Vectors and methods for increased non-coding RNA expression

Abstract

Provided herein are vectors with engineered regulatory sequences for expression of non-coding RNAs (ncRNAs). Regulatory sequences of expression vectors include a Pol III promoter and a binding site for a Pol II - associated transcription factor. Also provided are compositions comprising provided vectors and methods of using the same, e.g., for expression of ncRNAs, e.g., for gene editing.

Description

VECTORS AND METHODS FOR INCREASED NON-CODING RNA EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 669,437 filed July 10, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (JHV-16825_SL; size: 55.7 KB; and date of creation: July 9, 2025) has been submitted electronically in XML format and is herein incorporated by reference in its entirety.BACKGROUND

[0003] Prime editing (PE) is a nucleic acid editing platform that enables the targeted and programmable installation of defined changes in a nucleotide sequence at a desired locus. This advanced technique leverages a combination of a prime editor protein, which is a fusion of a Cas9 nickase and a reverse transcriptase, and a prime editing guide RNA (pegRNA) that directs the desired edit to the target site. Despite its revolutionary potential, there are several challenges and limitations with this mode of genome editing. One significant issue is the expression of pegRNAs, which are crucial for guiding the prime editor to the correct genomic location and ensuring accurate editing. Inefficient pegRNA expression can lead to suboptimal editing outcomes, reducing the overall effectiveness of the prime editing process.

[0004] There are a variety of gene regulatory platforms that rely on the expression of various non-coding RNAs (ncRNAs) including shRNAs, siRNAs, and pegRNAs. Despite the advancements in these platforms there is an ongoing need for innovative technologies that can improve the efficiency of genetic modifications through the modulation of ncRNA expression. Enhancements in the expression of ncRNAs could significantly boost the success rates of gene ablation procedures, paving the way for more reliable and effective genetic modifications in various applications.SUMMARY

[0005] The present disclosure is based, at least in part, on the discovery that vectors having a regulatory sequence that includes a binding site for a Pol II - associated transcription factor have improved expression of Pol Ill-expressed non-coding RNAs (ncRNAs) (i.e., non-protein encoding RNAs). Provided herein are vectors that include regulatory sequences comprising a Pol III promoter and a binding site for a Pol II - associated transcription factor, where the regulatory sequence is operably linked to a sequence encoding a ncRNA (e.g., a non-protein encoding RNA).

[0006] In some embodiments, provided regulatory sequences comprise a Pol III promoter that comprises or consists of a tRNA encoding sequence. In some embodiments, provided regulatory sequences comprise a Pol III promoter that comprises or consists of a U6 promoter, a U3 promoter, a 7SK promoter, or an Hl promoter. In some embodiments, provided regulatory sequences comprise a Pol III promoter that is a cell type-specific promoter. In some embodiments, provided regulatory sequences comprise a Pol III promoter that is a human promoter or derived from a human promoter.

[0007] In some embodiments, provided regulatory sequences comprise one or more binding sites for a Pol II - associated transcription factor. In some embodiments, the one or more binding sites for a Pol II - associated transcription factor are in the context of a tRNA promoter sequence. In some embodiments, provided regulatory sequences comprise a tRNA promoter sequence comprising one or more binding sites for a Pol II - associated transcription factor. In some embodiments, the tRNA promoter sequence can be a human tRNA promoter sequence or derived from it. In some embodiments, the tRNA promoter sequence corresponds to a sequence upstream of a tRNA. In some embodiments, the tRNA promoter sequence comprises a sequence 1-2000 bp upstream of a tRNA. In some embodiments, the tRNA promoter sequence comprises a sequence of at least 200 bp upstream of a tRNA. In some embodiments, the tRNA promoter sequence comprises a sequence 200-2000 bp upstream of a tRNA. In some embodiments, the tRNA promoter sequence comprises a sequence of at least 300 bp upstream of a tRNA. In some embodiments, the tRNA promoter sequence comprises a sequence 300-500 bp upstream of a tRNA.

[0008] In some embodiments, the one or more binding sites for a Pol II - associated transcription factor are or comprise a Pol II promoter. In some embodiments, the Pol II promoter can be a strong constitutive promoter. In some embodiments, a strong constitutive promoter is orincludes, e.g., CMV, EIFla, CVH, CAG, PGK, UBC, CXCL1, SV40, TRE, or human 0-actin. In some embodiments, the Pol II promoter is a cell type-specific promoter. In some embodiments, the Pol II promoter is a human promoter or derived from a human promoter. In some embodiments, the Pol II promoter can include one or more of a TATA box, initiator element (Inr), Downstream Promoter Element (DPE), Upstream Activating Sequence (UAS), TFIIB Recognition Element (BRE), Proximal Promoter Element (PPE), Hormone Response or Heat Schock Response Element, and / or an enhancer element.

[0009] In some embodiments, a Pol II promoter for use in accordance with the present technologies includes a TATA box motif. In some embodiments, a TATA box motif comprises a sequence of TATAAAA.

[0010] In some embodiments, provided regulatory sequences can include a tRNA promoter with Pol II - associated transcription factor binding signatures, a Pol II promoter, or a combination thereof.

[0011] In some embodiments, the expression vector includes a regulatory sequence with a tRNA promoter sequence that binds Pol III and a tRNA promoter sequence comprising one or more binding sites for a Pol II - associated transcription factor, wherein the regulatory sequence is operably linked to a sequence encoding a non-coding RNA.

[0012] In some embodiments, provided are uses for vectors as described herein, for expressing a ncRNA in a cell. In some embodiments, provided are uses for expression vectors as described herein, for increased expression of a ncRNA in a cell, wherein the expression of ncRNA is increased relative to a cell comprising an expression vector comprising regulatory sequence that does not include a Pol II binding sequence.

[0013] In some embodiments, provided are methods of increasing expression of a noncoding RNA (ncRNA), comprising expressing the ncRNA under the control of a regulatory sequence comprising a Pol III promoter sequence and a binding site for a Pol II - associated transcription factor.

[0014] In some embodiments, provided are methods of increasing expression of a noncoding RNA (ncRNA), comprising expressing the ncRNA under the control of a regulatory sequence comprising a tRNA encoding-sequence that binds Pol III and a binding site for a Pol II - associated transcription factor.

[0015] In some embodiments, provided are CRISPR-Cas systems comprising a vector for expression of a guide RNA and / or pegRNA, wherein the vector comprises a regulatory sequence as described herein.

[0016] In some embodiments, provided are methods of altering expression of one or more gene products in a eukaryotic cell. In some embodiments, a method of altering expression of one or more gene products in a eukaryotic cell comprises introducing into the cell a non-naturally occurring CRISPR-Cas system comprising a vector for expression of a ncRNA (e.g., guide RNA or pegRNA) as described herein. In some embodiments, a CRISPR-Cas system comprises a vector for expression a guide RNA and / or pegRNA that hybridizes with a target sequence, the vector comprising a regulatory sequence comprising a Pol III promoter and a binding site for a Pol II - associated transcription factor, operably linked to nucleotide sequence encoding the guide RNA and / or pegRNA. In some embodiments, the CRISPR-Cas system further comprises control elements that provide for transcription of a nucleotide sequence encoding a Type- II Cas9 protein. In some embodiments, the gRNA and / or pegRNA targets and hybridizes with the target sequence and the Cas9 protein cleaves the DNA molecule.

[0017] In some embodiments, provided are cells comprising vectors as described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell (e.g., a human cell). In some embodiments, provided are cells comprising vectors as described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, provided CRISPR-Cas systems (e.g., comprising vectors for expression of a gRNA or pegRNA as described herein) have improved editing efficiency.

[0018] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The Drawings included herein, which is composed of the following Figures, is for illustration purposes only and not for limitation.

[0020] FIG. 1A and FIG. IB are graphs displaying the editing efficiency at the target locus and quantification of pegRNA copy number, respectively, after transfection of components in human embryonic kidney (HEK) cells. FIG. 1A shows data, which presents results of editing efficiency (% insertion) using an fusion of wild type MLV reverse transcriptase to Cas9 (H840A) nickase. FIG. IB is similar to FIG. 1A, but shows data of the quantified number of pegRNAs targeting a specific genomic region of the HEK3 locus. Data was normalized to pegRNAs targeting a control genomic region of the housekeeping GAPDH locus. Specifically, the plasmids with the following architectures were assessed, going from top to bottom: a GFP negative control vector, a U6 promoter only (no coding sequence) vector, a pegRNA only negative control vector, a tRNA only negative control vector, a combined CMV and tRNA promoter operably linked to a pegRNA, a pegRNA flanked by tRNAs, a 500bp tRNA promoter and a tRNA operably linked to a pegRNA, a 300bp tRNA promoter and a tRNA operably linked to a pegRNA, a pegRNA flanked by a 500bp tRNA promoter and a tRNA, a pegRNA flanked by a 300bp tRNA promoter and a tRNA, a pegRNA flanked by a U6 promoter and a tRNA, a combined U6 and tRNA operably linked to a pegRNA, tRNA operably linked to a pegRNA, a U6 promoter operably linked to a pegRNA. Editing rates and pegRNA copy number were quantified by high throughput sequencing of genomic DNA amplicons.CERTAIN DEFINITIONS

[0021] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0022] Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

[0023] In this application, unless otherwise clear from context, (i) the terms “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and "including" may be understood to encompass itemized components or steps whether presented bythemselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

[0024] “Engineered” as used herein refers, in general, to the aspect of having been manipulated by the hand of man. For example, in some embodiments, a polynucleotide may be considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide. In some embodiments, an engineered polynucleotide may comprise a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Alternatively, or additionally, in some embodiments, first and second nucleic acid sequences that each encode polypeptide elements or domains that in nature are not linked to one another may be linked to one another in a single engineered polynucleotide. Comparably, in some embodiments, a cell or organism may be considered to be “engineered" if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, or previously present genetic material has been altered or removed). As is common practice and is understood by persons of skill in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity. Furthermore, as will be appreciated by persons of skill in the art, a variety of methodologies are available through which “engineering"" as described herein may be achieved. For example, in some embodiments, “engineering"" may involve selection or design (e.g., of nucleic acid sequences, polypeptide sequences, cells, tissues, and / or organisms) through use of computer systems programmed to perform analysis or comparison, or otherwise to analyze, recommend, and / or select sequences, alterations, etc.). Alternatively, or additionally, in some embodiments, “engineering"" may involve use of in vitro chemical synthesis methodologies and / or recombinant nucleic acid technologies such as, for example, nucleic acid amplification (e.g., via the polymerase chain reaction) hybridization, mutation, transformation, transfection, etc., and / or any of a variety of controlled mating methodologies. As will be appreciated by those skilled in the art, a variety of established such techniques (e.g., for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.)) are well known in the art and described invarious general and more specific references that are cited and / or discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 and Principles of Gene Manipulation: An Introduction to Genetic Manipulation, 5th Ed., ed. By Old, R.W. and S.B. Primrose, Blackwell Science, Inc., 1994, incorporated herein by reference in their entireties.

[0025] An “expression vector” or “expression construct” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and / or translation of a RNA and / or polypeptide, respectively. The expression vector may include a nucleic acid comprising a promoter sequence, with or without a sequence containing mRNA polyadenylation signals, and one or more restriction enzyme sites located downstream from the promoter allowing insertion of heterologous gene sequences. In some embodiments, the expression vector is capable of directing the expression of a heterologous non-coding RNA when the gene encoding the heterologous non-coding RNA is operably linked to the promoter by insertion into one of the restriction sites. In some embodiments, the recombinant expression construct allows expression of the heterologous non-coding RNA in a host cell when the expression construct containing the heterologous non-coding RNA is introduced into the host cell. Expression constructs can be derived from a variety of sources depending on the host cell to be used for expression. For example, an expression construct can contain components derived from a viral, bacterial, insect, plant, or mammalian source. In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) the inserted polynucleotide sequence need not be identical and can be "substantially identical" to a sequence of the gene from which it was derived.

[0026] As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas system and which associates with and directs the Cas protein to a specific sequence in a DNA molecule that includes complementarity to protospacer sequence of the guide RNA. This term includes guide RNAs that associate with any Cas protein, including but not limited to, Cas9, Casl2a (Cpfl), Casl3, Cas 14, as well as equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant). Without being bound by theory, a guide RNA serves to program the Cas protein to localize to a specific target nucleotide sequence. As used herein, the “guide RNA” may also be referred to as a “traditional guide RNA” to contrast it with the modifiedforms of guide RNA termed “prime editing guide RNAs” (or “pegRNAs”) which may be used in prime editing methods and compositions as described herein.

[0027] As used herein, the term “host cell” refers to a cell into which a nucleic acid or protein has been introduced. Persons of skill upon reading this disclosure will understand that such a term refers not only to the particular subject cell, but also is used to refer to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the phrase "host cell.” In some embodiments, a host cell is or comprises a prokaryotic or eukaryotic cell. In general, a host cell is any cell that is suitable for receiving and / or producing a heterologous nucleic acid or protein, regardless of the Kingdom of life to which the cell is designated. Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of Escherichia coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions such as, for example, hybridomas or quadromas. In some embodiments, a cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, a cell is eukaryotic and is selected from the following cells: Chinese Hamster Ovarian (CHO) (e.g., CHO KI, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, a cell comprises one or more viral genes, e.g., a retinal cell that expresses a viral gene (e.g., a PER.C6® cell). In some embodiments, a host cell is or comprises an isolated cell. In some embodiments, a host cell is part of a tissue. In some embodiments, a host cell is part of an organism.

[0028] As used herein, the term “non-coding RNA (ncRNA)”, also referred to herein as a non-protein encoding RNA (npcRNA) is an RNA molecule that is not translated into a protein. Less frequently, it may also be referred to in the art as non-messenger RNA (nmRNA), functional RNA (fRNA), or a small RNA (in the context of short bacterial ncRNAs). A ncRNA is usually afunctional RNA having a function other than encoding protein, but some may be non-functional or without a known function. The nucleic acid sequence from which a non-coding RNA is transcribed is often called an RNA gene. Non-coding RNA genes include highly abundant and functionally important RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs such as snoRNAs (including scRNA; for nucleotide modification of RNAs), snRNA (for splicing and other functions), gRNA (guide RNA; for mRNA nucleotide modification), pegRNAs (prime editing guide RNA; for mRNA nucleotide modification), RNase P (for tRNA maturation), RNase MRP (for rRNA maturation, and / or DNA replication), Y RNA (for RNA processing, and / or DNA replication), telomerase RNA (for Telomere synthesis), spliced leader RNA, SmY RNA (for mRNA transsplicing), antisense RNA, cis-natural antisense transcript, microRNA (for gene regulation), siRNA (including trans-acting siRNA; for gene regulation), exRNAs, and piRNA (including repeat associated siRNA; for transposon defense, and maybe other functions), 7SK RNA (for negatively regulating CDK9 / cyclin T complex), and long ncRNAs.

[0029] The term “operably linked” means that the regulatory sequence(s) necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. A regulatory sequence is “operably linked” to a coding sequence when it is in a correct functional location and orientation in relation to the coding sequence that expression (e.g., transcriptional initiation and / or expression) of the coding sequence is achieved under conditions compatible with the regulatory sequences. “Operably linked” sequences include both regulatory sequences that are contiguous with the gene of interest and regulatory sequences that act in trans or at a distance to control the gene of interest, and include, e.g., promoters, enhancers, polyadenylation signals, terminators, and the like, which provide for the expression of a coding sequence. In the context of a recombinant expression vector, “operably linked” is intended to mean that the coding sequence is linked to the regulatory element(s) in a manner that allows for expression of the coding sequence (e.g. in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0030] The terms “promoter” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns. Typically, a promoter is a nucleic acid regulatory region capable of binding RNA polymerase in a cell and control initiation and the rate of transcription of adownstream (3' direction) coding or non-coding sequence. The typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. A promoter may also contain subregions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissuespecific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter is referred to as an “endogenous promoter.” Examples of promoters that may be used in the present invention include, e.g., Pol II and Pol III promoters.

[0031] As used herein, the term “Pol II transcription factor binding element” refers to a nucleic acid sequence (e.g., a DNA sequence) that binds to a Pol Il-associated transcription factor. In some embodiments, a Pol II transcription factor binding element is part of a regulatory sequence as described herein.

[0032] As used herein, the term, “regulatory sequences” refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences that may be a combination of synthetic and natural sequences.

[0033] As used herein, the terms “upstream” and “downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5'-to-3' direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5' to the second element. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3' to the secondelement. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the “sense” or “coding” strand. In genetics, a “sense” strand is the segment within double-stranded DNA that runs from 5' to 3', and which is complementary to the antisense strand of DNA, or template strand, which runs from 3' to 5'.

[0034] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Recombinant expression vectors can comprise a nucleic acid of the presently disclosed subject matter in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operably-linked to the nucleic acid sequence to be expressed.DETAILED DESCRIPTION

[0035] The disclosure provides engineered vectors and methods and systems using the same to enhance the expression and production of non-coding RNAs (ncRNAs). Specifically, the presently disclosed expression vectors leverage engineered regulatory sequences comprising a Pol III promoter and one or more Pol II - associated transcription factor binding elements for expression of ncRNAs, such as, e.g., gRNA, pegRNA, siRNA, shRNAs, and related molecules. The present disclosure recognizes that provided vectors can be used to increase the expression of ncRNAs for various applications, such as, e.g., gene therapy.

[0036] In some embodiments, the disclosure provides an innovative approach to gene editing by expressing a gRNA and / or pegRNA under the control of a regulatory sequence comprising a Pol II - associated transcription factor binding element(s) upstream of Pol III promoters. The present disclosure recognizes that the efficiency and precision of CRISPR-based systems may be increased by improving gRNA and / or pegRNA expression. In some embodiments, provided vectors comprise regulatory sequences that enhance the expression of a Pol- III transcribed gRNA and / or pegRNA. In some embodiments, provided are CRISPR-based systems comprising vectors as described herein, which achieve improved targeting and cleavage of DNA sequences, thereby facilitating advanced genetic modifications with higher reliability and success rates.

[0037] The practice of the present technology will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning. A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies — A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1988; Freshney, R. I., “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw- Hill / Appleton & Lange 10thed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, available on the World Wide Web: http: / / www.ncbi.nlm.nih.gov / omim / and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), available on the World Wide Web: http: / / omia.angis.org.au / contact.shtml.Vectors

[0038] The present disclosure provides, inter alia, vectors useful for the expression of one or more RNA polymerase III (Pol III) transcribed sequences, such as, ncRNAs. In some embodiments, provided vectors comprise an engineered regulatory sequence comprising a Pol III promoter and one or more binding sites for a Pol II - associated transcription factor. In some embodiments, the engineered regulatory sequence is operably linked to a sequence encoding a ncRNA. In some embodiments, the Pol III promoter and one or more binding sites for a Pol II - associated transcription factor are upstream and operably linked to a sequence encoding a ncRNA.

[0039] In some embodiments, a provided expression vector is capable of driving expression of one or more Pol III expressed sequences in a cell (e.g., a eukaryotic cell, e.g., a mammalian cell).

[0040] In some embodiments, the expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver- specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid- specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989)EMBO J.8: 729-733) and immunoglobulins (Baneiji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546).

[0041] It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

[0042] In some embodiments, a provided expression vector is a viral vector. Advantageous viral vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells. Thus, as will be apparent to one of ordinary skill in the art, a variety of suitable viral expression vectors are available for transferring exogenous nucleic acid material into cells.

[0043] In some embodiments, a provided expression vector is a plasmid.

[0044] In some embodiments, physical methods to introduce a preselected DNA into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods to introduce the DNA of interest into a host cell include the use of DNA and RNA viral vectors. For mammalian gene therapy, as described hereinbelow, it is desirable to use an efficient means of inserting a copy gene into the host genome. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0045] To confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, PCR, and Next Generation Sequencing (NGS); “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0046] The optimization of the conditions for insertion of a provided expression vector into a cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation.Engineered regulatory sequences

[0047] In some embodiments, provided are engineered regulatory sequences for expression of a Pol III transcribed sequences, such as, ncRNAs. In some embodiments, provided are regulatory sequences comprising a Pol III promoter and one or more binding sites for a Pol II - associated transcription factor, operably linked to a sequence encoding a ncRNA.

[0048] In some embodiments, provided regulatory sequences comprise one or more a Pol II- associated transcription factor binding element(s) upstream of a Pol III promoter. In some embodiments, provided regulatory sequences comprise one or more a Pol II - associated transcription factor binding element(s) downstream of a Pol III promoter.

[0049] In some embodiments, provided regulatory sequences comprise two or more a Pol II- associated transcription factor binding elements. In some embodiments, at least one of the two or more Pol II - associated transcription factor binding elements are upstream of a Pol III promoter.

[0050] In some embodiments, provided regulatory sequences may further include enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Examples of additional regulatory elements are described, for example, in Goeddel (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.

[0051] In some embodiments, provided regulatory sequences may direct constitutive expression of a Pol III transcribed sequence (e.g., ncRNA) in many types of host cells.

[0052] In some embodiments, provided regulatory sequences may direct expression of a Pol III transcribed sequence (e.g., ncRNA) only in certain host cells (e.g., tissue-specific regulatorysequences). In some embodiments, a regulatory sequence may include a tissue-specific promoter that directs expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes).

[0053] In some embodiments, a regulatory sequence may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.

[0054] Regulatory sequences of the present disclosure can be produced using any methods known in the art. For example, regulatory sequences may be produced synthetically, using recombinant cloning, and / or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see U.S. Pat. Nos. 4,683,202 and 5,928,906). Any number of methods can be used to isolate fragments of regulatory sequences disclosed herein. For example, a PCR-based approach can be used to amplify flanking regions from a genomic library of a plant using publicly available sequence information. A number of methods are known to those of skill in the art to amplify unknown DNA sequences adjacent to a known sequence. Methods include, but are not limited to, inverse PCR (IPCR), vectorette PCR, Y-shaped PCR and genome walking approaches.

[0055] In some embodiments, a regulatory sequence of a vector described herein comprises one or more Pol III promoters and one or more binding sites for a Pol II - associated transcription factor, as described herein.

[0056] In some embodiments, a provided engineered regulatory sequence further comprises one or more enhancer elements. Enhancer elements can include, e.g., WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Takebe et al. (1988) Mol. Cell. Biol.S 466-412) SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (O’Hare et al. (1981) Proc. Natl. Acad. Sci. USA. 78(3): 1527-31).Binding sites for a Pol II - associated transcription factor

[0057] In some embodiments, an engineered regulatory sequence of a vector provided herein comprises one or more binding sites for a Pol II - associated transcription factor.

[0058] In some embodiments, a binding site for a Pol II - associated transcription factor is in the context of sequence that corresponds to a sequence upstream of tRNA encoding sequence. In some embodiments, provided vectors comprise a tRNA promoter sequence comprising one or more binding sites for a Pol II - associated transcription factor. In some embodiments, a sequenceupstream of tRNA encoding sequence comprising one or more binding sites for a Pol II - associated transcription factor corresponds to a sequence at least 100 bp, at least 200 bp, or at least 300 bp upstream of a tRNA transcription start site. In some embodiments, a sequence upstream of tRNA encoding sequence comprising one or more binding sites for a Pol II - associated transcription factor corresponds to a sequence 1 - 2000 bp, 200 - 2000 bp, 300 - 2000 bp, 100 - 1000 bp, 200 - 1000 bp, 300 - 1000 bp, 200 - 500 bp, or 300 - 500 bp upstream of a tRNA transcription start site.

[0059] In some embodiments, one or more binding sites for a Pol II - associated transcription factor provided in the context of a sequence that is upstream of tRNA- Arg encoding sequence. In some embodiments, a sequence upstream of a tRNA encoding sequence comprises a sequence that is at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24 or 28.

[0060] In some embodiments, a binding site for a Pol II - associated transcription factor comprises a consensus E2F sequence. In some embodiments, a consensus E2F sequence is TTTSSCGC.

[0061] In some embodiments, a binding site for a Pol II - associated transcription factor comprises a Pol II promoter. Pol II promoters are promoters that direct accurate initiation of transcription by an RNA Polymerase II. Examples of Pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (e.g., Boshart et al. (1985) Cell 41:521-530), the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. In some embodiments, a Pol II promoter comprises a human cytomegalovirus (CMV) promoter, a human ubiquitin C (UBC) promoters, a human histone H2A1 (HIST2H2AA3) promoter, a human inflammatory chemokine CXCL1 promoter, a Simian virus 40 (SV40) promoter, a elongation factor 1 -alpha (EFla) promoters, a phosphoglycerate kinase 1 (PGK) promoter, a human P-actin promoter, a tetracyclineresponsive (Tet) promoter, and / or a lactose operon (Lac) promoter. In some embodiments, a Pol II promoter comprises a promoter as set forth in Table 1.

[0062] Table 1: Example RNA Pol II promoter sequences

[0063] In some embodiments, a Pol II promoter sequence comprises a sequence that is at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1-7.

[0064] In some embodiments, also provided are variant Pol II promoter sequences. Variant Pol II promoter sequences contain changes in which one or more nucleotides of a native sequence is deleted, added, and / or substituted, preferably while substantially maintaining promoter function. Variant Pol II promoter sequences can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant DNA molecule or a portion thereof.

[0065] In some embodiments, the Pol II promoters of the disclosure may be one naturally associated with an endogenously regulated gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. In some embodiments,the Pol II promoter of the regulatory sequence can be, for example, the same Pol II promoter driving expression of the ncRNA of interest.

[0066] In some embodiments, the nucleic acid sequence encoding the ncRNA may be placed under the control of a recombinant or heterologous Pol II promoter, which refers to a promoter that is not normally associated with the targeted gene's natural environment. Such promoters include promoters isolated from any eukaryotic cell, and promoters not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression.

[0067] In some embodiments, an expression vector may contain a Pol II promoter that is operably linked to a nucleic acid sequence encoding a ncRNA.

[0068] In some embodiments, the Pol II promoter is bidirectional.

[0069] In some embodiments, the Pol II promoter is a naturally occurring constitutive promoter controlling the expression of essential cell functions. As a result, a nucleic acid sequence under the control of a constitutive promoter is expressed under all conditions of cell growth. Constitutive promoters include the promoters for the following genes which encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al. (1991)), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the beta-actin promoter (Lai et al. (1989)), and other constitutive promoters known to those of skill in the art.

[0070] In some embodiments, the regulatory sequences are associated with positions of a DNA binding and / or DNA occupying protein for a tissue or cell type.

[0071] In some embodiments, the DNA binding and / or DNA occupying protein is a transcription factor.Pol III promoters

[0072] In some embodiments, provided regulatory sequences of vectors described herein comprise RNA Pol III promoters. Promoters that direct accurate initiation of transcription by an RNA polymerase III are referred to as RNA Pol III promoters. Examples of RNA Pol III promoters for use in accordance with the present disclosure include, without limitation, a tRNA, a U6 promoter, a Hl promoter and promoters of transfer RNAs, 5S ribosomal RNA (rRNA), and the signal recognition particle 7SL RNA (7SL RNA).

[0073] In some embodiments, provided regulatory sequence comprise a tRNA as the Pol III promoter. In some embodiments, a tRNA includes a nucleic acid encoding any one tRNA known in the art such as but limiting to tRNA-Lysine (tRNA-Lys; see Acker et al. 2008. Nucleic acid res. 36(18): 5832-5844), a tRNA-Glutamine (tRNA-Glu), a tRNA- Valine (tRNA Vai; Marek et al. 2006. Nuceic Acid Res. 34(6): 1816-1835) or any other tRNA active in a cell, a tRNA-leucine (tRNA Leu, tRNA-leu(2), tRNA-leu(3)), a tRNA-isoleucine (tRNA-ile), a tRNA-tryptophan (tRNA-trp), a tRNA-tyrosine (tRNA-tyr), a tRNA-histidine (tRNA-his; tRNA-his).

[0074] In some embodiments, a Pol III promoter is or comprises a tRNA- Arg. In some embodiments, a Pol III promoter comprises a tRNA comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 27. In some embodiments, a Pol III promoter comprises a tRNA consisting or comprising a sequence of SEQ ID NO: 27.

[0075] tRNA-Arg: gggccagTGGCGCAATGGataacgcgtctgactacggatcagaagattccaGGTTCGACTCCtggctggctcg (SEQ ID NO: 27)

[0076] In some embodiments, a Pol III promoter is a U6 promoter, a Hl promoter, and / or a tRNA promoter. In some embodiments, a Pol III promoter is as set forth in Table 2.

[0077] Table 2: Example RNA Pol III sequences

[0078] In some embodiments, a Pol III promoter for use in the context of the provided regulatory sequences may contain a TATA box, a Proximal Sequence Element (PSE), and a Distal Sequence Element (DSE). The TATA box, which is named for its nucleotide sequence, is a major determinant of Pol III specificity. It is usually located at a position between nt. -23 and -30 relative to the transcribed sequence, and is a primary determinant of the beginning of the transcribed sequence. The PSE is usually located between nt. -45 and -66. The DSE is typically located upstream of the PSE and plays a crucial role in enhancing the transcriptional activity of the Pol III promoter by facilitating the binding of transcription factors and other regulatory proteins. Together, these elements position Pol III correctly so that it can transcribe the expressed sequence (Murphy et al. (1992) Mol. Cell Biol. 12:3247-3261; Mittal et al. (1996) Mol. Cell Biol. 16: 1955-1965; Ford and Hernandez (1997) J.Biol.Chem., 272: 16048-16055; Ford et al. (1998) Genes, Dev., 12:3528- 3540; Hovde et al. (2002) Genes Dev. 16:2772-2777).

[0079] In some embodiments, also provided are variant Pol III promoter sequences. Variant Pol III promoter sequences contain changes in which one or more nucleotides of a native sequence is deleted, added, and / or substituted, preferably while substantially maintaining promoter function.Variant Pol III promoter sequences can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant DNA molecule or a portion thereof.

[0080] In some embodiments, a Pol III promoter sequence comprises a sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 8-12.Non-coding RNAs (ncRNAs)

[0081] As described herein, the inventors unexpectedly discovered that including one or more binding sites for a Pol II - associated transcription factor in a regulatory sequence comprising a Pol III promoter enhanced expression of Pol- III transcribed ncRNAs.

[0082] In some embodiments, expression vectors provided herein may include more than one expressible ncRNA sequence. In some embodiments with more than one ncRNA encoding sequence, each is operably linked to a different promoter. In some embodiments, a ncRNA of interest includes, but is not limited to, tRNA, rRNA, snoRNAs, snRNA, gRNA, pegRNAs, RNase P, RNase MRP, Y RNA, telomerase RNA, spliced leader RNA, SmY RNA, antisense RNA, cis- natural antisense transcript, microRNA, siRNA, exRNAs, piRNA, 7SK RNA, and IncRNAs.

[0083] The present disclosure demonstrates that regulatory sequence comprising both a Pol III promoter and a Pol II binding elements have improved expression of ncRNAs, relative to a regulatory sequence comprising only the corresponding Pol III promoter. In some embodiments, an expression vector may contain a Pol II promoter that is operably linked to a nucleic acid sequence encoding a ncRNA. Thus, the Pol II promoter, i.e., a RNA polymerase II dependent promoter, initiates the transcription of the ncRNA.CRISPR

[0084] In some embodiments provided are CRISPR systems comprising vectors as described herein.

[0085] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) together with Cas (CRISPR-associated) genes comprise an adaptive immune system that provides acquired resistance against invading foreign nucleic acids in bacteria and archaea (Barrangou et al. (2007) Science 315:1709-12). CRISPR consists of arrays of short conserved repeat sequences interspaced by unique variable DNA sequences of similar size called spacers, which often originate from phageor plasmid DNA (Barrangou et al. (2007) Science 315: 1709-12; Bolotin et al. (2005) Microbiology 151 :2551-61 ; Mojica et al. (2005) J. Mol. Evol. 60:174-82). The CRISPR-Cas system functions by acquiring short pieces of foreign DNA (spacers) which are inserted into the CRISPR region and provide immunity against subsequent exposures to phages and plasmids that carry matching sequences (Barrangou et al. (2007) Science 315:1709-12; Brouns et al. (2008) Science 321:960-64). It is this CRISPR-Cas interference / immunity that enables crRNA-mediated silencing of foreign nucleic acids (Horvath & Barrangou (2010) Science 327:167-70; Deveau et al. (2010) Annu. Rev. Microbiol. 64:475-93; Marraffini & Sontheimer (2010) Nat. Rev. Genet. 11: 181-90; Bhaya et al. (2011) Annu. Rev. Genet. 45:273-97; Wiedenheft et al. (2012) Nature 482:331-338).

[0086] In some embodiments, the presently disclosed methods utilize a composition comprising a non-naturally occurring CRISPR system comprising one or more vectors comprising: a) a Pol II or Pol III promoter or combination thereof, operably linked to at least one nucleotide sequence encoding a CRISPR system prime editing guide RNA (pegRNA), wherein the pegRNA hybridizes with a target sequence of a DNA molecule in a cell, and wherein the DNA molecule encodes one or more gene products expressed in the cell; and b) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a Cas protein (e.g., Cas9), wherein components (a) and (b) are located on the same or different vectors of the system, wherein the pegRNA targets and hybridizes with the target sequence and the Cas protein cleaves the DNA molecule to alter expression of the one or more gene products.Cells

[0087] The present disclosure is further directed, in part, to cells comprising an expression vector as described herein. Any cell, e.g., cell line, e.g., a cell line suitable for expression of a ncRNA, known to one of skill in the art can be used. In some embodiments, a cell, e.g., cell line, may be used to express an expression vector, described herein. In some embodiments, a cell comprises an expression vector(s), described herein.

[0088] In some embodiments, a cell or cell line for expression of a vector and / or ncRNA as described herein in a mammalian cell or cell line.

[0089] Any suitable mammalian cell line known in the art can be engineered or screened in the context of the present disclosure. Mammalian cells for expression of viral vectors can include any mammalian cell type known in the art. Representative mammalian cells include, but are notlimited to, human embryonic kidney (HEK) cells (e.g., HEK 293 cells, HEK 293T cells, Expi293 cells), Chinese hamster ovary (CHO) cells, HeLa cells (e.g., HeLa S3 cells), PER.C6 cells, HKB1 1 cells, CAP cells, Baby Hamster Kidney fibroblasts (BHK cells) (e.g., BHK-21 cells), mouse myeloma cells (e.g., Sp2 / 0 cells and NSO cells), green African monkey kidney cells (e.g., COS cells and Vero cells), A549 cells, rhesus fetal lung cells (e.g., FRhL-2 cells), and any derivatives thereof. In some embodiments, mammalian cells of the present disclosure are highly transfectable.

[0090] In some embodiments, a mammalian cell line of the present disclosure is suitable for manufacturing of biologies. In some embodiments, a mammalian cell line is suitable for use in industrial-scale manufacturing of a biologic product. In some embodiments, a mammalian cell line is suitable for use in a method of manufacture that conforms with local regulatory standards (e.g., FDA and / or EMA regulatory standards). In some embodiments, a mammalian cell line is suitable for manufacturing of biologies (e.g., viral vectors) using current good manufacturing practices (cGMP). In some embodiments, a mammalian cell line is suitable for manufacturing of biologies using good manufacturing practices (GMP). In some embodiments, a mammalian cell line is suitable for manufacturing of biologies using non-good manufacturing practices (non-GMP).

[0091] In some embodiments, a cell comprising a vector described herein and / or for expression of a ncRNA as described herein is in a subject. In some embodiments, a cell comprising a vector described herein and / or for expression of a ncRNA as described herein is for administration to a subject.

[0092] In some embodiments, a subject is a mammal. In some embodiments, a mammal is a human.Compositions

[0093] In some embodiments, provided herein are compositions comprising vectors of the present disclosure. In some embodiments, a provided composition is a pharmaceutical composition comprising a vector of the present disclosure and a pharmaceutically acceptable carrier.

[0094] A “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically - acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically - acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or poly anhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0095] The pharmaceutical formulations of the present disclosure may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non-limiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions pH 7.0-8.0.Kits

[0096] Provided herein a pack or kit comprising one or more containers filled with at least one vector as described in the description, examples, and / or figures herein. Kits may be used in any applicable method (e.g., a research method). Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, (c) a contract that governs the transfer of materials and / or biological products between two or more entities and combinations thereof.MethodsMethods of expressing ncRNAsIn some embodiments, the present disclosure provides methods of expressing ncRNAs in a cell, comprising introducing into the cell a vector of the present disclosure. In some embodiments, provided are methods of increasing expression of ncRNA, comprising expressing the ncRNA using a vector of the present disclosure. In some embodiments, the expression of ncRNA using provided vectors is increased relative to expression using a standard comparator (e.g., a vector comprising a U6 promoter). In some embodiments, the expression of ncRNA using provided vectors is increased relative to expression using a comparable vector that does not include the binding site(s) for a Pol II - associated transcription factor.Methods of gene editing

[0097] In some embodiments, the present disclosure provides methods of genetically engineering one or more gene products in a cell, wherein the cell comprises a DNA molecule encoding the one or more gene products, the method comprising introducing into the cell a non- naturally occurring CRISPR-Cas system comprising one or more vectors comprising: a) a Pol II or Pol III promoter, or combination thereof, operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system prime editing guide RNA (pegRNA), wherein the pegRNA hybridizes with a target sequence of the DNA molecule; and b) a regulatory element operable in the cell operably linked to a nucleotide sequence encoding a Cas protein (e.g., Cas9), wherein components (a) and (b) are located on different vectors of the system, wherein the pegRNA targets and hybridizes with the target sequence and the Cas protein cleaves the DNA molecule.

[0098] In some embodiments, the Cas protein (e.g., Cas9) is codon optimized for expression in the cell. In some embodiments, the Cas protein is codon optimized for expression in the eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian or human cell. In some embodiments, the editing efficiency of the CRISPR-Cas system prime editing system is increased. In some embodiments, the presently disclosed subject matter provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and / or one or proteins transcribed therefrom, to a host cell. In someembodiments, the presently disclosed subject matter further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.Methods of delivery

[0099] In some embodiments, provided herein are methods for delivering the CRISPR / Cas- based editing system for providing genetic constructs and / or proteins of the CRISPR / Cas-based editing system. The delivery of the CRISPR / Cas-based editing system may be the transfection or electroporation of the CRISPR / Cas-based editing system as one or more nucleic acid molecules that is expressed in the cell and delivered to the surface of the cell. The CRISPR / Cas-based editing system protein may be delivered to the cell. The nucleic acid molecules may be electroporated using BioRad Gene Pulser Xcell or Amaxa Nucleofector lib devices or other electroporation device.Several different buffers may be used, including BioRad electroporation solution, Sigma phosphate- buffered saline product # D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N.V.). Transfections may include a transfection reagent, such as Lipofectamine 2000.

[0100] The vector encoding a CRISPR / Cas-based editing system protein may be delivered to the modified target cell in a tissue or subject by DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, and / or recombinant vectors. The recombinant vector may be delivered by any viral mode. The viral mode may be recombinant lentivirus, recombinant adenovirus, and / or recombinant adeno-associated virus.EXAMPLESExample 1: Conditional Pol II / Pol III promoter expression for regulating ribonucleoprotein enzymatic activity and expression of small non-coding RNAs.

[0101] The present example demonstrates that synthetic constructs with example engineered regulatory sequences as described herein efficiently express example ncRNAs (pegRNAs), which subsequently led to successful genomic modifications (insertions). Plasmids with different example Pol III promoters (U6 or endogenous tRNA), alone or in combination with one or more Pol II transcription factor binding elements (e.g., upstream region of tRNA and / or example Pol II promoters) were assessed for the ability to express pegRNAs targeting an example locus, the HEK3locus. Specifically, editing at the locus (FIG. 1A) and expression of pegRNA (FIG. IB) was assessed for plasmids having architectures as set forth in Table 3 below (SEQ ID NOs: 13-22). Results were normalized to a positive control construct with a U6 promoter operably linked to the example pegRNA.

[0102] Constructs with tRNA as the Pol III promoter were functional, but had reduced pegRNA expression and editing relative to the U6 control. In constructs where the tRNA is located downstream of the sequence encoding the guide RNA, the tRNA was not able to work as a promoter. Without wishing to be bound by theory, it is thought that a tRNA downstream of a guide RNA may still facilitate processing and potential multiplexing. Constructs including a 300 bp or 500 bp region upstream of an example tRNA, but without a Pol III promoter sequence were nonfunctional. In contrast, the incorporation of a 300 bp or 500 bp region upstream of an example tRNA and a tRNA promoter upstream of a pegRNA with a tRNA had significantly enhanced expression of the pegRNA, This enhanced pegRNA expression was correlated with improved editing efficiency. Additionally, the inclusion of a Pol II promoter (e.g., CMV) upstream of the tRNA promoter further augments pegRNA expression and enhances editing efficiency.

[0103] These results support that engineered regulatory regions including a Pol III promoter (e.g., a tRNA) and one or more Pol II transcription factor binding elements, can enhance the expression of a ncRNA (such as a guide RNA) and improve functions thereof (e.g., gene editing).

[0104] Table 3 - Example Vector SequencesMethods

[0105] Identification of endogenous Pol III promoters and vector design'. New Pol III promoter architectures were designed starting from endogenous tRNA and upstream sequences in the Hg38 reference genome. Sequences of tRNAs were identified from GtRNAdb (available from UCSC). Publicly available DNase I hypersensitive (DHS) and Assay for Transposase- AccessibleChromatin (ATAC) data from the ENCODE project was used to train a gkmSVM model to identify and rank regulatory motifs associated with tRNA expression. In silico plasmid design was performed in SnapGene and Benchling.

[0106] Plasmid construction: Expression vectors for ncRNAs were cloned using USER cloning or Gibson assembly. Primers were obtained from IDT as standard ssDNA oligos. Gene fragments were obtained from Twist as dsDNA. PCR was performed using Phusion U Polymerase (ThermoFisher). Enzymes for USER or Gibson cloning were obtained from NEB. Cloning was done in NEB5alpha E. coli cells. The HEK3+1CTT pegRNA plasmid (available on Addgene) with U6 promoter was used as the vector for cloning and positive / baseline control in dPCR and editing experiments. Plasmid sequencing was done by Sanger sequencing at GRCF or whole plasmid oxford nanopore sequencing at Plasmidsaurus or Quintara.

[0107] Cell culture: All cell culture work was done with human embryonic kidney (HEK) cell line 293T (Life Technologies, Grand Island, NY) supplied by ATCC. Cells were maintained at 37°C with 5% CO2 / 20% O2 in Dulbecco’s modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Grand Island, NY) and 2mM GlutaMAX (Invitrogen). For cell transfection, 1.6-1.8 x IO4cells were seeded in 96 well plates (Falcon, Corning, NY) 24 hours prior to transfection. Cells were transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer’s recommended protocol. For each well of a 96-well plate, 200ng of the Cas9 plasmid, 66ng of pegRNA vector, and 22ng of nicking sgRNA vector were mixed with 0.5pl of Lipofectamine 2000 and 4.5pl of Opti-MEM. Following 72h posttransfection, cells were lysed for DNA and RNA extraction. DNA was extracted following protocol from Doman et al. RNA was extracted using QIAgen’s miRNEasy advanced 96 kit (QIAgen) according to manufacturer’s instructions. Editing was assessed with Illumina sequencing.Expression was assessed by dPCR using a QIAgen QIAcuity Four and QIAgen’s QIAcuity Probe PCR Kit reagents (QIAgen) according to manufacturer’s instructions. Primers and probes for the dPCR assay were custom designed and ordered from IDT. Illumina data was analyzed with CRISPResso2. Downstream analysis was done in R and GraphPad Prism.INCORPORATION BY REFERENCE

[0108] All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control.

[0109] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.EQUIVALENTS

[0110] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. An expression vector for expression of a non-coding RNA (ncRNA), comprising:(a) an engineered regulatory sequence comprising a Pol III promoter sequence and one or more binding sites for a Pol II - associated transcription factor;(b) a sequence encoding the ncRNA, wherein the ncRNA is a non-protein encoding RNA.

2. The expression vector of claim 1 , wherein the Pol III promoter sequence comprises a tRNA encoding sequence.

3. The expression vector of claim 2, wherein the tRNA encoding-sequence is a human tRNA encoding-sequence or derived from a human tRNA encoding-sequence.

4. The expression vector of claim 1, wherein the Pol III promoter sequence comprises a U6 promoter, a U3 promoter, a 7SK promoter, a 5S rRNA promoter, or a Hl promoter.

5. The expression vector of claim 1, wherein the Pol III promoter sequence is a cell type specific promoter.

6. The expression vector of any one of claims 1-4, wherein the Pol III promoter is a human sequence or derived from a human sequence.

7. The expression vector of any one of claims 1-6, wherein the binding site for a Pol II - associated transcription factor sequence comprises a tRNA promoter.

8. The expression vector of any one of claims 1-7, wherein the one or more binding sites for a Pol II - associated transcription factor comprises a Pol II promoter.

9. The expression vector of any one of claims 1-8, wherein the one or more binding sites for a Pol II - associated transcription factor comprise a tRNA promoter comprising signatures of Pol II - associated transcription factors and a Pol II promoter.

10. The expression vector of claim 7 or 9, wherein the tRNA promoter comprises a sequence 1-2000 bp upstream of tRNA.

11. The expression vector of claim 7 or 9, wherein the tRNA promoter is operably linked to a sequence 300-500 bp upstream of tRNA.

12. The expression vector of claim 8 or 9, wherein the Pol II promoter is a strong constitutive Pol II promoter.

13. The expression vector of claim 12, wherein the Pol II promoter is selected from a CMV promoter, a EIF1 a promoter, a CVH promoter, a CAG promoter, a PGK promoter, a UBC promoter, a CXCL1 promoter, an SV40 promoter, a TRE promoter, a human P-actin promoter, a LacO promoter.

14. The expression vector of any one of claims 1-13, wherein the ncRNA is a guide RNA (gRNA).

15. The expression vector of any one of claims 1-13, wherein the ncRNA is a prime editing guide RNA (pegRNA).

16. The expression vector of any one of claims 1-13, wherein the ncRNA is a Pol III transcribed ncRNA.

17. Use of an expression vector of any one of claims 1-16, for expressing a ncRNA in a cell.

18. The use of claim 17, wherein the expression of ncRNA is increased relative to a cell comprising an expression vector comprising regulatory sequence that comprises the same Pol III promoter but does not include the one or more binding sites for a Pol II - associated transcription factor comprises a Pol II promoter.

19. A cell comprising the expression vector of any one of claims 1-16.

20. The cell of claim 19, wherein the cell is a eukaryotic cell.

21. The cell of claim 19 or 20, wherein the cell is a mammalian cell.

22. The cell of any one of claims 19-21, wherein the cell is a human cell.

23. A method of increasing expression of a non-coding RNA (ncRNA), comprising expressing the ncRNA using a vector of any one of claims 1-16.

24. The method of claim 23 wherein the expression of ncRNA is increased relative to expression by a vector comprising a regulatory sequence that comprises the same Pol III promoter but does not include the one or more binding sites for a Pol II - associated transcription factor comprises a Pol II promoter.

25. A method for gene editing in a cell, comprising contacting the cell with a CRISPR system, wherein the CRISPR system comprises a vector comprising (a) an engineered regulatory sequence comprising a Pol III promoter sequence and a binding site for a Pol II - associated transcription factor and (b) a sequence encoding a pegRNA, wherein the pegRNA hybridizes with a target sequence of a DNA molecule in the cell.

26. The method of claim 25, wherein the gene editing efficiency is improved relative to a method using a CRISPR system comprising a vector where the pegRNA is expressed by a U6 promoter.

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