Patent Application
20220228154
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5051.881CT2.ST25.txt, 16,634 bytes in size, generated on Jan. 7, 2022, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is incorporated by reference into the specification for its disclosures.
The invention relates to the use of CRISPR nucleic acids to screen for essential and non-essential genes and expendable genomic islands in bacteria, archaea, algae and/or yeast, to kill bacteria, archaea, algae and/or yeast, to identify the phenotype of a gene or genes, and/or to screen for reduced genome size and/or a gene deletion in bacteria, archaea, algae and/or yeast.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), in combination with associated sequences (cas), constitute the CRISPR-Cas system, which confers adaptive immunity in many bacteria. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and phages, as novel “spacers.”
CRISPR-Cas systems consist of arrays of short DNA repeats interspaced by hypervariable sequences, flanked by cas genes, that provide adaptive immunity against invasive genetic elements such as phage and plasmids, through sequence-specific targeting and interference (Barrangou et al. 2007. Science. 315:1709-1712; Brouns et al. 2008. Science 321:960-4; Horvath and Barrangou. 2010. Science. 327:167-70; Marraffini and Sontheimer. 2008. Science. 322:1843-1845; Bhaya et al. 2011. Annu. Rev. Genet. 45:273-297; Terns and Terns. 2011. Curr. Opin. Microbiol. 14:321-327; Westra et al. 2012. Annu. Rev. Genet. 46:311-339; Barrangou R. 2013. RNA. 4:267-278). Typically, invasive DNA sequences are acquired as novel “spacers” (Barrangou et al. 2007. Science. 315:1709-1712), each paired with a CRISPR repeat and inserted as a novel repeat-spacer unit in the CRISPR locus. Subsequently, the repeat-spacer array is transcribed as a long pre-CRISPR RNA (pre-crRNA) (Brouns et al. 2008. Science 321:960-4), which is processed into small interfering CRISPR RNAs (crRNAs) that drive sequence-specific recognition. Specifically, crRNAs guide nucleases towards complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonucleases (Garneau et al. 2010. Nature. 468:67-71; Haurwitz et al. 2010. Science. 329:1355-1358; Sapranauskas et al. 2011. Nucleic Acid Res. 39:9275-9282; Jinek et al. 2012. Science. 337:816-821; Gasiunas et al. 2012. Proc. Natl. Acad. Sci. 109:E2579-E2586; Magadan et al. 2012. PLoS One. 7:e40913; Karvelis et al. 2013. RNA Biol. 10:841-851). These widespread systems occur in nearly half of bacteria (˜46%) and the large majority of archaea (˜90%).
In general terms, there are two main classes (Makarova et al. Nat Rev Microbiol. 13:722-736 (2015)) of CRISPR-Cas systems, which encompass five major types and 16 different subtypes based on cas gene content, cas operon architecture, Cas protein sequences, and process steps (Makarova et al. Biol Direct. 6:38 (2011); Makarova and Koonin Methods Mol Biol. 1311:47-75 (2015); Barrangou, R. Genome Biology 16:247 (2015)). In types I and III, the specialized Cas endonucleases process the pre-crRNAs, which then assemble into a large multi-Cas protein complex capable of recognizing and cleaving nucleic acids complementary to the crRNA. Type I systems are the most frequent and widespread systems, which target DNA in a Cascade-driven and PAM-dependent manner, destroying target nucleic acids by using the signature protein Cas3. A different process is involved in Type II CRISPR-Cas systems. Here, the pre-CRNAs are processed by a mechanism in which a trans-activating crRNA (tracrRNA) hybridizes to repeat regions of the crRNA. The hybridized crRNA-tracrRNA are cleaved by RNase III and following a second event that removes the 5′ end of each spacer, mature crRNAs are produced that remain associated with the both the tracrRNA and Cas9. The mature complex then locates a target dsDNA sequence (‘protospacer’ sequence) that is complementary to the spacer sequence in the complex and cuts both strands. Target recognition and cleavage by the complex in the type II system not only requires a sequence that is complementary between the spacer sequence on the crRNA-tracrRNA complex and the target ‘protospacer’ sequence but also requires a protospacer adjacent motif (PAM) sequence located at the 3′ end of the protospacer sequence.
One aspect of the invention provides a method of screening a population of bacterial cells for essential genes, non-essential genes, and/or expendable genomic islands, comprising: introducing into said population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial cells of said population, thereby producing a population of transformed bacterial cells; determining the presence or absence of a deletion in the population of transformed bacterial, archaeal, algal or yeast cells, wherein the presence of a deletion in the population of transformed bacterial, archaeal or yeast cells means that the target region is comprised within a non-essential gene and/or an expendable genomic island, and the absence of a deletion in the population of transformed bacterial, archaeal, algal or yeast cells means that the target region is comprised within an essential gene. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
A second aspect of the invention provides a method of screening a population of bacterial, archaeal, algal or yeast cells for essential genes, non-essential genes, and/or expendable genomic islands, comprising: introducing into the population of bacterial, archaeal, algal or yeast cells (a) a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (chromosomal and/or plasmid) of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, thereby producing a population of transformed bacterial, archaeal, algal or yeast cells; and determining the presence or absence of a deletion in the population of transformed bacterial, archaeal, algal or yeast cells, wherein the presence of a deletion in the population of transformed bacterial, archaeal or yeast cells means that the target region is comprised within a non-essential gene and/or an expendable genomic island, and the absence of a deletion in the population of transformed bacterial, archaeal, algal or yeast cells means that the target region is comprised within an essential gene.
A third aspect of the invention provides a method of killing one or more bacterial cells within a population of bacterial cells, comprising: introducing into the population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial cells of said population, thereby killing one or more bacterial cells that comprise the target region within the population of bacterial cells. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
A fourth aspect of the invention provides a method of killing one or more cells within a population of bacterial, archaeal, algal or yeast cells, comprising: introducing into the population of bacterial, archaeal, algal or yeast cells (a) a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (chromosomal and/or plasmid) of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, thereby killing one or more cells within a population of bacterial, archaeal, algal or yeast cells that comprise the target region in their genome.
A fifth aspect of the invention provides a method of identifying a phenotype associated with a bacterial gene, comprising: introducing into a population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of the at least one repeat-spacer sequence and repeat-spacer-repeat sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial cells of said population, wherein the target region comprises at least a portion of an open reading frame encoding a polypeptide or functional nucleic acid, thereby killing the cells comprising the target region and producing a population of transformed bacterial cells without the target region; and analyzing the phenotype of the population. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
A sixth aspect of the invention provides a method of identifying a phenotype of a bacterial, archaeal, algal or yeast gene, comprising: introducing into a population of bacterial, archaeal, algal or yeast cells (a) a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (chromosomal and/or plasmid) of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, thereby killing the bacterial, archaeal or yeast cells comprising the target region and producing a population of transformed bacterial, archaeal, algal or yeast cells without the target region; and analyzing the phenotype of the population of transformed bacterial, archaeal, algal or yeast cells, and/or (i) growing individual bacterial, archaeal, algal or yeast colonies from the population of transformed bacterial, archaeal, algal or yeast cells and (ii) analyzing the phenotype of the individual colonies.
A seventh aspect of the invention provides a method of selecting one or more bacterial cells having a reduced genome size from a population of bacterial cells, comprising: introducing into a population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population, wherein the cells comprising the target region are killed, thereby selecting one or more bacterial cells without the target region and having a reduced genome size from the population of bacterial cells. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
An eighth aspect of the invention provides a method of selecting one or more bacterial cells having a reduced genome size from a population of bacterial cells, comprising: introducing into a population of bacterial cells (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial cells or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and (b) a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population, wherein the target region is located between the one or more heterologous nucleic acid constructs introduced into the genome and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, and cells comprising the target region are killed, thereby selecting one or more bacterial cells without the target region and having a reduced genome size from the population of transgenic bacterial cells. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
A ninth aspect of the invention provides a method of selecting one or more bacterial, archaeal, algal or yeast cells having a reduced the genome size from a population of bacterial, archaeal, algal or yeast cells, comprising: introducing into a population of bacterial, archaeal, algal or yeast cells (a) a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of the at least one repeat-spacer sequence and the at least one repeat-spacer-repeat sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (chromosomal and/or plasmid) of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein cells comprising the target region are killed, thereby selecting one or more bacterial, archaeal, algal or yeast cells without the target region and having a reduced genome size from the population of bacterial, archaeal, algal or yeast cells.
A tenth aspect of the invention provides a method of selecting one or more bacterial, archaeal, algal or yeast cells having a reduced the genome size from a population of bacterial, archaeal or yeast cells, comprising: introducing into a population of bacterial, archaeal, algal or yeast cells: (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial, archaeal, algal or yeast cells, or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial, archaeal, algal or yeast cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and (b)(i) a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid, (ii) a heterologous nucleic acid construct comprising a CRISPR array comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of the at least one repeat-spacer sequence and the at least one repeat-spacer-repeat sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (chromosomal and/or plasmid) of one or more bacterial, archaeal, algal or yeast cells of said population, and (iii) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein the target region is located between the one or more heterologous nucleic acid constructs incorporated into the genome and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, and cells comprising the target region are killed, thereby selecting one or more bacterial, archaeal, algal or yeast cells without the target region and having a reduced genome size from the population of transgenic bacterial, archaeal, algal or yeast cells.
An eleventh aspect of the invention provides a method of identifying in a population of bacteria at least one isolate having a deletion in its genome, comprising: introducing into a population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population, wherein cells comprising the target region are killed, thereby producing a population of transformed bacterial cells without the target region; and growing individual bacterial colonies from the population of transformed bacterial cells, thereby identifying at least one isolate from the population of transformed bacteria having a deletion in its genome. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
A twelfth aspect of the invention provides a method of identifying in a population of bacteria at least one isolate having a deletion in its genome, comprising: introducing into the population of bacterial cells (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial cells or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and b) a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population, wherein the target region is located between the one or more heterologous nucleic acid constructs introduced into the genome and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, and cells comprising the target region are killed, thereby producing a population of transformed bacterial cells without the target region; and growing individual bacterial colonies from the population of transformed bacterial cells, thereby identifying at least one isolate from the population of bacteria having a deletion in its genome. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
A thirteenth aspect of the invention provides a method of identifying in a population of bacterial, archaeal, algal or yeast cells at least one isolate having a deletion in its genome, comprising: introducing into a population of bacterial, archaeal, algal or yeast cells: (a) a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (chromosomal and/or plasmid) of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein cells comprising the target region are killed, thereby producing a population of transformed bacterial, archaeal, algal or yeast cells without the target region; and growing individual bacterial, archaeal or yeast colonies from the population of transformed bacterial, archaeal, algal or yeast cells, thereby identifying at least one isolate from the population of transformed bacterial, archaeal, algal or yeast cells having a deletion in its genome.
A fourteenth aspect of the invention provides a method of identifying in a population of bacterial, archaeal, algal or yeast cells at least one isolate having a deletion in its genome, comprising: introducing into the population of bacterial, archaeal, algal or yeast cells (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial, archaeal, algal or yeast cells, or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial, archaeal, algal or yeast cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and (b)(i) a heterologous nucleic acid construct comprising a trans-encoded CRISPR (tracr) nucleic acid, (ii) a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (chromosomal and/or plasmid) of one or more bacterial, archaeal, algal or yeast cells of said population, and (iii) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein the target region is located between the one or more heterologous nucleic acid constructs incorporated into the genome and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, and cells comprising the target region are killed, thereby producing a population of transformed bacterial, archaeal, algal or yeast cells without the target region; and growing individual bacterial, archaeal or yeast colonies from the population of transformed bacterial, archaeal, algal or yeast cells, thereby identifying at least one isolate from the population having a deletion in its genome.
Further provided herein are expression cassettes, cells and kits comprising the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules and/or nucleotide sequences of the invention.
These and other aspects of the invention are set forth in more detail in the description of the invention below.
(A) The location and distribution of putative essential ORFs (top row), insertion sequences (2nd row) and putative genomic islands (3rd row). Potential targets for CRISPR-Cas (4th row) mediated deletion were identified by mapping transposable elements of various families within the genome of Streptococcus thermophilus LMD-9. Genetic organization of putative genomic islands and the protospacer/PAM combinations corresponding to each. (B) provides four panels. The upper panel provides Genomic island 1, encoding an oligopeptide transport system (SEQ ID NO:6). The second panel from the top provides Genomic island 2, containing the cell-envelope proteinase PrtS (SEQ ID NO:7). The third panel from the top provides Genomic island 3, encoding an ATPase copper-efflux protein (SEQ ID NO:8) and the bottom panel provides the. (E) The genomic island encoding selected ORFs including the Lac operon (from left to right, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11.
The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
The term “about,” as used herein when referring to a measurable value such as a dosage or time period and the like refers to variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.
As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y” and phrases such as “from about X to Y” mean “from about X to about Y.”
The term “comprise,” “comprises” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
As used herein, the transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
“Cas9 nuclease” refers to a large group of endonucleases that catalyze the double stranded DNA cleavage in the CRISPR Cas system. These polypeptides are well known in the art and many of their structures (sequences) are characterized (See, e.g., WO2013/176772; WO/2013/188638). The domains for catalyzing the cleavage of the double stranded DNA are the RuvC domain and the HNH domain. The RuvC domain is responsible for nicking the (−) strand and the HNH domain is responsible for nicking the (+) strand (See, e.g., Gasiunas et al. PNAS 109(36):E2579-E2586 (Sep. 4, 2012)).
As used herein, “chimeric” refers to a nucleic acid molecule or a polypeptide in which at least two components are derived from different sources (e.g., different organisms, different coding regions).
“Complement” as used herein can mean 100% complementarity or identity with the comparator nucleotide sequence or it can mean less than 100% complementarity (e.g., about 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%, and the like, complementarity).
The terms “complementary” or “complementarity,” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A.” Complementarity between two single-stranded molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
As used herein, “contact,” contacting,” “contacted,” and grammatical variations thereof, refers to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction (e.g., integration, transformation, screening, selecting, killing, identifying, amplifying, and the like). The methods and conditions for carrying out such reactions are well known in the art (See, e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M. R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
A “deletion” as used herein can comprise the loss or deletion of genetic material including but not limited to a deletion of a portion of a chromosome or a plasmid, a deletion of a gene or a portion of a gene from a chromosome or a plasmid. In some embodiments, a deletion can comprise one gene or more than one gene. In some embodiments, a deletion may also comprise the loss of non-protein-coding regions that may encode small non-coding RNAs. In some embodiments, a deletion can comprise the loss of an entire plasmid or of an entire mobile genetic element. In some embodiments, the loss of a mobile genetic element may be defined as, for example, an inability to replicate or persist.
In some embodiments, a phasmid of the invention may comprise a CRISPR array from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, and/or a Type V CRISPR-Cas system (see, Makarova et al. Nature Reviews Biotechnology 13:722736 (2015)).
Thus, in some embodiments, in addition to a Type I crRNA, a phasmid of the invention may comprise Type I polypeptides and/or Type I Cascade polypeptides (i.e., a Type I CRISPR-Cas system).
As used herein, “Type I polypeptide” refers to any of a Cas3 polypeptide, Cas3′ polypeptide, a Cas3″ polypeptide, fusion variants thereof, and any one or more of the Type I Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated complex for antiviral defense (“Cascade”) polypeptides. Thus, the term “Type I polypeptide” refers to the polypeptides that make up a Type I-A CRISPR-Cas system, a Type I-B CRISPR-Cas system, a Type I-C CRISPR-Cas system, a Type I-D CRISPR-Cas system, a Type I-E CRISPR-Cas system, a Type I-F CRISPR-Cas system, and/or a Type I-U CRISPR-Cas system. Each Type-I CRISPR-Cas system comprises at least one Cas3 polypeptide. Cas3 polypeptides generally comprise both a helicase domain and an HD domain. However, in some Type I CRISPR-Cas systems, the helicase and HD domain are found in separate polypeptides, Cas3′ and Cas3″. In particular, Cas3′ encodes the helicase domain whereas Cas3″ encodes the HD domain. Consequently, because both domains are required for Cas3 function, Type I subtypes either encode Cas3 (I-C, I-D, I-E, I-F, I-U) or Cas3′ and Cas3″ (I-A, I-B).
As used herein, “Type I Cascade polypeptides” refers to a complex of polypeptides involved in processing of pre-crRNAs and subsequent binding to the target DNA in Type I CRISPR-Cas systems. These polypeptides include, but are not limited to, the Cascade polypeptides of Type I subtypes I-A, I-B, I-C, I-D, I-E and I-F. Non-limiting examples of Type I-A Cascade polypeptides include Cas7 (Csa2), Cas8a1 (Csx13), Cas8a2 (Csx9), Cas5, Csa5, Cas6a, Cas3′ and/or a Cas3″. Non-limiting examples of Type I-B Cascade polypeptides include Cas6b, Cas8b (Csh1), Cas7 (Csh2) and/or Cas5. Non-limiting examples of Type I-C Cascade polypeptides include Cas5d, Cas8c (Csd1), and/or Cas7 (Csd2). Non-limiting examples of Type I-D Cascade polypeptides include Cas10d (Csc3), Csc2, Csc1, and/or Cas6d. Non-limiting examples of Type I-E Cascade polypeptides include Cse1 (CasA), Cse2 (CasB), Cas7 (CasC), Cas5 (CasD) and/or Cas6e (CasE). Non-limiting examples of Type I-F Cascade polypeptides include Cys1, Cys2, Cas7 (Cys3) and/or Cas6f (Csy4). Non-limiting examples of Type I-U Cascade polypeptides include Cas8c, Cas7, Cas5, Cas6 and/or Cas4.
In some embodiments, a phasmid of the invention may comprise may comprise a Type II CRISPR-Cas system in addition to a Type II crRNA. Type II CRISPR-Cas systems comprise three subtypes: Type II-A, Type II-B and Type II-C, each of which comprise the multidomain protein, Cas9, in addition to the adaptation polypeptides, Cas1, Cas2 and optionally, Csn2 and/or Cas4. Most Type II loci also encode a tracrRNA. Organisms comprising exemplary Type II CRISPR-Cas systems include Legionella pneumophila str. Paris, Streptococcus thermophilus CNRZ1066 and Neisseria lactamica 020-06.
In additional embodiments, a phasmid of the invention may comprise may comprise a Type III CRISPR-Cas system in addition to a Type III crRNA. Similar to Type I CRISPR-Cas systems, in Type III systems processing and interference is mediated by multiprotein CRISPR RNA (crRNA)-effector complexes (Makarova et al. Nature Reviews Biotechnology 13:722736 (2015))—“CASCADE” in Type I and “Csm” or “Cmr” in Type III. Thus, in some embodiments, a Type III CRISPR-Cas system can comprise a Csm complex (e.g., Type III-A Csm) and/or a Cmr complex (e.g., Type III-B Cmr), and optionally a Cas6 polypeptide. In representative embodiments, a Csm complex may comprise Cas10 (or Csm1), Csm2, Csm3, Csm4, Csm5, and Csm6 polypeptides and a Cmr complex may comprise Cmr1, Cas10 (or Cmr2), Cmr3, Cmr4, Cmr5, and Cmr6 polypeptides. In addition to the Csm complex or Cmr complex, a Type III CRISPR-Cas system may further comprise a Cas7 polypeptide. Four subtypes of a Type III CRISPR-Cas system have been characterized, III-A, III-B, III-D. In some embodiments, a Type III-A CRISPR-Cas system comprises Cas6, Cas10, Csm2, Cas7 (Csm3), Cas5 (Csm4), Cas7 (Csm5), and Csm6 polypeptides. In some embodiments, a Type III-B CRISPR-Cas system comprises Cas7 (Cmr1), Cas10, Cas5 (Cmr3), Cas7 (Cmr4), Cmr5, Cas6, and Cas7 (Cmr6) polypeptides. In some embodiments, a Type III-C CRISPR-Cas system comprises Cas7 (Cmr1), Cas7 (Cmr6), Cas10, Cas7 (Cmr4), Cmr5 and Cas5 (Cmr3), polypeptides. In some embodiments, a Type III-D CRISPR-Cas system comprises Cas10, Cas7 (Csm3), Cas5 (Csx10), Csm2, Cas7 (Csm3), and all1473 polypeptides.
In some embodiments, a phasmid of the invention may comprise a Type IV CRISPR-Cas system, in addition to a Type IV crRNA. Type IV CRISPR-Cas systems can comprise a Csf4 polypeptide (dinG) and/or a Csf1, Cas7 (Csf2) and/or Cas5 (csf3) polypeptide. (Makarova et al. Nature Reviews Microbiology 13:722-736 (2015)).
In some embodiments, a phasmid of the invention further comprises a Type V CRISPR-Cas system, in addition to a Type V crRNA. Type V CRISPR-Cas systems can comprise a Cpf1 polypeptide and/or a Cas1, Cas2 and/or Cas4 polypeptide. (Makarova et al. Nature Reviews Microbiology 13:722-736 (2015)).
A “fragment” or “portion” of a nucleotide sequence of the invention will be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or substantially identical (e.g., 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% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Thus, hybridizing to (or hybridizes to, and other grammatical variations thereof), for example, at least a portion of a target DNA (e.g., target region in the genome), refers to hybridization to a nucleotide sequence that is identical or substantially identical to a length of contiguous nucleotides of the target DNA. In some embodiments, a repeat of a repeat spacer sequence or a repeat-spacer-repeat sequence can comprise a fragment of a repeat sequence of a wild-type CRISPR locus or a repeat sequence of a synthetic CRISPR array, wherein the fragment of the repeat retains the function of a repeat in a CRISPR array of hybridizing with the tracr nucleic acid.
In some embodiments, the invention may comprise a functional fragment of a Cas9, Cas3, Cas3′, Cas3″, or Cpf1 nuclease. A Cas9 functional fragment retains one or more of the activities of a native Cas9 nuclease including, but not limited to, HNH nuclease activity, RuvC nuclease activity, DNA, RNA and/or PAM recognition and binding activities. A functional fragment of a Cas9 nuclease may be encoded by a fragment of a Cas9 polynucleotide. A Cas3, Cas3′ or Cas3″ functional fragment retains one or more of the activities of a native Cas9 nuclease including, but not limited to, nickase activity, exonuclease activity, DNA-binding, and/or RNA binding. A functional fragment of a Cas3, Cas3′ or Cas3″ nuclease may be encoded by a fragment of a Cas3, Cas3′ or Cas3″ polynucleotide, respectively.
As used herein, the term “gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, RNAi (miRNA, siRNA, shRNA), anti-microRNA antisense oligodeoxyribonucleotide (AMO), and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5′ and 3′ untranslated regions). A gene may be “isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
The term “genome” as used herein includes an organism's chromosomal/nuclear genome as well as any mitochondrial, and/or plasmid genome.
A “hairpin sequence” as used herein, is a nucleotide sequence comprising hairpins (e.g., that forms one or more hairpin structures). A hairpin (e.g., stem-loop, fold-back) refers to a nucleic acid molecule having a secondary structure that includes a region of complementary nucleotides that form a double strand that are further flanked on either side by single stranded-regions. Such structures are well known in the art. As known in the art, the double stranded region can comprise some mismatches in base pairing or can be perfectly complementary. In some embodiments of the present disclosure, a hairpin sequence of a nucleic acid construct can be located at the 3′end of a tracr nucleic acid.
A “heterologous” or a “recombinant” nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.
Different nucleic acids or proteins having homology are referred to herein as “homologues.” The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the compositions and methods of the invention further comprise homologues to the nucleotide sequences and polypeptide sequences of this invention.
“Orthologous,” as used herein, refers to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a nucleotide sequence of this invention has a substantial sequence identity (e.g., at least about 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%, and/or 100%) to said nucleotide sequence of the invention. Thus, for example, a homologue of a Type I, Type II, Type III, Type IV, or Type V polynucleotide or polypeptide can be about 70% homologous or more to any one of any known or later identified Type I, Type II, Type III, Type IV, or Type V polynucleotide or polypeptide.
As used herein, hybridization, hybridize, hybridizing, and grammatical variations thereof, refer to the binding of two fully complementary nucleotide sequences or substantially complementary sequences in which some mismatched base pairs may be present. The conditions for hybridization are well known in the art and vary based on the length of the nucleotide sequences and the degree of complementarity between the nucleotide sequences. In some embodiments, the conditions of hybridization can be high stringency, or they can be medium stringency or low stringency depending on the amount of complementarity and the length of the sequences to be hybridized. The conditions that constitute low, medium and high stringency for purposes of hybridization between nucleotide sequences are well known in the art (See, e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M. R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
As used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a “wild type mRNA” is a mRNA that is naturally occurring in or endogenous to the organism. A “homologous” nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.
Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence” and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2′-hydroxy in the ribose sugar group of the RNA can also be made. The nucleic acid constructs of the present disclosure can be DNA or RNA, but are preferably DNA. Thus, although the nucleic acid constructs of this invention may be described and used in the form of DNA, depending on the intended use, they may also be described and used in the form of RNA.
As used herein, the term “nucleotide sequence” refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5′ to 3′ end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded. The terms “nucleotide sequence” “nucleic acid,” “nucleic acid molecule,” “oligonucleotide” and “polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides. Except as otherwise indicated, nucleic acid molecules and/or nucleotide sequences provided herein are presented herein in the 5′ to 3′ direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§ 1.821-1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.
As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.
A “protospacer sequence” refers to the target double stranded DNA and specifically to the portion of the target DNA (e.g., or target region in the genome) that is fully or substantially complementary (and hybridizes) to the spacer sequence of the CRISPR repeat-spacer sequences, CRISPR repeat-spacer-repeat sequences, and/or CRISPR arrays.
As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” “suppress,” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In particular embodiments, the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount of the component being measured (e.g., the population of cells or a genome size). Thus, for example, a reduced genome size can mean a reduction in the size of a genome of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control.
A control as used herein may be, for example, a population of bacterial, archaeal, algal or yeast cells that has not been transformed with a heterologous nucleic acid construct of this invention. In some embodiments, a control may be a wild-type population of bacterial, archaeal, algal or yeast cells, or it may be a population of bacterial, archaeal or yeast cells transformed with a heterologous construct comprising a CRISPR array comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is not complementary to a target region in the genome of the bacterial, archaeal or yeast cells of said population (i.e., non-self targeting/“scrambled spacer”). In additional aspects, a control may be, for example, a wild-type population of bacterial, archaeal, algal or yeast cells, or a population of bacterial, archaeal, algal or yeast cells transformed with a heterologous construct comprising a CRISPR array comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial, archaeal, algal or yeast cells of said population that is not located adjacent to a protospacer adjacent motif (PAM).
A “repeat sequence” as used herein refers, for example, to any repeat sequence of a wild-type CRISPR locus or a repeat sequence of a synthetic CRISPR array that are separated by “spacer sequences” (e.g., a repeat-spacer sequence or a repeat-spacer-repeat sequence of the invention). A repeat sequence useful with this invention can be any known or later identified repeat sequence of a CRISPR locus. Accordingly, in some embodiments, a repeat-spacer sequence or a repeat-spacer-repeat comprises a repeat that is substantially identical (e.g. at least about 70% identical (e.g., at least about 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%, or more)) to a repeat from a wild-type Type II CRISPR array. In some embodiments, a repeat sequence is 100% identical to a repeat from a wild type Type I CRISPR array, a wild type Type II CRISPR array, wild type Type III CRISPR array, wild type Type IV CRISPR array, or wild type Type V CRISPR array. In additional embodiments, a repeat sequence useful with this invention can comprise a nucleotide sequence comprising a partial repeat that is a fragment or portion of a consecutive nucleotides of a repeat sequence of a CRISPR locus or synthetic CRISPR array of any of a Type I crRNA, Type II crRNA, Type III crRNA, Type IV crRNA, or Type V crRNA.
As used herein, “CRISPR array” of a Type I, Type II, Type III, Type IV, or Type V CRISPR-Cas system refers to a nucleic acid construct that comprises from 5′ to 3′ a repeat-spacer-repeat sequence or comprises from 5′ to 3′ at least one repeat-spacer sequence (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 repeat-spacer sequences, and any range or value therein). When more than one repeat-spacer is comprised in a CRISPR array, the spacer of the prior (5′ to 3′) repeat-spacer sequence can be linked to the repeat of the following repeat-spacer (e.g., the spacer of a first repeat-spacer sequence is linked to the repeat of a second repeat-spacer sequence). In some embodiments, a CRISPR array can comprise two repeats (or two partial repeats) separated by a spacer (e.g., a repeat-spacer-repeat sequence).
As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).
A “spacer sequence” as used herein is a nucleotide sequence that is complementary to a target DNA (i.e., target region in the genome or the “protospacer sequence”, which is adjacent to a protospacer adjacent motif (PAM) sequence). The spacer sequence can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 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%, or more)) to a target DNA. In representative embodiments, the spacer sequence has 100% complementarity to the target DNA. In additional embodiments, the complementarity of the 3′ region of the spacer sequence to the target DNA is 100% but is less than 100% in the 5′ region of the spacer and therefore the overall complementarity of the spacer sequence to the target DNA is less than 100%. Thus, for example, the first 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and the like, nucleotides in the 3′ region of a 20 nucleotide spacer sequence (seed sequence) can be 100% complementary to the target DNA, while the remaining nucleotides in the 5′ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In some embodiments, the first 7 to 12 nucleotides of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5′ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In other embodiments, the first 7 to 10 nucleotides of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5′ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In representative embodiments, the first 7 nucleotides (within the seed) of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5′ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
As used herein, a “target DNA,” “target region” or a “target region in the genome” refers to a region of an organism's genome that is fully complementary or substantially complementary (e.g., at least 70% complementary (e.g., 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%, or more)) to a spacer sequence in a repeat-spacer sequence or repeat-spacer-repeat sequence. In some embodiments, a target region may be about 10 to about 40 consecutive nucleotides in length located immediately adjacent to a PAM sequence (PAM sequence located immediately 3′ of the target region) in the genome of the organism (e.g., Type I CRISPR-Cas systems and Type II CRISPR-Cas systems). In the some embodiments, e.g., Type I systems, the PAM is on the alternate side of the protospacer (the 5′ end). There is no known PAM for Type III systems. Makarova et al. describes the nomenclature for all the classes, types and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)). Guide structures and PAMs are described in by R. Barrangou (Genome Biol. 16:247 (2015)).
In some embodiments, a target region useful with this invention is located within an essential gene or a non-essential gene.
In representative embodiments, a target region can be randomly selected or can be specifically selected. In some embodiments, a randomly selected target region may be selected from any at least 10 consecutive nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and the like, and any range or value therein) located immediately adjacent to a PAM sequence in a bacterial, archaeal, algal or yeast genome. In some embodiments, the target region can be about 10 to about 20 consecutive nucleotides, about 10 to about 30 consecutive nucleotides, and/or about 10 to about 40 consecutive nucleotides and the like, or any range or value therein, located immediately adjacent to a protospacer adjacent motif (PAM) sequence in a bacterial, archaeal, algal or yeast genome. In some embodiments, specifically selecting a target region can comprise selecting two or more target regions that are located about every 100 nucleotides to about every 1000 nucleotides, about every 100 nucleotides to about every 2000, about every 100 nucleotides to about every 3000, about every 100 nucleotides to about every 4000, and/or about every 100 nucleotides to about every 5000 nucleotides, and the like, from one another in the genome of the one or more bacteria, archaea, algal or yeast cells. In particular embodiments, specifically selecting a target region comprises specifically selecting a target region from a gene, open reading frame, a putative open reading frame or an intergenic region comprising at least about 10 to about 40 consecutive nucleotides immediately adjacent to a PAM sequence in a bacterial, archaeal, algal or yeast genome.
A “trans-activating CRISPR (tracr) nucleic acid” or “tracr nucleic acid” as used herein refers to any tracr RNA (or its encoding DNA). A tracr nucleic acid comprises from 5′ to 3′ a lower stem, an upper stem, a bulge, a nexus hairpin and terminal hairpins (See, Briner et al. (2014) Molecular Cell. 56(2):333-339). A trans-activating CRISPR (tracr) nucleic acid functions in hybridizing to the repeat portion of mature or immature crRNAs, recruits Cas9 protein to the target site, and may facilitate the catalytic activity of Cas9 by inducting structural rearrangement. The functional composition of tracrRNA molecules is listed above. Sequences for tracrRNAs are specific to the CRISPR-Cas system and can be variable. Any tracr nucleic acid, known or later identified, can be used with this invention.
As used herein, the phrase “substantially identical,” or “substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, refers to two or more sequences or subsequences that have at least about 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%, and/or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, the substantial identity exists over a region of the sequences that is at least about 50 residues to about 150 residues in length. Thus, in some embodiments of the invention, the substantial identity exists over a region of the sequences that is at least about 3 to about 15 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 residues in length and the like or any value or any range therein), at least about 5 to about 30, at least about 10 to about 30, at least about 16 to about 30, at least about 18 to at least about 25, at least about 18, at least about 22, at least about 25, at least about 30, at least about 40, at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, or more residues in length, and any range therein. In representative embodiments, the sequences can be substantially identical over at least about 22 nucleotides. In some particular embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, sequences of the invention can be about 70% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In some embodiments, sequences of the invention can be about 75% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In further embodiments, sequences of the invention can be about 80% to about 100% identical over at least about 16 nucleotides to about 25 nucleotides. In further embodiments, sequences of the invention can be about 80% to about 100% identical over at least about 7 nucleotides to about 25 nucleotides. In some embodiments, sequences of the invention can be about 70% identical over at least about 18 nucleotides. In other embodiments, the sequences can be about 85% identical over about 22 nucleotides. In still other embodiments, the sequences can be 100% identical over about 16 nucleotides. In a further embodiment, the sequences are substantially identical over the entire length of a coding region. Furthermore, in exemplary embodiments, substantially identical nucleotide or polypeptide sequences perform substantially the same function (e.g., the function or activity of a crRNA, tracr nucleic acid, repeat sequence, Cas9 nuclease (nickase, DNA, RNA and/or PAM recognition and binding), Cas3, Cas3′, Cas3″ or any other CRISPR-Cas polynucleotide or polypeptide).
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.). An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention “percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.
Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. In some representative embodiments, two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions.
“Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.
The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the invention. In one embodiment, a reference nucleotide sequence hybridizes to the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. In another embodiment, the reference nucleotide sequence hybridizes to the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C. or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C. In still further embodiments, the reference nucleotide sequence hybridizes to the “test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.
Any nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in any species of interest. Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences. As is understood in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity (e.g., 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%, and the like) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original, native nucleotide sequence. Thus, in representative embodiments of the invention, the nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in the particular species of interest.
In some embodiments, the heterologous or recombinant nucleic acids molecules, nucleotide sequences and/or polypeptides of the invention are “isolated.” An “isolated” nucleic acid molecule, an “isolated” nucleotide sequence or an “isolated” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide. In representative embodiments, the isolated nucleic acid molecule, the isolated nucleotide sequence and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.
In other embodiments, an isolated nucleic acid molecule, nucleotide sequence or polypeptide may exist in a non-native environment such as, for example, a recombinant host cell. Thus, for example, with respect to nucleotide sequences, the term “isolated” means that it is separated from the chromosome and/or cell in which it naturally occurs. A polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur (e.g., a different host cell, different regulatory sequences, and/or different position in the genome than as found in nature). Accordingly, the heterologous nucleic acid constructs, nucleotide sequences and their encoded polypeptides are “isolated” in that, by the hand of man, they exist apart from their native environment and therefore are not products of nature, however, in some embodiments, they can be introduced into and exist in a recombinant host cell.
In some embodiments, the heterologous or recombinant nucleic acid constructs of the invention are “synthetic.” A “synthetic” nucleic acid molecule, a “synthetic” nucleotide sequence or a “synthetic” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that is not found in nature but is created by the hand of man and is therefore not a product of nature.
In any of the embodiments described herein, the nucleotide sequences and/or heterologous nucleic acid constructs of the invention can be operatively associated with a variety of promoters and other regulatory elements for expression in various organisms cells. Thus, in representative embodiments, a nucleic acid construct of this invention can further comprise one or more promoters operably linked to one or more nucleotide sequences.
By “operably linked” or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term “operably linked” or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Thus, a first nucleotide sequence that is operably linked to a second nucleotide sequence, means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For instance, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered “operably linked” to the nucleotide sequence.
A “promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter. The coding sequence may encode a polypeptide and/or a functional RNA. Typically, a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5′, or upstream, relative to the start of the coding region of the corresponding coding sequence. The promoter region may comprise other elements that act as regulators of gene expression. These include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants, the CAAT box may be substituted by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).
Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for use in the preparation of heterologous nucleic acid constructs, i.e., “chimeric genes” or “chimeric polynucleotides.” These various types of promoters are known in the art.
The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.
In some embodiments, a nucleic acid construct of the invention can be an “expression cassette” or can be comprised within an expression cassette. As used herein, “expression cassette” means a heterologous nucleic acid construct comprising a nucleotide sequence of interest (e.g., the nucleic acid constructs of the invention (e.g., a synthetic tracr nucleic acid construct, a synthetic CRISPR nucleic acid construct, a synthetic CRISPR array, a chimeric nucleic acid construct; a nucleotide sequence encoding a polypeptide of interest, a Type I polypeptide, Type II polypeptide, Type III polypeptide, Type IV polypeptide, and/or Type V polypeptide)), wherein said nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter). Thus, some aspects of the invention provide expression cassettes designed to express the nucleotides sequences of the invention.
An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in the selected host cell. A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host cell, or may be derived from another source (i.e., foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).
An expression cassette also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed host cell. As used herein, “selectable marker” means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., fluorescence). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.
In addition to expression cassettes, the nucleic acid molecules and nucleotide sequences described herein can be used in connection with vectors. The term “vector” refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell. A vector comprises a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered or introduced. Vectors for use in transformation of host organisms are well known in the art. Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable. A vector as defined herein can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Additionally included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells). In some representative embodiments, the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell. Accordingly, the nucleic acid molecules of this invention and/or expression cassettes can be comprised in vectors as described herein and as known in the art.
“Introducing,” “introduce,” “introduced” (and grammatical variations thereof) in the context of a polynucleotide of interest means presenting the polynucleotide of interest to the host organism or cell of said organism (e.g., host cell) in such a manner that the polynucleotide gains access to the interior of a cell. Where more than one polynucleotide is to be introduced these polynucleotides can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different expression constructs or transformation vectors. Accordingly, these polynucleotides can be introduced into cells in a single transformation event, in separate transformation/transfection events, or, for example, they can be incorporated into an organism by conventional breeding protocols. Thus, in some aspects, one or more nucleic acid constructs of this invention can be introduced singly or in combination into a host organism or a cell of said host organism. In the context of a population of cells, “introducing” means contacting the population with the heterologous nucleic acid constructs of the invention under conditions where the heterologous nucleic acid constructs of the invention gain access to the interior of one or more cells of the population, thereby transforming the one or more cells of the population.
The term “transformation” or “transfection” as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, in some embodiments, a host cell or host organism is stably transformed with a nucleic acid construct of the invention. In other embodiments, a host cell or host organism is transiently transformed with a nucleic acid construct of the invention. Thus, in representative embodiments, a heterologous nucleic acid construct of the invention can be stably and/or transiently introduced into a cell.
“Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
By “stably introducing” or “stably introduced,” in the context of a polynucleotide, means that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.
“Stable transformation” or “stably transformed” as used herein means that a nucleic acid construct is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid construct is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein includes the nuclear, mitochondrial and plasmid genome, and therefore may include integration of a nucleic acid construct into, for example, the plasmid or mitochondrial genome. Stable transformation as used herein may also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome or a plasmid.
Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a bacterium, an archaea, a yeast, an algae, and the like). Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.
Accordingly, in some embodiments, the nucleotide sequences, constructs, expression cassettes can be expressed transiently and/or they can be stably incorporated into the genome of the host organism.
A heterologous nucleic acid construct of the invention can be introduced into a cell by any method known to those of skill in the art. In some embodiments of the invention, transformation of a cell comprises nuclear transformation. In still further embodiments, the heterologous nucleic acid construct (s) of the invention can be introduced into a cell via conventional breeding techniques.
Procedures for transforming both eukaryotic and prokaryotic organisms are well known and routine in the art and are described throughout the literature (See, for example, Jiang et al. 2013. Nat. Biotechnol. 31:233-239; Ran et al. Nature Protocols 8:2281-2308 (2013))
A nucleotide sequence therefore can be introduced into a host organism or its cell in any number of ways that are well known in the art. The methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into the organism, only that they gain access to the interior of at least one cell of the organism. Where more than one nucleotide sequence is to be introduced, they can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, the nucleotide sequences can be introduced into the cell of interest in a single transformation event, or in separate transformation events, or, alternatively, where relevant, a nucleotide sequence can be incorporated into an organism as part of a breeding protocol.
Mobile genetic elements (MGEs) present bacteria with continuous challenges to genomic stability, promoting evolution through horizontal gene transfer. The term MGE encompasses plasmids, bacteriophages, transposable elements, genomic islands, and many other specialized genetic elements (1). MGEs encompass genes conferring high rates of dissemination, adaptive advantages to the host, and genomic stability, leading to their near universal presence in bacterial genomes. To cope with the permanent threat of predatory bacteriophages and selfish genetic elements, bacteria have evolved both innate and adaptive immune systems targeting exogenous genetic elements. Innate immunity includes cell-wall modification, restriction/modification systems, and abortive phage infection (2). Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas) are an adaptive immune system targeted against invasive genetic elements in bacteria (3). CRISPR-Cas mediated immunity relies on distinct molecular processes, categorized as acquisition, expression, and interference (3). Acquisition occurs via molecular ‘sampling’ of foreign genetic elements, from which short sequences, termed spacers, are integrated in a polarized fashion into the CRISPR array (4). Expression of CRISPR arrays is constitutive and inducible by promoter elements within the preceding leader sequence (5-6). Interference results from a corresponding transcript that is processed selectively at each repeat sequence, forming CRISPR RNAs (crRNAs) that guide Cas proteins for sequence-specific recognition and cleavage of target DNA complementary to the spacer (7). CRISPR-Cas technology has applications in strain typing and detection (8-10), exploitation of natural/engineered immunity against mobile genetic elements (11), programmable genome editing in diverse backgrounds (12), transcriptional control (13-14), and manipulation of microbial populations in defined consortia (15).
The various CRISPR systems are known in the art. For example, see Makarova et al., which describes the nomenclature for all the classes, types and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)); see also, R. Barrangou (Genome Biol. 16:247 (2015)).
Although sequence features corresponding to CRISPR arrays were described previously in multiple organisms (16-17), Streptococcus thermophilus was the first microbe where the roles of specific cas genes and CRISPR-array components were elucidated (4). S. thermophilus is a non-pathogenic thermophilic Gram-positive bacterium used as a starter culture that catabolizes lactose to lactic acid in the syntrophic production of yogurt and various cheeses (18). S. thermophilus encodes up to four CRISPR-Cas systems, two of them (SthCRISPR1 and SthCRISPR3) are classified as Type II-A systems that are innately active in both acquisition and interference (4, 19). Accordingly, genomic analysis of S. thermophilus and its bacteriophages established a likely mechanism of CRISPR-Cas systems for phage/DNA protection. Investigation of CRISPR-Cas systems in S. thermophilus led to bioinformatic analysis of spacer origin (4, 20), discovery of the proto-spacer adjacent motif (PAM) sequences (19; 21), understanding of phage-host dynamics (22-23), demonstration of Cas9 endonuclease activity (7, 24-25), and recently, determination of the tracrRNA structural motifs governing function and orthogonality of Type II systems (26). Genomic analysis of S. thermophilus revealed evolutionary adaptation to milk through loss of carbohydrate catabolism and virulence genes found in pathogenic streptococci (18). S. thermophilus also underwent significant acquisition of niche-related genes, such as those encoding including cold-shock proteins, copper resistance proteins, proteinases, bacteriocins, and lactose catabolism proteins (18). Insertion sequences (ISs) are highly prevalent in S. thermophilus genomes and contribute to genetic heterogeneity between strains by facilitating dissemination of islands associated with dairy adaptation genes (18). The concomitant presence of MGEs and functional CRISPR-Cas systems in S. thermophilus suggests that genome homeostasis is governed at least in part by the interplay of these dynamic forces. Thus, S. thermophilus constitutes an ideal host for investigating the genetic outcomes of CRISPR-Cas targeting of genomic islands.
CRISPR-Cas systems have recently been the subject of intense research in genome editing applications (12), but the evolutionary roles of most endogenous microbial systems remain unknown (27). Even less is known concerning evolutionary outcomes of housing active CRISPR-Cas systems beyond the prevention of foreign DNA uptake (7), spacer acquisition events (4), and mutation caused by chromosomal self-targeting (28-32). Thus, the present inventors sought to determine the outcomes of targeting integrated MGEs with endogenous Type II CRISPR-Cas systems. Four islands were identified in S. thermophilus LMD-9, with lengths ranging from 8 to 102 kbp and totaling approximately 132 kbp, or 7% of the genome. In order to target genomic islands, plasmid-based expression of engineered CRISPR arrays with self-targeting spacers were transformed into S. thermophilus LMD-9. Collectively, our results elucidate fundamental genetic outcomes of self-targeting events and show that CRISPR-Cas systems can direct genome evolution at the bacterial population level.
Utilizing these discoveries, the present inventors have developed novel methods for screening populations of bacterial, archaeal, algal or yeast cells for essential genes, non-essential genes, and/or expendible genomic islands; for killing one or more cells within a population of bacterial, archaeal, algal or yeast cells; for identifying a phenotype of a bacterial, archaeal, algal or yeast gene; for selecting one or more bacterial, archaeal, algal or yeast cells having a reducing the genome size from a population of bacterial, archaeal or yeast cells; and/or for identifying in a population of bacterial, archaeal, algal or yeast cells at least one isolate having a mutation (e.g., deletion) in its genome.
Thus in one aspect, the present inventors, have developed methods for identifying genetic variants in a population that have altered genetic content that provides them the ability to escape targeting. Here, the target sequence has been modified, and one looks for survivors that have that modification. In some aspects, the modification (i.e., mutation) is a deletion. Further, if the target sequence has been modified, then the wild type genotype is not essential.
Accordingly, in one aspect of the invention a method of screening a population of bacterial cells for essential genes, non-essential genes, and/or expendable genomic islands is provided, comprising: introducing into said population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial cells of said population, thereby producing a population of transformed bacterial cells; and determining the presence or absence of a deletion in the population of transformed bacterial cells, wherein the presence of a deletion in the population of transformed bacterial cells indicates that the target region is comprised within a non-essential gene and/or an expendable genomic island, and the absence of a deletion in the population means that the target region is comprised within an essential gene. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
In additional aspects, the invention provides a method of screening a population of bacterial, archaeal, algal or yeast cells for essential genes, non-essential genes, and/or expendable genomic islands, comprising: introducing into the population of bacterial, archaeal, algal or yeast cells: (a) a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, thereby producing a population of transformed bacterial, archaeal, algal or yeast cells; and determining the presence or absence of a deletion in the population of transformed bacterial, archaeal, algal or yeast cells, wherein the presence of a deletion in the population of transformed bacterial, archaeal or yeast cells means that the target region is comprised within a non-essential gene and/or an expendable genomic island, and the absence of a deletion in the population of transformed bacterial, archaeal, algal or yeast cells means that the target region is comprised within an essential gene.
In other aspects, a method of killing one or more bacterial cells within a population of bacterial cells is provided, comprising: introducing into the population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial cells of said population, thereby killing one or more bacterial cells that comprise the target region within the population. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
In an additional aspect, a method of killing one or more cells within a population of bacterial, archaeal, algal or yeast cells is provided, the method comprising: introducing into the population of bacterial, archaeal or yeast cells (a) a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of the at least one repeat-spacer sequence and repeat-spacer-repeat sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, thereby killing one or more cells that comprise the target region in their genome within the population of bacterial, archaeal, algal or yeast cells.
Transformation of bacterial genome-targeting CRISPR RNAs can be used to selectively kill bacterial cells on a sequence-specific basis to subtract genetically distinct subpopulations, thereby enriching bacterial populations lacking the target sequence. This distinction can occur on the basis of the heterogeneous distribution of orthogonal CRISPR-Cas systems within genetically similar populations. Thus, in some embodiments, an CRISPR array that is introduced into a population of cells can be compatible (i.e., functional) with a CRISPR-Cas system in the one or more bacterial cells to be killed but is not compatible (i.e., not functional) with the CRISPR Cas system of at least one or more bacterial cells in the population. For instance, Escherichia coli and Klebsiella pneumoniae can exhibit either Type I-E or Type I-F CRISPR-Cas systems; Clostridium difficile encodes Type I-B systems, and different strains of S. thermophilus exhibit both Type II-A and Type I-E systems or just Type II-A systems. Depending on the specific CRISPR RNA transformed into a mixture of bacteria, it can specifically target that subset of the population based on its functional compatibility with its cognate system. This can be applied to diverse species containing endogenous CRISPR-Cas systems such as, but not limited to: Pseudomonas spp. (such as: P. aeruginosa), Escherichia spp. (such as: E. coli), Enterobacter spp. (such as: E. cloacae), Staphylococcus spp. (such as: S. aureus), Enterococcus spp. (such as: E. faecalis, E. faecium), Streptomyces spp. (such as: S. somaliensis), Streptococcus spp. (such as: S. pyogenes), Vibrio spp. (such as: V. cholerae), Yersinia spp. (such as: Y. pestis), Francisella spp. (such as: F. tularensis, F. novicida), Bacillus spp. (such as: B. anthracis, B. cereus), Lactobacillus spp. (such as: L. casei, L. reuteri, L. acidophilus, L. rhamnosis), Burkholderia spp. (such as: B. mallei, B. pseudomallei), Klebsiella spp. (such as: K. pneumoniae), Shigella spp. (such as: S. dysenteriae, S. sonnei), Salmonella spp. (such as: S. enterica), Borrelia spp. (such as: B. burgdorfieri), Neisseria spp. (such as: N. meningitidis), Fusobacterium spp. (such as: F. nucleatum), Helicobacter spp. (such as: H. pylori), Chlamydia spp. (such as: C. trachomatis), Bacteroides spp. (such as: B. fragilis), Bartonella spp. (such as: B. quintana), Bordetella spp. (such as: B. pertussis), Brucella spp. (such as: B. abortus), Campylobacter spp. (such as: C. jejuni), Clostridium spp. (such as: C. difficile), Bifidobacterium spp. (such as: B. infantis), Haemophilus spp. (such as: H. influenzae), Listeria spp. (such as: L. monocytogenes), Legionella spp. (such as: L. pneumophila), Mycobacterium spp. (such as: M. tuberculosis), Mycoplasma spp. (such as: M. pneumoniae), Rickettsia spp. (such as: R. rickettsia), Acinetobacter spp. (such as: A. calcoaceticus, A. baumanii), Rumincoccus spp. (such as: R. albus), Propionibacterium spp. (such as: P. freudenreichii), Corynebacterium spp. (such as: C. diphtheriae), Propionibacterium spp. (such as: P. acnes), Brevibacterium spp. (such as: B. iodinum), Micrococcus spp. (such as: M. luteus), and/or Prevotella spp. (such as: P. histicola).
CRISPR targeting can remove specific bacterial subsets on the basis of the distinct genetic content in mixed populations. Support for this claim is presented in examples 4, 5 where Lac− bacteria are selected for while Lac+ are removed from the population. The genetic distinction between the Lac+ and Lac− strains is presented in examples 8 and 10, where sequencing of the surviving clones revealed up to 5.5% difference in genetic content compared to the reference wild-type S. thermophilus strain. CRISPR-targeting spacers can thus be tuned to various levels of bacterial relatedness by targeting conserved or divergent genetic sequences. Thus, in some embodiments, the bacterial cells in the population can comprise the same CRISPR Cas system and the introduced CRISPR array thus may be functional in the bacterial population as a whole but the genetic content of the different strains or species that make up the bacterial population is sufficiently distinct such that the target region for the introduced CRISPR array is found only in the one or more bacterial species of the population that is to be killed. This can be applied to diverse species containing endogenous CRISPR-Cas systems such as, but not limited to: Pseudomonas spp. (such as: P. aeruginosa), Escherichia spp. (such as: E. coli), Enterobacter spp. (such as: E. cloacae), Staphylococcus spp. (such as: S. aureus), Enterococcus spp. (such as: E. faecalis, E. faecium), Streptomyces spp. (such as: S. somaliensis), Streptococcus spp. (such as: S. pyogenes), Vibrio spp. (such as: V. cholerae), Yersinia spp. (such as: Y. pestis), Francisella spp. (such as: F. tularensis, F. novicida), Bacillus spp. (such as: B. anthracis, B. cereus), Lactobacillus spp. (such as: L. casei, L. reuteri, L. acidophilus, L. rhamnosis), Burkholderia spp. (such as: B. mallei, B. pseudomallei), Klebsiella spp. (such as: K. pneumoniae), Shigella spp. (such as: S. dysenteriae, S. sonnei), Salmonella spp. (such as: S. enterica), Borrelia spp. (such as: B. burgdorfieri), Neisseria spp. (such as: N. meningitidis), Fusobacterium spp. (such as: F. nucleatum), Helicobacter spp. (such as: H. pylori), Chlamydia spp. (such as: C. trachomatis), Bacteroides spp. (such as: B. fragilis), Bartonella spp. (such as: B. quintana), Bordetella spp. (such as: B. pertussis), Brucella spp. (such as: B. abortus), Campylobacter spp. (such as: C. jejuni), Clostridium spp. (such as: C. difficile), Bifidobacterium spp. (such as: B. infantis), Haemophilus spp. (such as: H. influenzae), Listeria spp. (such as: L. monocytogenes), Legionella spp. (such as: L. pneumophila), Mycobacterium spp. (such as: M. tuberculosis), Mycoplasma spp. (such as: M. pneumoniae), Rickettsia spp. (such as: R. rickettsia), Acinetobacter spp. (such as: A. calcoaceticus, A. baumanii), Rumincoccus spp. (such as: R. albus), Propionibacterium spp. (such as: P. freudenreichii), Corynebacterium spp. (such as: C. diphtheriae), Propionibacterium spp. (such as: P. acnes), Brevibacterium spp. (such as: B. iodinum), Micrococcus spp. (such as: M. luteus), and/or Prevotella spp. (such as: P. histicola).
The extent of killing within a population using the methods of this invention may be affected by the amenability of the particular population to transformation in addition to whether the target region is comprised in a non-essential gene, an essential gene or an expendable island. The extent of killing in a population of bacterial, archaeal or yeast cells can vary, for example, by organism, by genus and species. Accordingly, as used herein “killing” means eliminating 2 logs or more of the cells in a population (1% survival or less). Less than 1 log of killing would be a small reduction in the population; whereas 2-3 logs of killing results in a significant reduction of the population; and more than 3 logs of killing indicates that the population has been substantially eradicated.
In another aspect, a method of identifying a phenotype associated with a bacterial gene is provided, comprising: introducing into a population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of the at least one repeat-spacer sequence and repeat-spacer-repeat sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial cells of said population, wherein the target region comprises at least a portion of an open reading frame encoding a polypeptide or functional nucleic acid, thereby killing the cells comprising the target region and producing a population of transformed bacterial cells without the target region (i.e., surviving cells do not comprise the target region); and (i) analyzing the phenotype of the population of cells, or (ii) growing individual bacterial colonies from the population of transformed bacterial cells and analyzing the phenotype of the individual colonies. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
In another aspect, a method of identifying the phenotype of a bacterial, archaeal, algal, or yeast gene is provided, comprising: introducing into a population of bacterial, archaeal, algal or yeast cells (a) a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, thereby killing the bacterial, archaeal, algal or yeast cells comprising the target region and producing a population of transformed bacterial, archaeal, algal or yeast cells without the target region; and (i) analyzing the phenotype of the population of cells, and/or (ii) growing individual bacterial, archaeal, or yeast colonies from the population of transformed bacterial, archaeal, algal or yeast cells; and analyzing the phenotype of the individual colonies.
In some embodiments, the analysis comprises PCR, optical genome mapping, genome sequencing, restriction mapping and/or restriction analysis to identify and characterize the mutation, and complementation analysis and/or phenotypic assays to analyze the phenotype.
In some embodiments of the invention determining the extent of killing or a reduction in a population can comprise any method for determining population number, including, but not limited to, (1) plating the cells and counting the colonies, (2) optical density, (3) microscope counting, (4) most probable number, and/or (5) methylene blue reduction. In some embodiments, 16S rDNA sequencing can be used to profile a composition of mixed populations. This can be done, for example, by purifying DNA from the sample as a whole, and performing either whole-genome shotgun sequencing using high-throughput technologies or, for example, by PCR amplifying the 16S gene and sequencing the products in the same manner. The sequences can then be computationally assigned to certain bacterial taxa. In other embodiments, quantitative PCR methods may also be used to quantify bacterial levels. Such techniques are well known in the art. For example, primers for qPCR can be designed to amplify specifically from a strain species, genus, or group of organisms that share the sequence. Thus, a threshold number (ct) may be used to quantify said organism or group of organisms. In additional embodiments, any bacterial activity (phenotype) specific to the target population may also be used as a metric to determine depletion of a population.
In further embodiments, a method of selecting one or more bacterial cells having a reduced genome size from a population of bacterial cells is provided, comprising: introducing into a population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population, wherein the cells comprising the target region are killed, thereby selecting one or more bacterial cells without the target region and having a reduced genome size from the population of bacterial cells. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
In some embodiments, a method of selecting one or more bacterial cells having a reduced genome size from a population of bacterial cells, comprising: introducing into a population of bacterial cells: (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial cells, or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and (b) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population, the target region is located between the one or more heterologous nucleic acid constructs introduced into the genome, and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, wherein cells comprising the target region are killed and cells not comprising the target region survive, thereby selecting one or more bacterial cells without the target region and having a reduced genome size from the population of transgenic bacterial cells. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
As is well known in the art, transposons can be created via, for example, PCR amplification or through designed DNA synthesis, and may be introduced via any method of transformation.
In some embodiments, the invention provides a method of selecting one or more bacterial, archaeal, algal or yeast cells having a reduced genome size from a population of bacterial, archaeal, algal or yeast cells, comprising: introducing into a population of bacterial, archaeal or yeast cells (a) a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid, (b) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of the at least one repeat-spacer sequence and the at least one repeat-spacer-repeat sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial, archaeal, algal or yeast cells of said population, and (c) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein cells comprising the target region are killed, thereby selecting one or more bacterial, archaeal, algal or yeast cells without the target region and having a reduced genome size from the population of bacterial, archaeal, algal or yeast cells.
In other embodiments, a method of selecting one or more bacterial, archaeal, algal or yeast cells having a reduced genome size from a population of bacterial, archaeal, algal or yeast cells is provided, comprising: introducing into a population of bacterial, archaeal, algal or yeast cells: (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial, archaeal, algal or yeast cells, or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial, archaeal, algal or yeast cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and (b)(i) a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid, (ii) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of the at least one repeat-spacer sequence or the at least one repeat-spacer-repeat sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial, archaeal, algal or yeast cells of said population, and (iii) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein the target region is located between the one or more heterologous nucleic acid constructs incorporated into the genome, and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, wherein cells comprising the target region are killed and cells not comprising the target region survive, thereby selecting one or more bacterial, archaeal, algal or yeast cells without the target region and having a reduced genome size from the population of transgenic bacterial, archaeal, algal or yeast cells.
In some aspects, the reduced genome size may be reduced as compared to a control. In some aspects, a control may be a wild-type population of bacterial, archaeal, algal or yeast cells, or a population of bacterial, archaeal, algal or yeast cells transformed with a heterologous construct comprising a CRISPR array (e.g., a Type I, Type II, Type III, Type IV or Type V CRISPR array) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is not complementary to a target region in the genome of the bacterial, archaeal, algal or yeast cells of said population (i.e., non-self targeting/“scrambled spacer”). In additional aspects, a control may be a population of bacterial, archaeal, algal or yeast cells transformed with a heterologous construct comprising a CRISPR array comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of the bacterial, archaeal, algal or yeast cells of said population but lacks a protospacer adjacent motif (PAM).
In some embodiments, a method of identifying in a population of bacteria at least one isolate having a deletion in its genome (e.g., a chromosomal and/or plasmid deletion) is provided, comprising: introducing into a population of bacterial cells a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising (5′ to 3′) a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or said at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population and cells comprising the target region are killed, thereby producing a population of transformed bacterial cells without the target region; and growing individual bacterial colonies from the population of transformed bacterial cells, thereby identifying at least one isolate from the population of transformed bacteria having a deletion in its genome. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
In additional embodiments, the invention provides a method of identifying in a population of bacteria at least one isolate having a deletion in its genome, comprising: introducing into the population of bacterial cells: (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial cells, or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and b) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial cells of said population, wherein the target region is located between the one or more heterologous nucleic acid constructs introduced into the genome and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, and cells comprising the target region are killed [and cells not comprising the target region survive], thereby producing a population of transformed bacterial cells without the target region; and growing individual bacterial colonies from the population of transformed bacterial cells, thereby identifying at least one isolate from the population of bacteria having a deletion in its genome. A CRISPR array useful with this invention may be Type I, Type II, Type III, Type IV or Type V CRISPR array.
In further embodiments, a method of identifying in a population of bacterial, archaeal, algal or yeast cells at least one isolate having a deletion in its genome is provided, comprising: introducing into a population of bacterial, archaeal, algal or yeast cells: (a) a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid; (b) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer of said repeat-spacer-repeat sequence or at least one repeat-spacer sequence comprises a nucleotide sequence that is substantially complementary to a target region in the genome (e.g., chromosomal, mitochondrial and/or plasmid genome) of the bacterial, archaeal, algal or yeast cells of said population; and (c) a Cas9 polypeptide or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein cells comprising the target region are killed, thereby producing a population of transformed bacterial, archaeal, algal or yeast cells without the target region; and growing individual bacterial, archaeal or yeast colonies from the population of transformed bacterial, archaeal, algal or yeast cells, thereby identifying at least one isolate from the population of transformed bacterial, archaeal, algal or yeast cells having a deletion in its genome.
In still further embodiments, the invention provides a method of identifying in a population of bacterial, archaeal, algal or yeast cells at least one isolate having a deletion in its genome, comprising: introducing into the population of bacterial, archaeal, algal or yeast cells: (a)(i) one or more heterologous nucleic acid constructs comprising a nucleotide sequence having at least 80 percent identity to at least 300 consecutive nucleotides present in the genome of said bacterial, archaeal, algal or yeast cells, or (ii) two or more heterologous nucleic acid constructs comprising at least one transposon, thereby producing a population of transgenic bacterial, archaeal, algal or yeast cells comprising a non-natural site for homologous recombination between the one or more heterologous nucleic acid constructs integrated into the genome and the at least 300 consecutive nucleotides present in the genome, or between a first and a second transposon integrated into the genome; and (b)(i) a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid, (ii) a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) comprising a repeat-spacer-repeat sequence or at least one repeat-spacer sequence, wherein the spacer comprises a nucleotide sequence that is substantially complementary to a target region in the genome of one or more bacterial, archaeal, algal or yeast cells of said population; and (iii) a Cas9 polypeptide and/or a heterologous nucleic acid construct comprising a polynucleotide encoding a Cas9 polypeptide, wherein the target region is located between the one or more heterologous nucleic acid constructs incorporated into the genome and the at least 300 consecutive nucleotides present in the genome and/or between the first transposon and second transposon, and cells comprising the target region are killed and cells not comprising the target region survive, thereby producing a population of transformed bacterial, archaeal, algal or yeast cells without the target region; and growing individual bacterial, archaeal or yeast colonies from the population of transformed bacterial, archaeal, algal or yeast cells, thereby identifying at least one isolate from the population having a deletion in its genome.
In some embodiments, fitness/growth rate can be increased by reducing genome size or by deleting select genes (encoding polypeptides or functional nucleic acids (e.g., transcriptional regulators)) that require high energy input for transcription and translation. Thus, in some embodiments, a method of increasing the fitness or growth rate of a population of bacterial, archaeal, algal or yeast cells is provided, comprising: selecting for a reduced genome size (e.g., selecting for the absence of a portion of the genome) and/or deletion in the genomes of the bacterial, archaeal, algal or yeast cells of the populations as described herein. In some embodiments, the deletion may comprise one gene or more than one gene. Therefore, through reducing the genome size or deleting a particular gene or genes, the cells of the population no longer expend energy on the transcription/translation of the portion of the genome that is absent or the deleted gene or genes, thereby having reduced energy needs and increased fitness as compared to a control population still comprising said portion of the genome and/or said gene or genes.
In other embodiments, a method of increasing the amount of a product produced from a population of bacterial, archaeal, algal or yeast cells is provided, comprising increasing the fitness or growth rate of the cell by selecting for a deletion in the genomes of the bacterial, archaeal, algal or yeast cells as described herein. In some embodiments, the products can include, but are not limited to, antibiotics, secondary metabolites, vitamins, proteins, enzymes, acids, and pharmaceuticals.
In some embodiments, a CRISPR array (crRNA, crDNA) useful with this invention may be an array from any Type I CRISPR-Cas system, Type II CRISPR-Cas system, Type III CRISPR-Cas system, Type IV CRISPR-Cas system, or a Type V CRISPR-Cas system.
With regard to the preceding embodiments, a heterologous nucleic acid construct comprising a tracr nucleic acid and a heterologous nucleic acid construct comprising a CRISPR array may be comprised in and introduced in the same construct (e.g., expression cassette or vector) or in different constructs. In particular embodiments, a heterologous nucleic acid construct comprising a tracr nucleic acid and a heterologous nucleic acid construct comprising a CRISPR array may be comprised in single construct (e.g., expression cassette and/or vector) that may optionally further comprise a polynucleotide encoding Cas9 polypeptide. In some embodiments, the heterologous nucleic acid construct comprising a tracr nucleic acid and the heterologous nucleic acid construct comprising a CRISPR array may be operably linked to a single promoter and/or to separate promoters.
In some embodiments, a heterologous nucleic acid construct comprising a trans-activating CRISPR (tracr) nucleic acid and a heterologous nucleic acid construct comprising a CRISPR array (crRNA, crDNA) may be comprised in a CRISPR guide (gRNA, gDNA). In some embodiments, a CRISPR guide may be operably linked to a promoter.
In some embodiments, a Cas9 polypeptide useful with this invention comprises at least 70% identity (e.g., about 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%, and the like) to an amino acid sequence of a Cas9 nuclease. Exemplary Cas9 nucleases useful with this invention can be any Cas9 nuclease known to catalyze DNA cleavage in a CRISPR-Cas system. As known in the art, such Cas9 nucleases comprise a HNH motif and a RuvC motif (See, e.g., WO2013/176772; WO/2013/188638). In some embodiments, a functional fragment of a Cas9 nuclease can be used with this invention.
CRISPR-Cas systems and groupings of Cas9 nucleases are well known in the art and include, for example, a Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, a Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, and a Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases. Additional Cas9 nucleases include, but are not limited to, those of Lactobacillus curvatus CRL 705. Still further Cas9 nucleases useful with this invention include, but are not limited to, a Cas9 from Lactobacillus animalis KCTC 3501, and Lactobacillus farciminis WP 010018949.1.
Furthermore, in particular embodiments, the Cas9 nuclease can be encoded by a nucleotide sequence that is codon optimized for the organism comprising the target DNA. In still other embodiments, the Cas9 nuclease can comprise at least one nuclear localization sequence.
In some embodiments, a Type I polypeptide useful with this invention comprises at least 70% identity (e.g., about 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%, and the like) to an amino acid sequence of a Cas3, Cas3′ nuclease, a Cas3″ nuclease, fusion variants thereof. In some embodiments, a Type I Cascade polypeptide useful with this invention comprises at least 70% identity (e.g., about 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%, and the like) to an amino acid sequence of a Cas7 (Csa2), Cas8a1 (Csx13), Cas8a2 (Csx9), Cas5, Csa5, Cas6a, Cas6b, Cas8b (Csh1), Cas7 (Csh2), Cas5, Cas5d, Cas8c (Csd1), Cas7 (Csd2), Cas10d (Csc3), Csc2, Csc1, Cas6d, Cse1 (CasA), Cse2 (CasB), Cas7 (CasC), Cas5 (CasD), Cas6e (CasE), Cys1, Cys2, Cas7 (Cys3), Cas6f (Csy4), Cas6 and/or Cas4
Type I CRISPR-Cas systems are well known in the art and include, for example, Archaeoglobus fulgidus comprises an exemplary Type I-A CRISPR-Cas system, Clostridium kluyveri DSM 555 comprises an exemplary Type I-B CRISPR-Cas system, Bacillus halodurans C-125 comprises an exemplary Type I-C CRISPR-Cas system, Cyanothece sp. PCC 802 comprises an exemplary Type I-D CRISPR-Cas system, Escherichia coli K-12 comprises an exemplary Type I-E CRISPR-Cas system, Geobacter sulfurreducens comprises an exemplary Type I-U CRISPR-Cas system and Yersinia pseudotuberculosis YPIII comprises an exemplary Type I-F CRISPR-Cas system.
In some embodiments, a Type II polypeptide useful with this invention comprises at least 70% identity (e.g., about 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%, and the like) to an amino acid sequence of a Cas9. Type II CRISPR-Cas systems well known in the art and include, for example, Legionella pneumophila str. Paris, Streptococcus thermophilus CNRZ1066 and Neisseria lactamica 020-06.
In some embodiments, a Type III polypeptide useful with this invention comprises at least 70% identity (e.g., about 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%, and the like) to an amino acid sequence of a Cas6, Cas10 (or Csm1), Csm2, Csm3, Csm4, Csm5, and Csm6, Cmr1, Cas10 (or Cmr2), Cmr3, Cmr4, Cmr5, and Cmr6, Cas7, Cas10, Cas7 (Csm3), Cas5 (Csm4), Cas7 (Csm5), Csm6, Cas7 (Cmr1), Cas5 (Cmr3), Cas7 (Cmr4), Cas7 (Cmr6), Cas7 (Cmr6), Cmr5, Cas5 (Cmr3), Cas5 (Csx10), Csm2, Cas7 (Csm3), and all1473. Type III CRISPR-Cas systems are well known in the art and include, for example, Staphylococcus epidermidis RP62A, which comprises an exemplary Type III-A CRISPR-Cas system, Pyrococcus furiosus DSM 3638, which comprises an exemplary Type III-B CRISPR-Cas system, Methanothermobacter thermautotrophicus str. Delta H, which comprises an exemplary Type III-C CRISPR-Cas system, and Roseiflexis sp. Rs-1, which comprises an exemplary Type III-D CRISPR-Cas system.
In some embodiments, a Type IV polypeptide useful with this invention comprises at least 70% identity (e.g., about 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%, and the like) to an amino acid sequence of a Csf4 (dinG), Csf1, Cas7 (Csf2) and/or Cas5 (csf3). Type IV CRISPR-Cas systems are well known in the art, for example, Acidithiobacillus ferrooxidans ATCC 23270 comprises an exemplary Type IV CRISPR-Cas system.
In some embodiments, a Type V polypeptide useful with this invention comprises at least 70% identity (e.g., about 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%, and the like) to an amino acid sequence of a Cpf1, Cas1, Cas2, or Cas4. Type V CRISPR-Cas systems are well known in the art and include, for example, Francisella cf. novicida Fx1 comprises an exemplary Type V CRISPR-Cas system.
Additionally provided herein are expression cassettes and vectors comprising the nucleic acid constructs, the nucleic acid arrays, nucleic acid molecules and/or the nucleotide sequences of this invention, which can be used with the methods of this disclosure.
In further aspects, the nucleic acid constructs, nucleic acid arrays, nucleic acid molecules, and/or nucleotide sequences of this invention can be introduced into a cell of a host organism. Any cell/host organism for which this invention is useful with can be used. Exemplary host organisms include bacteria, archaea, algae and fungi (e.g., yeast).
The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.
All bacterial strains are listed in Table 1. Bacterial cultures were cryopreserved in an appropriate growth medium with 25% glycerol (vol/vol) and stored at −80° C. S. thermophilus was propagated in Elliker media (Difco) supplemented with 1% beef extract (wt/vol) and 1.9% (wt/vol) β-glycerolphosphate (Sigma) broth under static aerobic conditions at 37° C., or on solid medium with 1.5% (wt/vol) agar (Difco), incubated anaerobically at 37° C. for 48 hours. Concentrations of 2 μg/mL of erythromycin (Em) and 5 μg/mL of chloramphenicol (Cm) (Sigma) were used for plasmid selection in S. thermophilus, when appropriate. E. coli EC1000 was propagated aerobically in Luria-Bertani (Difco) broth at 37° C., or on brain-heart infusion (BHI) (Difco) solid medium supplemented with 1.5% agar. Antibiotic selection of E. coli was maintained with 40 μg/mL kanamycin (Kn) and 150 μg/mL of Em for recombinant E. coli, when appropriate. Screening of S. thermophilus derivatives for β-galactosidase activity was assessed qualitatively by supplementing a synthetic Elliker medium with 1% lactose, 1.5% agar, and 0.04% bromo-cresol purple as a pH indicator.
All kits, enzymes, and reagents were used according to the manufacturers' instructions. DNA purification and cloning were performed as described previously (41). Briefly, purification of genomic DNA from S. thermophilus employed a ZR Fungal/Bacterial MiniPrep kit (Zymo). Plasmid DNA was isolated from E. coli using Qiagen Spin miniprep kit (Qiagen). High fidelity PCR amplification of DNA was performed with PFU HS II DNA polymerase (Stratagene). Routine PCRs were conducted with Choice-Taq Blue polymerase (Denville). Primers for PCR amplification were purchased from Integrated DNA Technologies (Coralville, Iowa). DNA extraction from agarose gels was performed with a Zymoclean DNA gel recovery kit (Zymo). Restriction endonucleases were acquired from Roche Molecular Biochemicals. Ligations were performed with New England Biolabs quick T4 ligase. Sequencing was performed by Davis Sequencing Inc. (Davis, Calif.). Cryopreserved rubidium chloride competent E. coli cells were prepared as previously described (41). Plasmids with lacZ targeting arrays were constructed with each consisting sequentially of the (1) native leader sequence specific to SthCRISPR1 or SthCRISPR3 (2) native repeats specific to CRISPR 1 or CRISPR 3 (3) spacer sequence specific to the 5′ end of lacZ (4) another native repeat (
The programmable specificity of chromosomal cleavage hinges upon selection of a desired spacer sequence unique to the target allele. Specificity is further compounded by the requisite PAM, a short conserved sequence that must be proximate to the proto-spacer in the target sequence (21, 43). Thus, strict criteria for selection and design of spacers were the location of consensus PAM sequences and incidental sequence identity to extraneous genomic loci. Putative protospacers were constrained by first defining the location of all putative PAM sequences in the sense and antisense strands of lacZ. Within the 3,081 nt gene, there were 22 CRISPR1 (AGAAW) and 39 CRISPR3 (GGNG) PAM sites that were identical to their bioinformatically derived consensus sequences (21). After potential spacers were identified, the complete proto-spacer, seed, and PAM sequence were subjected to BLAST analysis against the genome of S. thermophilus LMD-9 to prevent additional targeting of non-specific loci. The spacers for CRISPR1 and CRISPR3 were disparate in sequence and corresponding PAM sites, but were designed to target the 5′ end of lacZ, resulting in predicted cleavage sites residing 6 nt apart. Therefore, the leader sequences, repeats, and spacers on each plasmid represented orthogonal features unique to CRISPR1 or CRISPR3, respectively. To assess target locus-dependent mutations, an additional CRISPR3 plasmid was created with a spacer to the metal cation-binding residue essential for β-galactosidase activity. A CRISPR1 array plasmid containing a non-self-spacer was used as a control to quantify lethality of self-targeting.
Plasmids were electroporated into competent S. thermophilus containing the temperature-sensitive helper plasmid, pTRK669, according to methods described previously (44). Briefly, an overnight culture of S. thermophilus was inoculated at 1% (vol/vol) into 50 mL of Elliker medium supplemented with 1% beef extract, 1.9% β-glycerophosphate and Cm selection. When the culture achieved an OD600 nm of 0.3, penicillin G was added to achieve a final concentration of 10 μg/mL, in order to promote electroporation efficiency (45). Cells were harvested by centrifugation and washed 3× in 10 mL cold electroporation buffer (1 M sucrose and 3.5 mM MgCl2). The cells were concentrated 100-fold in electroporation buffer and 40 μL of the suspension was aliquoted into 0.1 mm electroporation cuvettes. Each suspension was combined with 700 ng of plasmid. Electroporation conditions were set at 2,500 V, 25 μFd capacitance, and 200 Ohms resistance. Time constants were recorded and ranged from 4.4 to 4.6 ms. The suspensions were immediately combined with 950 μL of recovery medium and incubated for 8 hours at 37° C. Cell suspensions were plated on selective medium and electroporation cuvettes were washed with medium to ensure recovery of cells.
Transformants generated from both CRISPR1 and CRISPR3 were initially screened for the β-galactosidase deficient phenotype by restreaking colonies on semi-synthetic Elliker medium supplemented with 1% lactose as the sold carbohydrate source. Loss of β-galactosidase activity was confirmed by performing Miller assays (o-nitrophenyl-β-D-galactoside (ONPG) (46). Briefly, cultures were propagated to late-log phase (OD600 nm of 1.2) in 5 mL of medium and harvested by centrifugation (4,000×g for 10 min). Cells were washed and resuspended in 0.5 mL phosphate-buffered saline (Gibco-Invitrogen). Each suspension was combined with 100 uL of 0.1 mm glass beads (Biospec) and then subjected to five 60 s cycles of homogenization in a Mini-Beadbeater (Biospec). Samples were then centrifuged (15,000×g for 5 min) to remove debris and intact cells. Cell lysates (10 μL aliquots) were combined with 600 μL of substrate solution (60 mM Na2HPO4; 40 mM NaH2PO4; 1 mg/mL ONPG; 2.7 μL/mL (3-mercaptoethanol) and incubated for 5 min at room temperature, at which point 700 μL stop solution was added (1 M NaCO3). The absorbance at 420 nm was recorded and activity of β-galactosidase was reported as Miller units, calculated as previously described (46).
Cultures were preconditioned for growth assays by subculturing for 12 generations in a semi-synthethic Elliker medium deficient in lactose. Fresh medium was inoculated with an overnight culture at 1% (vol/vol) and incubated at 37° C. statically. OD600 monitored hourly until the cultures achieved stationary phase. Acidification of milk was assessed by inoculating skim milk with an overnight culture to a level of 108 cfu/mL and incubating at 42° C. The pH was subsequently monitored using a Mettler Toledo Seven Easy pH meter and Accumet probe. Skim milk was acquired from the NCSU Dairy plant and Pasteurized for 30 min at 80° C.
In silico prediction of mobile and expendable loci for CRISPR-Cas targeting was performed on the basis of i) location, orientation, and nucleotide identity of IS elements, and ii) location of essential ORFs. In Bacillus subtilis, 271 essential ORFs were identified by determining the lethality of genome-wide gene knockouts (33). The S. thermophilus genome was queried for homologues to each essential gene from B. subtilis using the BLASTp search tool under the default scoring matrix for amino acid sequences. Homologues to about 239 essential ORFs were identified in S. thermophilus, all of which were chromosomally encoded (Table 4). Proteins involved in conserved cellular processes including DNA replication/homeostasis, translation machinery, and core metabolic pathways were readily identified. No homologues corresponding to cytochrome biosynthesis/respiration were observed, in accordance with the metabolic profile of fermentative bacteria. Each putative essential ORF was mapped to the reference genome using SnapGene software, facilitating visualization of their location and distribution in S. thermophilus LMD-9 (
IS elements within the S. thermophilus genome were grouped by aligning transposon coding sequences using Geneious® software (
By combining the location of predicted essential ORFs and IS elements, expendable islands flanked by IS elements of high fidelity were identified (
In Type II systems, Cas9 interrogates DNA and binds reversibly to PAM sequences with activation of Cas9 at the target occurring via formation of the tracrRNA::crRNA duplex (37), ultimately resulting in dsDNA cleavage (
Transformation with plasmids eliciting chromosomal self-targeting by CRISPR-Cas systems appeared cytotoxic as measured by the relative reduction in surviving transformants compared to non-self-targeting plasmids (15, 29). Targeting the lacZ gene in S. thermophilus resulted in about a 2.5-log reduction in recovered transformants (
To determine if the target locus was mutated in response to Cas9-induced cleavage, transformants were first screened for loss of β-galactosidase activity. Clones deficient in activity were genotyped at the lacZ locus. No mutations due to classical or alternative end joining, nor any spontaneous single nucleotide polymorphisms were observed in any of the clones sequenced. The absence of single nucleotide polymorphisms may be attributed to a low transformation efficiency compounded by low incidence of point mutations, and the absence of Ku and LigaseIV homologs correlated with an absence of non-homologous end joining (38). PCR screening indicated that the wild-type lacZ was not present, but the PCR amplicons did not correspond to the native lacZ locus; rather, an IS element-flanked sequence at another genomic locus was amplified. To investigate the genotype responsible for the loss of β-galactosidase activity, Single Molecule Real Time sequencing was performed on two clones; one generated from CRISPR3 targeting the 5′ end of lacZ, and one generated from CRISPR3 targeting the sequence encoding the ion-binding pocket necessary for (3-galactosidase catalysis (
The 102 kbp segments deleted constitute approximately 5.5% of the 1.86 Mbp genome of S. thermophilus. The region contained 102 putative ORFs (STER_1278-1379), encoding ABC transporters, two-component regulatory systems, bacteriocin synthesis genes, phage related proteins, lactose catabolism genes, and several cryptic genes with no annotated function (35). The effect of the deletion on growth phenotype was assessed in broth culture by measuring OD 600 nm over time (
In order to investigate the mechanism of deletion, the nucleotide sequences flanking the segment were determined. The only homologous sequences observed at the junctions were two truncated IS1193 insertion sequences exhibiting 91% nucleotide sequence identity globally over 727 bp. Accordingly, a primer pair flanking the two IS elements was designed to amplify genomic DNA of surviving clones exhibiting the deletion. Each of the deletion strains exhibited a strong band of the predicted size (about 1.2 kb), and confirmed the large genomic deletion event (
Next, CRISPR-Cas targeting was evaluated for the ability to facilitate isolation of deletions for each locus with the same genetic architecture. For this purpose, three CRISPR3 repeat-spacer arrays, one targeting the oligonucleotide transporter in the first locus, prtS from the second locus, and the ATPase copper efflux gene from the third locus were generated and cloned into pORI28 (
In this study, native Type IIA systems harbored in S. thermophilus were repurposed for defining spontaneous deletions of large genomic islands. By independently targeting four islands in S. thermophilus, stable mutants collectively lacking a total of 7% of the genome were generated. Characterization of the deletion junctions suggested that an IS-dependent recombination mechanism contributes to population heterogeneity and revealed deletion events ranging from 8 to 102 kbp. Precise mapping of the chimeric IS elements indicated that natural recombination events are likely responsible for the large chromosomal deletions in S. thermophilus and could potentially be exploited for targeted genome editing.
Our results demonstrate that wild-type clones were removed from the population while mutants without CRISPR-Cas targeted features survived. Thus, adaptive islands were identified and validated, showing that precise targeting by an endogenous Cas9 can be exploited for isolating large deletion variants in mixed populations.
Genome evolution of bacteria occurs through horizontal gene transfer, intrinsic mutation, and genome restructuring. Genome sequencing and comparative analysis of S. thermophilus strains has revealed significant genome decay, but also indicates that adaptation to nutrient-rich food environments occurred through niche-specific gene acquisition (18; 35). The presence of MGEs including integrative and conjugative elements, prophages, and IS elements in S. thermophilus genomes is indicative of rapid evolution to a dairy environment (38-39). Mobile genetic features facilitate gene acquisition and conversely, inactivation or loss of non-essential sequences. Consequently, MGEs confer genomic plasticity as a means of increasing fitness or changing ecological lifestyles. Our results strongly indicate that CRISPR-Cas targeting of these elements may influence chromosomal rearrangements and homeostasis. This is in contrast to experiments targeting essential features, which resulted in selection of variants with inactivated CRISPR-Cas machinery (Jiang 2013). Mutation of essential ORFs is not a viable avenue for circumvention of CRISPR-Cas targeting, and thus only those clones with inactivated CRISPR-Cas systems remain. By design, targeting genetic elements predicted to be hypervariable and expendable demonstrated that variants with altered loci were viable, maintaining active CRISPR-Cas systems during self-targeting events.
Despite the near ubiquitous distribution of IS elements in bacterial genomes they remain an enigmatic genetic entity, largely due to their diversity and plasticity in function (34). Our results suggest it is possible to predict recombination between related IS elements by analyzing their location, orientation, and sequence conservation (
CRISPR-Cas systems generally limit genetic diversity through interference with genetic elements, but acquired MGEs can also provide adaptive advantages to host bacteria. Thus, the benefit of maintaining genomically integrated MGEs despite CRISPR-Cas targeting is an important driver of genome homeostasis. Collectively, our results establish that in sit/co prediction of GEIs can be coupled with CRISPR-Cas targeting to isolate clones exhibiting large genomic deletions. Chimeric insertion sequence footprints at each deletion junction indicated a common mechanism of deletion for all four islands. The high prevalence of self-targeting spacers exhibiting identity to genomic loci, combined with experimental demonstrations of genomic alterations, suggest that CRISPR-Cas self-targeting may contribute significantly to genome evolution of bacteria (28;30). Collectively, studies on CRISPR-Cas induced large deletions substantiate this approach as a rapid and effective means to assess the essentiality and functionality of gene clusters devoid of annotation, and define minimal bacterial genomes based on chromosomal deletions occurring through transposable elements.
Both orthogonal type II systems (CRISPR1 and CRISPR3) were tested; CRISPR1 targets in dark grey, CRISPR3 targets in light grey. Specific genetic features were selected to test (i) intergenic regions (INT), (ii) mobile genetic elements (IS, GEI1-GEI3, PRO, lacZ, EPS), (iii) essential genes (dltA, LTA), (iv) poles of the replichore (ORI, TER), and forward versus reverse strands of DNA (outer targets versus inner targets).
Furthermore, RNA sequencing data supports the boundaries of the deletions by using read coverage mapping and transcriptional value comparisons and additionally supports the discernment of phenotype using comparative transcriptomics generated using the same data set. Specifically, the lack of transcriptional activity present at the expected deletion regions using high-throughput RNA-sequencing is confirmed as shown in
In addition, RNA-sequencing data was used to compare the transcriptional levels of genes not encoded on the deleted island (GEI4), i.e., other genes still present in the chromosome, and identifying phenotypes associated with genomic deletions. Genes that are differentially transcribed in the deletion strain suggest that cellular processes were impacted by the genes that were lost or that there is compensation for the loss of the activity of these genes. Thus, inferences can be made about the pathways to which these genes or genomic regions are relevant. Table 5 provides a list of differentially expressed genes identified in deletion strain GEI4. Many of the genes observed to be differentially expressed relate to the biosynthetic capacity of Streptococcus thermophilus, including aromatic amino acid and purine biosynthesis.
Exemplary CRISPR-Cas Type II guides of L. Casei are provided in
Exemplary Type II guides for targeted killing of L. gasseri are provided in
Plasmids were transformed into L. gasseri each carrying different constructs as follows: an empty pTRK563 vector, a construct with the correct protospacer but an incorrect PAM, the correct PAM but a protospacer that is not in the array, and the correct protospacer with the PAM. The results are shown in
Exemplary Type II guides for targeted killing of L. pentosus are provided in
Plasmids were transformed into L. pentosus, each plasmid carrying different constructs as follows: a construct with the correct protospacer but an incorrect PAM, a construct with a correct PAM but a protospacer that is not in the array, an empty pTRK563 vector, and a correct protospacer with a correct PAM. The results are shown in
Plasmids were transformed into L. jensenii each carrying different constructs as follows: a construct comprising an empty pTRK563 vector, a construct with the correct protospacer but an incorrect PAM, a construct with a correct PAM but a protospacer that is not in the array, and a construct having a correct protospacer with a correct PAM. The results are shown in
CRISPR-Cas systems as described herein may be used for, for example, (i) targeted reduction of pathogens in the case of either medical intervention (e.g., pathogens including but not limited to, fungi, nematodes, protozoa (e.g., malaria), cestodes, coccidia (microsporidia), trematodes, pentastomids, acanthocephalans, arthropods, and the like); (ii) for protection of consumables (food systems, animals, crops); (iii) for control and/or removal of undesirable organisms from industrial fermentative processes (raw materials, processing equipment, starter cultures) and (iv) for control of environmental microbial consortia to impact ecosystems and/or chemical cycles as well as for remediation.
E. coli EC1000
S. thermophilus
S thermophilus
TGTACTCTCAAGATTTAAGTAACTGTACAACATTAAGAGATTGTCTTAACTT SEQ ID NO: 51
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.
This application is a continuation application of U.S. patent application Ser. No. 16/153,052, filed on Oct. 5, 2018, which is a continuation application of U.S. patent application Ser. No. 15/167,727, filed on May 27, 2016, now U.S. Pat. No. 10,136,649, issued on Nov. 27, 2018, which claims the benefit, under 35 U.S.C. § 119 (e), of U.S. Provisional Application No. 62/168,355 filed on May 29, 2015, and U.S. Provisional Application No. 62/296,853, filed on Feb. 18, 2016, the entire contents of each of which is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 62296853 | Feb 2016 | US | |
| 62168355 | May 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16153052 | Oct 2018 | US |
| Child | 17575948 | US | |
| Parent | 15167727 | May 2016 | US |
| Child | 16153052 | US |
