plasmid pools stored in Escherichia coli and also in the infected human cell pools was quantified using Illumina HiSeq sequencing (
To confirm the function of gRNAs to edit endogenous genes in OVCAR8-ADR-Cas9 cells, Surveyor assays were performed to estimate the cleavage efficiency at 8 randomly picked loci targeted by the gRNAs from the library. Indel generation efficiencies ranging from 1.9% to 26.2% were observed at day 12 post-infection (
Indel generation efficiency was also estimated by performing deep sequencing at targeted genomic loci. Large variations in the rates of generating indels were observed (i.e., 14 to 93%;
A pooled combinatorial genetic screen with OVCAR8-ADR-Cas9 cells was initiated to identify gRNA combinations that regulate cancer cell proliferation. A mathematical model was constructed to map out how relative changes in abundances of each library member within a population depend on various parameters (see Methods below;
In the experimental screen, to identify gRNA combinations that regulate cancer cell proliferation, the OVCAR8-ADR-Cas9 cell populations infected with the two-wise combinatorial gRNA library were cultured for 15 and 20 days, then genomic DNA was isolated from the cells for unbiased amplification and quantification of the integrated barcodes (
Hits from the screen were validated by evaluating the ability of the gRNA pair to inhibit the proliferation of OVCAR8-ADR-Cas9 cells (i.e., by ˜33% within 5 days) in individual (non-pooled) cell growth assays using the corresponding gRNA pairs delivered via lentiviruses (
Many gRNAs targeting epigenetic genes exhibited stronger anti-proliferative effects when used in combination with other epigenetic-gene-targeting gRNAs than when used in combination with control gRNAs (
Off-target activity of the gRNAs was assessed by deep sequencing, which revealed a low indel generation rate (i.e., 0.15 to 0.38%) at all exonic off-target genomic loci computationally predicted by the CRISPR design and CCTop tools for the two gRNAs (
The gRNA pairs were confirmed with validation assays (
The methods described herein allow for the identification of novel epigenetic target gene pairs that inhibit cancer cell proliferation and the potential development of synergistic drug therapies. The methods also expand the utility of CRISPR-Cas9-based systems for performing systemic multiplexed genetic perturbation screens in a high-throughput capacity.
These methods can also help identify new areas for biological inquiry, such as studies into the mechanisms that underlie observed phenotypes. For example, gene expression patterns were evaluated in cell populations infected with lentiviruses encoding gRNAs targeting both KDM4C and BRD4, or KDM6B and BRD4 (
The vectors were constructed using standard molecular cloning techniques, including restriction enzyme digestion, ligation, PCR, and Gibson assembly (Table 3). Custom oligonucleotides were purchased from Integrated DNA Technologies. The vector constructs were transformed into E. coli strain DH5a, and 50 μg/ml of carbenicillin (Teknova) was used to isolate colonies harboring the constructs. DNA was extracted and purified using Plasmid Mini or Midi Kits (Qiagen). Sequences of the vector constructs were verified with Genewiz's DNA sequencing service.
To generate a lentiviral vector encoding an shRNA that targeted a specific gene, oligonucleotide pairs harboring the sense and antisense sequences were synthesized, annealed, and cloned in the AgeI- and EcoRI-digested pLKO.1 vector29 (Addgene plasmid #10879) by ligation. The shRNA sense and antisense sequences were designed and constructed based on the siRNA Selection Program (sirna.wi.mit.edu/) (Table 4).
To generate the pAWp30 lentiviral expression vector encoding Cas9 protein and Zeocin resistance as the selection marker, the EFS promoter and Cas9 sequences were amplified from Addgene plasmid #49535, while the Zeocin sequence was amplified from Addgene plasmid #25736, by PCR using Phusion DNA polymerase (New England Biolabs). The PCR products were cloned into the pAWp11 lentiviral vector backbone using Gibson Assembly Master Mix (New England Biolabs).
To construct a storage vector containing U6 promoter (U6p)-driven expression of sgRNA that targeted a specific gene, oligo pairs with the 20 bp sgRNA target sequences were synthesized, annealed, and cloned in the BbsI-digested pAWp28 vector using T4 ligase (New England Biolabs). To construct a lentiviral vector for U6p-driven expression of single or combinatorial sgRNA(s), U6p-sgRNA expression cassettes were prepared from digestion of the storage vector with BglII and MfeI enzymes (Thermo Scientific), and inserted into the pAWp12 vector backbone or the single sgRNA expression vector, respectively, using ligation via the compatible sticky ends generated by digestion of the vector with BamHI and EcoRI enzymes (Thermo Scientific). To express the sgRNAs together with the dual RFP and GFP fluorescent protein reporters, the U6p-driven sgRNA expression cassettes were inserted into the pAWp9, instead of pAWp12, lentiviral vector backbone using the same strategy described above. The pAWp9 vector was modified from the pAWp7 vector backbone by introducing unique BamHI and EcoRI sites into the vector to enable the insertion of the U6p-sgRNA expression cassettes.
Assembly of the Barcoded Combinatorial sgRNA Library Pool
An array of 153 oligo pairs (Oligo F-(x) and Oligo R-(x), where x=1 to 153) harboring the barcoded sgRNA sequences were synthesized, and annealed to generate double-stranded inserts harboring the 20 bp sgRNA target sequences, two BbsI restriction sites, 8 bp barcodes unique to each sgRNA while differed from each other by at least two bases, and 5′ overhangs at their ends. To generate the pooled storage vector library, the 153 annealed inserts were mixed at equal ratios and cloned in the pAWp28 storage vector (digested with BbsI and MfeI) via a single pot of ligation reaction via their compatible ends. To build the barcoded sgRNA library, another one-pot ligation reaction was performed with the pooled storage vector library digested with BbsI, and an insert containing the sgRNA scaffold sequence, BamHI and EcoRI restriction sites, and 5′ overhangs at their ends that was prepared via synthesis and annealing of an oligo pair S1 and S2. The pooled storage vector and the barcoded sgRNA libraries were both prepared in Endura competent cells (Lucigen) and purified by the Plasmid Midi kit (Qiagen).
Pooled lentiviral vector libraries harboring single or combinatorial gRNA(s) were constructed with same strategy as for the generation of single and combinatorial sgRNA constructs described above, except that the assembly was performed with pooled inserts and vectors, instead of individual ones. Briefly, the pooled U6p-sgRNA inserts were generated by a single-pot digestion of the pooled storage vector library with BglII and MfeI. The destination lentiviral vector (pAWp12) was digested with BamHI and EcoRI. The digested inserts and vectors were ligated via their compatible ends (i.e., BamHI+BglII & EcoRI+MfeI) to create the pooled one-wise sgRNA library (153 sgRNAs) in lentiviral vector. The one-wise sgRNA vector library was digested again with BamHI and EcoRI, and ligated with the same U6p-sgRNA insert pool to assemble the two-wise sgRNA library (153×153 sgRNAs=23,409 total combinations). After the pooled assembly steps, the sgRNAs were localized to one end of the vector construct and their respective barcodes were concatenated at the other end. The lentiviral sgRNA library pools were prepared in XL10-Gold ultracompetent cells (Agilent Technologies) and purified by Plasmid Midi kit (Qiagen).
HEK293T cells were obtained from ATC, and were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1× antibiotic-antimycotic (Life Technologies) at 37° C. with 5% CO2. OVCAR8-ADR cells were a gift from T. Ochiya (Japanese National Cancer Center Research Institute, Japan). The identity of the OVCAR8-ADR cells was authenticated (Genetica DNA Laboratories). OVCAR8-ADR cells stably expressing Cas9 protein (OVCAR-ADR-Cas9) were generated by lentiviral infection of OVCAR8-ADR cells with the pAWp30 vector and selected for three weeks in the presence of 200m/ml Zeocin (Life Technologies). OVCAR8-ADR and OVCAR8-ADR-Cas9 cells were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum and 1× antibiotic-antimycotic at 37° C. with 5% CO2. For drug treatment, SD70 (Xcessbio #M60194), GSK-J4 (Cayman Chemical #12073), and/or (+)-JQ1 (Cayman Chemical #11187) were used to treat OVCAR8-ADR cells at indicated drug doses prior to the cell viability assays.
Lentiviruses were produced and packaged in HEK283T cells in 6-well format. HEK293T cells were maintained at ˜70% confluency before transfection. FuGENE HD transfection reagents (Promega) were mixed with 0.5 μg of lentiviral vector, 1 μg of pCMV-dR8.2-dvpr vector, and 0.5 μg of pCMV-VSV-G vector in 100 μl of OptiMEM medium (Life Technologies), and were incubated for 15 minutes at room temperature before adding to cell culture. Culture medium was replaced the next day. Supernatant containing newly produced viruses were collected at 48-hour and 96-hour post-transfection, and filtered through a 0.45 μm polyethersulfone membrane (Pall). 500 μl of filtered viral supernatant was used to infect 250,000 cells in the presence of 8 μg/ml polybrene (Sigma) overnight for transduction with individual vector constructs. For pooled lentiviral library production used in the screens, lentivirus production and transduction were scaled up using the same experimental procedures. Filtered viral supernatant was concentrated using Amicon Ultra Centrifugal Filter Unit (Millipore). Cells were infected in the presence of 8 μg/ml polybrene at a multiplicity of infection of 0.3 to 0.5 to ensure single copy integration in most cells, which corresponded to an infection efficiency of 30-40%. The total number of cells used in the screening was approximately 300-fold more than the library sizes in order to maintain library coverage and reduce any spurious effects due to random lentiviral integration into the genome. Cell culture medium was replaced the next day after infection and cultured for indicated time periods prior to experiments.
To prepare samples from cultured cells for barcode sequencing, genomic DNA was extracted and prepared using DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's protocol. For the barcoded sgRNA plasmid libraries, plasmid DNA transformed into E. coli was extracted using the Plasmid Midi Kit (Qiagen). DNA concentrations were determined using Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies).
A ˜360 bp fragment containing unique barcode representing each combination within the pooled vector and infected cell libraries was PCR amplified from the plasmid/genomic DNA samples using Kapa Hotstart Ready Mix (Kapa Biosystems). For plasmid DNA, 1 ng of DNA template was added for a 25 μl PCR reaction. For genomic DNA, 800 ng of DNA was added for a 50-μl PCR reaction and a total of 64 PCR reactions were performed for each genomic DNA sample to ensure that the number of cell genomes being amplified was more than 100 times the library size. Moreover, the PCR parameters were optimized to ensure that PCR amplification steps were maintained in the exponential phase to avoid PCR bias. The Illumina anchor sequences and an 8 base-pair indexing barcode were added during the PCR for multiplexed sequencing. The primer pair sequences used to amplify barcode sequence were: 5′-AATGATACGGCGACCACCGAGATCTACACGGATCCGCAACGGAATTC-3′ (SEQ ID NO: 1) and 5′CAAGCAGAAGACGGCATACGAGATNNNNNNNNGGTTGCGTCAGCAAACACAG-3′ (SEQ ID NO: 2), where NNNNNNNN indicates a specific indexing barcode assigned for each experimental sample.
The PCR products containing the barcode sequences were then purified based on fragment size by running on a 1.5% agarose gel and further extracted using the QIAquick Gel Extraction Kit (Qiagen). The PCR product concentrations were determined by quantitative PCR using KAPA SYBR Fast qPCR Master Mix (Kapa Biosystems) and the Illumina Library Quantification Kit (Kapa Biosystems). The forward and reverse primer used for quantitative PCR were 5′-AATGATACGGCGACCACCGA-3′ (SEQ ID NO: 3) and 5′-CAAGCAGAAGACGGCATACGA-3′ (SEQ ID NO: 4), respectively. The PCR products from different samples were then pooled at a desired ratio for multiplexed sample sequencing and loaded on the Illumina HiSeq system with CombiGEM barcode primer (5′-CCACCGAGATCTACACGGATCCGCAACGGAATTC-3′ (SEQ ID NO: 5)) and indexing barcode primer (5′-GTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACC-3′ (SEQ ID NO: 6)).
Barcode reads for each sgRNA combinations were processed from the Illumina sequencing data. Barcode reads representing each combination were normalized per million reads for each sample categorized by the indexing barcodes. As measures of cell proliferation, barcode count ratios of normalized barcode reads comparing day 20 against day 15 groups were calculated as fold changes. Pro-proliferation and anti-proliferation phenotypes had fold changes of normalized barcode reads of >1 and <1 respectively, while no phenotypic change resulted in a fold change=1. Barcodes that gave less than ˜100 absolute reads in the day 15 group were filtered out to improve data reliability. The fold changes of the different possible orders of each same sgRNA combination were averaged, and high consistency in the fold-changes was observed (i.e., coefficient of variation (CV)<0.2 and <0.4 for over 82% and 95% of the combinations, respectively (
The MTT colorimetric assay was performed to assess cell viability. For each 96 well, 100 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (Sigma) was added to the cell cultures. Cells were incubated for 3 hours at 37° C. with 5% CO2. Viable cells convert the soluble MTT salt to insoluble blue formazan crystals. Formazan crystals were dissolved with 100 μl of solubilization buffer at 37° C. Absorbance reading at an optical density (OD) of 570 nm and 650 nm (reference) were measured using a Synergy H1 Microplate Reader (BioTek).
The Bliss independence (BI) model (Bliss Ann. Appl. Biol. (1939) 26: 585-615) and the Highest Single Agent (HSA) model (Borisy, et al. PNAS (2003) 100: 7977-7982) are commonly used methods to evaluate synergy between drug combinations.
Based on the BI model, the expected effect (EExp) is given by:
where EA is the growth inhibition effect observed at a certain concentration of drug A alone, and EB is the growth inhibition effect observed at a certain concentration of drug B alone. EObs is the observed growth inhibition effect for the drug combination (A+B), each at the same concentration as in EA and EB, respectively. Each effect is expressed as a fractional inhibition between 0 and 1. When EObs−EExp>0, the two drugs are considered to be interacting synergistically.
The HSA model is similar to the BI model except, according to the HSA model, EExp is equal to the larger of the growth inhibition effect produced by the combination's single drug agents (EA or EB) at the same concentrations as in the drug combination (A+B).
Two drugs were considered to be synergistic if EObs−EExp>0.1 (i.e. >10% of excess inhibition over the predicted BI and HSA models in
Cells were collected at 4-day and 8-day post infection. Samples were washed and resuspended in 1×PBS supplemented with 2% fetal bovine serum. To remove any clumps of cells, the resuspended cells were passed through cell strainers before loading onto the LSRII Fortessa flow cytometer (Becton Dickinson). At least 20,000 events were acquired per sample. Proper laser sets and filters were selected based on cell samples. Forward scatter and side scatter were used to identify appropriate cell populations. Data were analyzed using manufacturer's build-in software.
Cultured cell were directly observed under an inverted fluorescent microscope (Zeiss) three days post-lentiviral infection. Images were captured using the Zeiss built-in software.
Cells were lysed in 2×RIPA buffer supplemented with protease inhibitors. Lysates were homogenized using a pestle motor mixer (Agros) for 30 seconds, and then centrifuged at 15,000 rpm for 15 min at 4° C. Supernatants were quantified using the BCA assay (Thermo Scientific). Protein was denatured at 99° C. for 5 minutes before gel electrophoresis on a 4-15% polyacrylamide gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes at 80V for 2 hours at 4° C. Primary antibodies used were: anti-BRD4 (1:2,000, Cell Signaling #13440), anti-KDM4C (1:1,000, Abcam ab85454) anti-KDM6B (1:1,000, Abcam ab85392), and anti-beta-actin (1:4,000, Abcam ab6276). Secondary antibodies used were: HRP-linked anti-rabbit IgG (1:2,000, Cell Signaling #7074), and HRP-linked anti-mouse IgG (1:4,000, Cell Signaling #7076). Membranes were developed by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and imaged using a ChemiDoc Touch imaging system (BioRad).
The Surveyor assay was carried out to evaluate DNA cleavage efficiency. Genomic DNA was extracted from cell cultures using QuickExtract DNA extraction solution (Epicentre) according to the manufacturer's protocol. Amplicons harboring the targeted loci were generated by PCR using Phusion DNA polymerase and primers listed in Table 5. About 200 ng of the PCR amplicons were denatured, self-annealed, and incubated with 1.5 μl of Surveyor Nuclease (Transgenomic) at 42° C. for 30 minutes. The samples were then analyzed on a 2% agarose gel. The DNA band intensities were quantified using ImageJ software, and the indel occurrence was estimated with the following formula (Ran et al. Nat. Protoc. (2013) 8:2281-2308):
Indel (%)=100×(1−square root of (1−fcut)),
with fcut=(b+c)/(a+b+c), where a is the band intensity for the uncleaved PCR amplicon while b and c are the intensities for each cleaved band. The expected uncleaved and cleaved bands for the targeted alleles are listed in
Sanger sequencing was performed to analyze the genome modifications generated by the expression of combinatorial sgRNAs. Cells infected with the combinatorial sgRNA constructs were cultured for 12 days, and re-plated in 96-well plates as single cells by serial dilution of the cultures. Genomic DNA was extracted from the isolated single cell-expanded clones after culturing for 5 to 21 days using QuickExtract DNA extraction solution, and amplicons harboring the targeted alleles were prepared by PCR as described above. The PCR amplicons were cloned into a TOPO vector using TA Cloning Kit (Life Technologies) according to the manufacturer's protocol, and the nucleotide mutations, insertions, and deletions were identified using Sanger sequencing.
ATATACTATATATATAGTGGTCTGGTCTTGTGAACTTGGACATCA
ATATACTATATATATAGTGGTCTGGTCTTGTGAACTTGGACATCA
ACTTAGTGGATCTTCAAAGGGGCATGATACACAAGGGGAAACCAG
ACTTAGTGGATCTTCAAAGGGGCATGATACACAAGGGGAAACCAG
AA--------------GTGGTCTGGTCTTGTGAACTTGGACATCA
GCCCCCGTGGATCTTCAAAGGGGCATGATACACAAGGGGAAACCA
GCCCCCGTGGATCTTCAAAGGGGCATGATACACAAGGGGAAACCA
AAGCCCTGGATCTTCAAAGGGGCATGATACACACAAGGGGAAACC
AAGCCCTGGATCTTCAAAGGGGCATGATACACACAAGGGGAAACC
ACGCCGTGGATCTTCAAAGGGGCATGATACACACAAGGGGAAACC
TATAATATATTCATAGTGGTCTGGTCTTGTGAACTTGGACATCAC
TATAATATATTCATAGTGGTCTGGTCTTGTGAACTTGGACATCAC
GTAGTGAATCTGAATAGTGGTCTGGTCTTGTGAACTTGGACATCA
TTCAAAAGGATCTTCAAAGGGGCATGATACACACAAGGGGAAACC
TTCAAAAGGATCTTCAAAGGGGCATGATACACACAAGGGGAAACC
RNA Extraction and Quantitative RT-PCR (qRT-PCR)
RNA were extracted from cells using TRIzol Plus RNA Purification Kit (Life Technologies) according to manufacturer's protocol and treated with PureLink on-column DNase kit (Life Technologies). RNA quality and concentration was determined using NanoDrop Spectrophotometer. RNA samples were reverse-transcribed using SuperScript III Reverse Transcriptase (Life Technologies), Random Primer Mix (New England Biolabs) and RNAse OUT (Invitrogen). To evaluate gene expression level, quantitative PCR was conducted using SYBR FAST qPCR MasterMix (KAPA) on the LightCycler480 system (Roche). Data was quantified and analyzed using LifeCyler480 SW 1.1 build-in software. PCR primers were designed and evaluated using PrimerBlast (NCBI). Primer sequences are listed in Table 7.
RNA was extracted from cells using TRIzol Plus RNA Purification Kit (Life Technologies) according to the manufacturer's protocol and treated with PureLink on-column DNase kit (Life Technologies). RNA quality and concentration was determined using NanoDrop Spectrophotometer. RNA samples were reverse-transcribed using SuperScript III Reverse Transcriptase (Life Technologies), Random Primer Mix (New England Biolabs) and RNAse OUT (Invitrogen). Sequencing libraries were prepared using the Illumina Library Prep Kit, starting with an input amount of 1 μg total RNA and following the manufacturer's recommendations. After PCR amplification, the libraries were size-selected to 300+/−25 bp on a 2% agarose gel (E-Gel EX, Invitrogen) and submitted to single-end sequencing on an Illumina HiSeq 2000 instrument. RNA-Seq experiments were performed in two biological replicates.
Raw single-end reads of the cDNA fragments were aligned to the human transcriptome (RefSeq, hg19) using TopHat2 (Kim, et al. Genome Biol (2013) 14:R36) and Bowtie (Langmead, et al. Genome Biol (2009) 10:R25). Differentially expressed genes between samples were called using Cuffdiff2 (Trapnell, et al. Nat Biotechnol (2013) 31:46-53) with the bias correction option, masking reads mapping to mitochondrial and ribosomal RNA transcripts. Genes were called differentially expressed if they met a minimum of 0.1 fragments per kilobase per million reads (FPKM) in at least one of the conditions tested, the absolute log2-fold-change was at least 0.5, and the P-value after multiple hypothesis correction (Q-value) was less than 0.05. Gene set enrichment analysis was performed using MSigDB database (broadinstitute.org/gsea/index.jsp) (Subramanian, et al. Proc Natl Acad Sci USA (2005) 102:15545-50).
To estimate how different parameters affect results in our pooled screens, we simulated cell proliferation in a mixed cell population harboring gRNA combinations that exhibit different growth rates. At a given time (t), the total cell count (CN) in the population is represented by the summation of individual cell counts containing different combinations (Ci; 1 to N, where N is the total number of combinations), such that CN(t)=Σi=1NCi(t).
For each individual gRNA combination, cell growth is represented by Eq. (1). Based on an exponential cell growth model, cells with each gRNA combination consist of two populations: one with a modified growth rate (km) due to gene disruption by the CRISPR-Cas9 system, and the other (unmodified cells) with the wild-type growth rate (kwt). The former population is defined as a fraction of cells, p, which is limited by the cleavage efficiency of the CRISPR-Cas9 system. For simplicity, we assumed that p was constant throughout the duration of the assay.
where p represents the fraction of mutated cells with a modified growth rate, and C0 represents the initial number of cells carrying the same barcoded gRNA combination. The cell growth rate (k) is evaluated from the cell's doubling time (Tdoubling) following Eq. 2. The doubling time for wild-type OVCAR8-ADR-Cas9 cells was experimentally determined to be ˜24 hours (data not shown).
For simplicity in modeling, we segregated the total cell population into three sub-populations with different growth phenotypes as described in Eq. (3).
where Ci,slow(t) and Ci,fast(t) represent the average growth profiles of cells with anti-proliferative gRNAs and pro-proliferative gRNAs, respectively, which are determined by Eq. (1). At the start of the experiment, the percentages of the overall population that behave as wild type or that contain anti-proliferative gRNAs and pro-proliferative gRNAs are represented by fwt, fs and ff, respectively.
Based on this mixed cell growth model, we modeled the relative frequency (R. F) of a pro-proliferative gRNA's and an anti-proliferative gRNA's representation in the whole population. The relative frequency is defined as the barcode abundance at a given time compared to the initial time point
The total number of combinations in a pool, N and the initial number of cells, C0, do not impact the relative frequency results. After running the simulation with defined parameters, we observed enrichment and depletion of a pro-proliferative gRNA and anti-proliferative gRNA in the population, respectively (
Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or,” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
This application is a divisional of U.S. application Ser. No. 15/521,931, filed Apr. 26, 2017, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2015/058304, filed Oct. 30, 2015, which was published under PCT Article 21(2) in English, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/073,126, filed Oct. 31, 2014 and U.S. provisional application No. 62/166,302, filed May 26, 2015, each of which is incorporated by reference herein in its entirety.
This invention was made with government funding support under Grant No. OD008435 awarded by National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62073126 | Oct 2014 | US | |
| 62166302 | May 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15521931 | Apr 2017 | US |
| Child | 17338027 | US |
