Abstract:
The invention provides compositions and methods for repeatable directed endonucleases (RDEs) and methods for repeatedly, and specifically cleaving DNA offset from the RDE&#39;s DNA recognition sequence on the target nucleic acid rather than within the DNA recognition sequence. Conservation of the recognition sequence of the target nucleic acid enables for re-localization of an RDE back to the DNA recognition sequence for further cleavage. The RDEs and methods of the invention are useful in applications including, but not limited to, recording data into a genome, timing the order of biochemical pathway events, efficient genome engineering and encoding lagged cellular death.

Description:
RELATED APPLICATION 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 62/026,577, filed on Jul. 18, 2014. The entire teachings of the above application are incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Directed endonucleases localize on a specific DNA recognition sequence where they cleave one of both strands of DNA to create a single-strand break (SSB) or double-strand break (DSB). Examples of directed endonucleases include Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs), and proteins, like Cas9, associated with Clustered Regularly Interspaced Palindromic Repeats (CRISPR). Zinc Fingers and Transcription Activator Like Effectors are proteins with peptide sequences designed for binding to its corresponding DNA recognition sequence. The native repair pathways of such breaks in cells as well as identification and engineering of more efficient directed endonucleases have allowed recent art to advance the field of genome engineering and synthetic biology. 
         [0003]    Recently the prior art has provided new artificial combinations of directed endonucleases fused to less specific nuclease domains (Tsai et al. (2014)  Nature Biotechnology ; doi:10.1038/nbt.2908; Guilinger et al.,  Nature Biotechnology  (2014) doi:10.1038/nbt.2909; Sun and Zhao (2014)  Mol. BioSyst.,  10:446; Minczuk et al.,  Nucleic Acids Research,  2008, 36:3926-3938; Ramirez et al.,  Nucleic Acids Research,  2012, doi.1093/nar/gks179. 
         [0004]    It would be desirable to use existing and new directed endonuclease variants and directed endonuclease fusion proteins for use in designing and using self-repeating and highly specific, sequence dependent changes to a targeted region of DNA. 
       SUMMARY OF THE INVENTION 
       [0005]    The invention provides compositions and methods for repeatable directed endonucleases (RDEs) and methods for repeatedly, and specifically cleaving DNA offset from the RDE&#39;s DNA recognition sequence on the target nucleic acid rather than within the DNA recognition sequence. Conservation of the recognition sequence of the target nucleic acid enables for re-localization of an RDE back to the DNA recognition sequence for further cleavage. The RDEs and methods of the invention are useful in applications including, but not limited to, recording data into a genome, timing the order of biochemical pathway events, efficient genome engineering and encoding lagged cellular death. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0006]      FIG. 1  is a diagram of single chain dCas9-FokI-FokI for repeatable cleavage offset to recognition sequence. 
           [0007]      FIG. 2  is a diagram of single chain dCas9-FokI-FokI for repeatable cleavage offset to recognition sequence including a gene for a component of the DRD within the deletable region to halt extension of the deletion. The “deletable region” as used herein should be taken to mean the nucleic acid sequence in the direction of the offset that is positioned between the recognition sequence and the nearest genetic element that is essential for further targeting on the recognition sequence or replication of DNA containing the recognition sequence. 
           [0008]      FIG. 3  is a diagram of single chain dCas9-FokI-FokI for repeatable cleavage offset to recognition sequence showing sequential control of gene expression by including ordered functional non-essential DNA in the deletable region. 
           [0009]      FIG. 4  is a diagram of single chain dCas9-FokI-FokI for repeatable cleavage offset to recognition sequence including essential DNA within the deletable region for delayed cell death, reduction of fitness, or replication. 
           [0010]      FIG. 5  is a diagram of a system for improving efficiency in biomanufacturing through limiting the number of mutations an industrial strain can propagate by programming delayed cell death. 
           [0011]      FIG. 6  is a diagram of single chain dCas9-FokI-FokI for repeatable cleavage offset to recognition sequence showing repeatable asymmetric cleavage for efficient homologous directed recombination. 
           [0012]      FIGS. 7A and 7B  are diagrams of single chain RDEs for efficient homologous recombination induced by one or a pair of single-strand breaks. 
           [0013]      FIGS. 8A and 8B  are diagrams of single chain RDEs for efficient genomic excisions induced by pairs of single-strand breaks or a pair of double-strand breaks. 
           [0014]      FIG. 9  is a diagram of alternative embodiments of the invention using any DRD with HEs and REs that activate upon homodimerization to create either SSBs or DSBs in DNA. 
           [0015]      FIG. 10  is a diagram of alternative embodiments of the invention using any DRD with HEs and REs that activate upon heterodimerization to create either SSBs or DSBs in DNA. 
           [0016]      FIG. 11  is a diagram of alternative embodiments of the invention using any DRD with HEs and REs that do not require dimerization to create either single or double-strand breaks in DNA. 
           [0017]      FIG. 12  is the amino acid sequence of dCas9 (SEQ ID NO: 1). 
           [0018]      FIG. 13  is the amino acid sequence of FokI (SEQ ID NO: 2). 
           [0019]      FIG. 14  is a plasmid map of one preferred RDE of the invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    The invention provides compositions and methods using RDEs for repeatedly cleaving DNA at a roughly fixed number of base pairs offset from a RDE&#39;s DNA recognition sequence. Cutting events can thereby initiate DNA repair end joining pathways that result in a short deletion of base pairs from the position of cleavage. The break in DNA is repaired by native end joining processes, such as Non-Homologous End Joining (NHEJ) or Alternative End Joining (AEJ), which often remove a base from the DNA break. Thus, additional localization events cause additional removal of bases adjacent to the recognition sequence. 
         [0021]    In one embodiment, the present invention provides repeatable directed endonucleases (RDEs) comprising all or a functional fragment of a directed endonuclease fused to all or a functional fragment of a nuclease domain via an amino acid linker sequence. Any directed endonuclease or directed endonuclease binding domain known to those skilled in the art may be used as a DNA-recognition domain (DRD) in the practice of the present invention. Examples of DRDs include, but are not limited to, Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs), and proteins, like Cas9 when associated with a specific guide RNA (Cas9-gRNA), of the Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system. Cas9 of the CRISPER system contains wild type DNA-cleaving activity when complexed with a specific guide RNA (referred to herein as “gRNA” or “sgRNA”) molecule for targeted DNA-binding. An engineered variant of Cas9 called deactivated (dCas9) has a loss of DNA-cleaving function. dCas9 in combination with its gRNA is a particularly preferred DRD of the present invention. 
         [0022]    As used herein, a “functional fragment” of a DRD, nuclease domain or any other protein, polypeptide or nucleic acid as referenced in this application, is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same or has enhanced function as compared to the full-length protein, polypeptide or nucleic acid. Additionally, a functional fragment may have lesser function than the full-length protein, polypeptide or nucleic acid, but still have adequate function as defined by the user. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. Functional fragments of DRDs may comprise at least about 50% sequence similarity with a native binding domain sequence, at least about 60-70%, and at least about 80%-90% or greater sequence similarity with, a native binding domain to retain sufficient binding activity. Such a variant binding domain may include one or more of: an N- or C-terminal truncation, one or more amino acid substitutions, deletions or insertions, or modification of an amino acid, for example, modification of an amino acid side chain entity. 
         [0023]    In a preferred embodiment, the DRD is selected from the group consisting of Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs), or dCas9 associated with a guide RNA. In a preferred embodiment the DRD is dCAS9 associated with a guide RNA.  FIG. 10  shows an amino acid sequence dCas9 (SEQ ID NO: 1; Guilinger et al.,  Nature Biotechnology  (2014) doi:10.1038/nbt.2909). 
         [0024]    As used herein, “nuclease domain” (also referred to herein as a catalytic domain or an induction domain) is a domain responsible for physical cleavage of DNA strands and may introduce either single stranded or double-stranded breaks. The introduction of a single stranded break in DNA is also referred to as a “nick” or “nicking” of the DNA. Examples of nuclease domains include homing endonucleases (HEs) or restriction enzymes (REs). HEs include, but are not limited to, NucA, TevI, I-SceI and ColE7. REs include, but are not limited to, for example FokI, PvuII, NdeI, BsrBI, BsaI, and MMeI. Engineered derivatives and functional fragments of any of these REs and HEs can work as monomers, heterodimers, or homodimers for cleaving on one or both strands of DNA. In a preferred embodiment the nuclease domain is FokI. An exemplary amino acid sequence for FokI is SEQ ID NO: 2; Guilinger et al.,  Nature Biotechnology  (2014) doi:10.1038/nbt.2909). 
         [0025]    Examples of variant nuclease domains include N- or C-terminal truncated nuclease domains, for example, N-terminal truncations of up to about 20 amino acid residues and C-terminal truncations of up to about 15 amino acid residues, and one or more amino acid substitutions, insertions or deletions which do not adversely affect nuclease activity. Suitable amino acid substitutions include conservative amino acid substitutions, for example, substitution of an amino acid with a hydrophobic side chain with a like amino acid, e.g. alanine, valine, leucine, isoleucine, phenylalanine and tyrosine; substitution of an amino acid with an uncharged polar side chain with a like amino acid, e.g. serine, threonine, asparagine and glutamine; substitution of an amino acid having a positively charged side chain with a like amino acid, e.g. arginine, histidine and lysine; or substitution of an amino acid having a negatively charged side chain with a like amino acid, e.g. aspartic and glutamic acid. Variant nuclease domains may also include one or more modified amino acids, for example, amino acids including modified side chain entities which do not adversely affect nuclease activity. 
         [0026]    At least a first nuclease domain is linked to a DRD via a first linking domain. The first linking domain will generally be a polypeptide of a length sufficient to permit the first nuclease domain to retain nuclease function when linked to the DRD, and also sufficient to permit the DNA-binding domain to bind the endonuclease to a target substrate but at a sufficient distance from the cleavage site of the nuclease such that the DNA recognition sequence of the DRD is preserved after cleavage of the target DNA by the nuclease. The first linking domain is not limited to, but may be from 1 amino acid residue to about 125 amino acid residues, from about 1 amino acid residue to about 95 amino acid residues, from about 1 amino acid residue to about 60 amino acid residues, from about 1 amino acid residue to about 70, from about 1 to about 60 amino acid residues, from about 1 to about 50 amino acid residues, from about 1 to about 40 amino acid residues, from about 1 to about 30 amino acid residues, or from about 1 amino acid residue to about 25 amino acid residues. The first linking domain may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42, 44, 45, 46, 47, 48, 49, or 50 amino acid residues in length. The length of the linker domain may be adjusted depending on the distance between the binding and cleavage sites on a target nucleic acid molecule. By including an appropriately sized linker, RDEs of the invention can cleave nucleic acid molecules where the binding and cleavage sites are separated by varying numbers of base pairs. 
         [0027]    The linking domain may be a random sequence, for example, may be one or more glycine residues. The linking domain may be a simple repeat of amino acids, for example, GGS, which may be repeated multiple times. As used herein, such a repeat will be indicated by placing the amino acids in parenthesis and using a subscript to indicate the number of times repeated. Thus (GGS) 6  indicates a linking domain of six repeats of the amino acids glycine-glycine-serine. In some embodiments, the linker domain may comprise one or more glycine residues in addition to one or more other amino acid residues. The linking domain may be flexible or may comprise one or more regions of secondary structure that impart rigidity, for example, alpha helix forming sequences. Table 1 provides example of suitable first linking domains (Guilinger et al.,  Nature Biotechnology  (2014) doi:10.1038/nbt.2909). 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Linker Amino Acid Sequence 
                 SEQ ID NO: 
               
               
                   
                   
               
             
             
               
                   
                 GGSGGSGGS 
                  3 
               
               
                   
                   
               
               
                   
                 GGSGGSGGSGGSGGSGGS 
                  4 
               
               
                   
                   
               
               
                   
                 MKIIEQLPSA 
                  5 
               
               
                   
                   
               
               
                   
                 VRHKLRKRVGS 
                  6 
               
               
                   
                   
               
               
                   
                 VPFLLEPDNINGKTC 
                  7 
               
               
                   
                   
               
               
                   
                 GHGTGSTGSGSS 
                  8 
               
               
                   
                   
               
               
                   
                 MSRPDPA 
                  9 
               
               
                   
                   
               
               
                   
                 GSAGSAAGSGEF 
                 10 
               
               
                   
                   
               
               
                   
                 SGSETPGTSESA 
                 11 
               
               
                   
                   
               
               
                   
                 SGSETPGTSESATPES 
                 12 
               
               
                   
                   
               
               
                   
                 SGSETPGTSESATPEGGSGGS 
                 13 
               
               
                   
                   
               
               
                   
                 GGSM 
                 14 
               
               
                   
                   
               
               
                   
                 SGGGSGGGSGGGSS 
                 15 
               
               
                   
                   
               
             
          
         
       
     
         [0028]    In one preferred embodiment, the RDE of the invention comprises a second nuclease domain fused to the C-terminus of the first nuclease domain via a second linking domain. This creates an RDE construct that is a single-chain quasi-dimeric RDE which is particularly useful when using nuclease domains that require dimer pairs for cleavage such as Fok1 nucleases. The second linking domain between the pair of nuclease domains is to be long enough and flexible enough to allow the formation of a intra-molecular FokI dimer for appropriate cleavage of the target nucleic acid. 
         [0029]    In one embodiment the second linking domain may be from 1 amino acid residue to about 150 amino acid residues, from about 25 amino acid residue to about 125 amino acid residues, from about 30 amino acid residue to about 95 amino acid residues, from about 35 amino acid residue to about 70, 80, 90 or 100 amino acid residues. In one embodiment the seconding linking domain is at least 30 amino acid residues in length, preferably at least 40 amino acid residues in length and preferably at least about 50 amino acid residues in length. Other embodiments may include linkers outside of these ranges. Examples of suitable amino acid sequences for second linking domains are found in Table 2 (Sun and Zhao, 2014 , Mol. BioSyst.,  10:446; Minczuk et al.,  Nucleic Acids Research,  2008, 36:3926-3938). 
         [0000]    
       
         
               
               
             
           
               
                   
               
               
                 Amino Acid Sequence 
                 SEQ ID NO 
               
               
                   
               
             
             
               
                 SSGGGGSGGGGSGGGGSG 
                 16 
               
               
                   
               
               
                 SSGGGGSGGGGSGGGGSGGGGS 
                 17 
               
               
                   
               
               
                 SSGGGGSGGGGSGGGGSGGGGSGGGGS 
                 18 
               
               
                   
               
               
                 SSGGGSGGGSGGGSGGGSGGGSGGGS 
                 19 
               
               
                   
               
               
                 SSGGGSGGGSGGGSGGGSSGGGGSGGGSGGGS 
                 20 
               
               
                   
               
               
                 GSGSRSAPEAMYAPDAVKMVHEFGGSNYTGMTIKMS 
                 21 
               
               
                 TSLTVTDEGSSHTTQPHAENTRRDDQNIGSRFAGES 
                   
               
               
                 HVNNTTKTTTLEEGSGSG G 
                   
               
               
                   
               
               
                 GSGSGSTPWKPIIARHDRRRPLSTGSGSGSG 
                 22 
               
               
                   
               
               
                 GSGSGSITRTTNPRNVVPKIYMSAGSIPLTTHITNS 
                 23 
               
               
                 IQPTLWTIGSINGVAPLAKSIKLGIPVTGSAYTDQT 
                   
               
               
                 TAMVRKKVSVFMGSGSGSG 
                   
               
               
                   
               
               
                 GSGSGSMNKMQPNWTFTWQANSLIGSGSGSG 
                 24 
               
               
                   
               
               
                 GSRFAGESHVNNTTKTTKLEGSGSSGSGSS 
                 25 
               
               
                   
               
               
                 GSGSGSTHRKRHPPMTKEVIPPTAGSKVVKPNLPTN 
                 26 
               
               
                 NARIRWNIGSTLTVWTSVTNMQQEFTTTGSGSGSG 
                   
               
               
                   
               
               
                 GSGSGSNYAAKPIPSAGQLETSHNGSGSGSG 
                 27 
               
               
                   
               
               
                 GSGSGSTTRRWPPGRPNQLRNLTTGSGSGSG 
                 28 
               
               
                   
               
               
                 GSGSGSATMTANFQVNSKPITSPDGSGSG 
                 29 
               
               
                   
               
               
                 GSGSGSAPEAMYAPDAVKMVHEFG GSNYTGMTIKM 
                 30 
               
               
                 STSLTVTDEESSHTTQPRAENTRRD DQNIGSRFAG 
                   
               
               
                 ESHVNNTTKTTKLEGSGSGKRLEPLPDPSD 
                   
               
               
                   
               
               
                 GSGSGSTILKRYSLKNEQPRALHIGSGSG 
                 31 
               
               
                   
               
               
                 GSGSGSVLMTLIDRATASTKPKDAGSAHKGMPHARP 
                 32 
               
               
                 RPWASSTVGSGTLTKIF 
                   
               
               
                   
               
               
                 GGGGSGGGGSGGGGSGGGGSGGGGS 
                 37 
               
               
                   
               
               
                 GGGGS GGGSGGGGSGGGSGGGGS 
               
               
                   
               
             
          
         
       
     
         [0030]    In some embodiments, an RDE of the invention may comprise one or more additional domains. Examples of additional domains include, but are not limited to, linking domains and functional domains. Typically, linking domains may be disposed between two functional domains, for example, between a nuclease domain and a DNA-recognition domain and also between two nuclease domains. Other functional domains include domains comprising nuclear localization signals (NLS), transcription activating domains, dimerization domains, and other functional domains known to those skilled in the art. A suitable NLS, for example is the amino acid sequence PKKKRKV (SEQ ID NO: 33); KRX 10 KKKL (SEQ ID NO: 34); PKKNRLRRP (SEQ ID NO:35); and PLLKKIKQ (SEQ ID NO:36). 
         [0031]    Preferred RDEs of the invention include, but are not limited to, a FokI nuclease or a FokI nuclease dimer pair linked to a dCas9 targeting domain, a zinc finger DNA targeting domain, or a TALEN DNA targeting domain wherein the nuclease is linked at a position that is sufficiently offset from the DNA recognition site of the target nucleic acid such that the DNA recognition site is preserved after cleavage of the target DNA by the nuclease. This allows the RDE to repeatedly relocate to the preserved recognition site and continue to cleave the target DNA until terminated in accordance with the methods of the invention. 
         [0032]    In one preferred embodiment, the RDE comprises a single chain fusion protein comprising dCas9 linked to first FokI protein via a first linker and a second FokI protein linked to the first FokI via a second linker wherein the first linker comprises at least 5 amino acids and the second linker comprises at least 30 amino acids and wherein the first FokI protein is capable of forming a dimer pair with the second FokI protein for cleaving a target nucleic acid. 
         [0033]    The present invention also provides nucleic acid molecules encoding the RDEs of the invention. Such molecules may be DNA or RNA. Typically, DNA molecules will comprise one or more promoter regions operably linked to a nucleic acid sequence encoding all or a portion of an RDE of the invention. Nucleic acid molecules of the invention may be provided as part of a larger nucleic acid molecule, for example, an expression vector. Suitable expression vectors include, but are not limited to, plasmid vectors, viral vectors, and retroviral vectors. Nucleic acid molecules of the invention may be provided as part of a composition, for example, a pharmaceutical composition.  FIG. 14  shows a plasmid map of a preferred RDE of the invention. 
         [0034]    The present invention also provides cells, cell lines and transgenic organisms (e.g., plants, fungi, animals) comprising one or more nucleic acid molecules of the invention. Suitable cells include, but are not limited to, mammalian cells (e.g., mouse cells, human cells, rat cells, etc.) which may be stem cells, avian cells, plant cells, insect cells, bacterial cells, fungal cells (e.g., yeast cells), and any other type of cell known to those skilled in the art. 
         [0035]    Recombinant technology may be used to prepare the RDEs of the invention. In this regard, a DNA construct comprising DNA encoding the selected nuclease, first linking domain, second linking domain (if present), DNA-targeting domain, and any functional domains if present may be inserted into a suitable expression vector which is subsequently introduced into an appropriate host cell (such as bacterial, yeast, algal, fungal, insect, plant and mammalian) for expression. Suitable expression vectors are those vectors which will drive expression of the inserted DNA in the selected host. Typically, expression vectors are prepared by site-directed insertion of a DNA construct therein. The DNA construct is prepared by replacing a coding region, or a portion thereof, within a gene native to the selected host, or in a gene originating from a virus infectious to the host, with the endonuclease construct. In this way, regions required to control expression of the endonuclease DNA, which are recognized by the host including a promoter and a 3′ region to terminate expression, are inherent in the DNA construct. To allow selection of host cells stably transformed with the expression vector, a selection marker is generally included in the vector which takes the form of a gene conferring some survival advantage on the transformants such as antibiotic resistance. Cells stably transformed with endonuclease DNA-containing vector are grown in culture media and under growth conditions that facilitate the growth of the particular host cell used. One of skill in the art would be familiar with the media and other growth conditions. 
         [0036]    The RDEs of the invention may be made using well-established peptide synthetic techniques, for example, FMOC and t-BOC methodologies. In addition, polynucleotides disclosed herein, for example, DNA substrates and DNA encoding the present chimeric endonucleases may also be made based on the known sequence information using well-established techniques. Peptides and oligonucleotides are also commercially available. 
         [0037]    In a further embodiment of the invention, a method of repeatedly cleaving a target nucleic acid is provided comprising the step of contacting a target nucleic acid with a repeatable directed endonuclease (RDE), wherein the RDE binds to its recognition sequence on the target nucleic acid and cleaves the target nucleic acid at a position that is offset from the RDE&#39;s recognition sequence thereby preserving the recognition sequence and wherein after the first cleavage of the target nucleic acid, the preserved recognition sequence is bound by an RDE which makes a second cleavage of the target nucleic acid sequence at a position that is offset from the RDE&#39;s recognition sequence thereby preserving the recognition sequence on the target nucleic acid. Using this method two or more relocalization events of the targeting domain of the RDE cause additional removal of bases adjacent to the recognition sequence. Placement of a gene—by means of synthetic assembly or site-specific homologous recombination—that expresses a protein or nucleic acid component of the RDE complex within the target nucleic acid can allow this process to terminate once the deleted area extends into said gene. If essential DNA is included in the detectable region of the target nucleic acid, then this process can be used to program a delayed cell death with limited cost to fitness before the deleted region extends into essential DNA. Similarly, non-essential functional DNA can be added to the detectable region for sequential control of a biochemical pathway. 
         [0038]    The terms “DNA recognition sequence” and “DNA recognition site” are used synonymously herein and refer to a polynucleotide of a particular sequence which can be bound and cut by a given endonuclease. A polynucleotide of a given sequence may therefore be a DNA recognition sequence or DNA recognition site for one endonuclease, but may or may not be a DNA recognition sequence or DNA recognition site for another endonuclease. 
         [0039]    Therefore, another embodiment provides a method of repeatedly cleaving a target nucleic acid comprising the step of contacting a target nucleic acid with a repeatable directed endonuclease (RDE), wherein the RDE binds to its recognition sequence on the target nucleic acid cleaves the target nucleic acid at a position that is offset from the RDE&#39;s recognition sequence thereby preserving the recognition sequence and wherein after the first cleavage of the target nucleic acid, the preserved recognition sequence is bound by an RDE which makes a second cleavage of the target nucleic acid sequence at a position that is offset from the RDE&#39;s recognition sequence thereby preserving the recognition sequence on the target nucleic acid and wherein the target nucleic acid comprises a promoter or gene for an essential element of the RDE such that when the RDE cleaves the promoter or gene, the cleaving activity of the RDE is terminated. 
         [0040]    In one embodiment of the method, the nuclease domain is a dimer pair of nuclease domains fused in a single chain to the DRD of the RDE. 
         [0041]    In some embodiments, the target nucleic acid may be a gene of interest in a cell. Thus, methods of the invention may be used in genomic editing applications. Typically a method of this type will comprise introducing, into the cell, one or more one RDEs of the invention that bind to a target nucleic acid sequence in the gene (or nucleic acid molecules encoding such chimeric endonuclease under conditions resulting in expression of the chimeric endonucleases), wherein the DNA-targeting domain of the endonuclease binds to the target nucleic acid sequence and the nuclease domain cleaves the target nucleic acid at a position that is offset from the DNA recognition sequence of the target nucleic acid. In some embodiments, cleavage of the gene results in disrupting the function of the gene as repair of the double-stranded break or single stranded break introduced by the RDE of the invention may result in one or more insertions and or deletions of nucleotides at the site of the break. 
         [0042]    Turning now to  FIG. 1 , in a preferred embodiment of the invention, there is constitutive expression of a fusion protein consisting of dCas9 linked to two FokI catalytic homodimeric domains as a single chain. Upon the expression of single guide RNA (sgRNA), the Cas9-sgRNA complex binds to the recognition sequence and positions the linked FokI dimer at an asymmetrically offset adjacent position (Top of Figure). At this offset position, the FokI dimer cleaves DNA to leave a DSB (Middle of Figure), which is repaired by native end joining processes. These end joining processes, like NHEJ and AEJ, often result in a short deletion at the breakage (Betermier et al., 2014, PLOS Genetics 10(1):1-9) that would not extend into the recognition sequence (Bottom of Figure). 
         [0043]    As shown in  FIG. 2 , the preferred embodiment of  FIG. 1  continues to cut asymmetrically offset at roughly the same distance from the recognition sequence of the DRD as expression continues for the entire active DRD-RE/HE complex. Extension of the resulting deleted region is constrained by including in the deletable region the genetic element transcribing a DNA-recognition conferring component in the complex. For the preferred embodiment illustrated in  FIG. 1 , the simplest example would be to include a constitutively driven guide RNA under the U6 promoter. As shown, this can be a promoter and sgRNA pair for the illustrated preferred embodiment. Thus, deletions extending into this element prevent continued localization of the DRD-RE/HE complex to its target recognition sequence. Before deletions extend into the described additional element, use of pathway inducible or repressible promoters in this element can provide a correlation between the length of the deleted region and the activity of the pathway&#39;s associated signals. Measurement of the deleted region can be accomplished through fragment size analysis, NGS sequencing, fluorescent in situ hybridization or sequencing, as well as through expression patterns encoded in the deletable region. The inherent stochastic nature of DNA-binding guarantees variability in the length of the deleted region in individual cells of a population. The average length can be calculated for the population to measure the time integral of RDE expression. However, variability can also be used to generate sequence diversity among single-cells and can be applied to cell barcoding. 
         [0044]    As shown in  FIG. 3  the deletable region can be an endogenous or synthetic sequence. Furthermore, the deletable region can include multiple functional genetic elements. Such functional elements can be used for programmable progression of gene expression. As shown, the deletable region contains RFP and GFP under separate promoters. Partial deletion of the RFP gene, which comes earlier in the deletable region than that of GFP, eliminates further RFP expression and therefore provides optical feedback for an upper bound on the length of the deleted region. Similarly, when the deleted region extends to GFP, observed loss of GFP expression lowers the upper bound of the length of the deletion. 
         [0045]    As shown in  FIG. 4 , without including a genetic element to prevent further cleavage, the deleted region ultimately extends into DNA essential for the cell or for the replication of plasmid DNA. Such essential DNA can be endogenous, such as a polymerase gene or a plasmid&#39;s origin of replication. Essential DNA can also be inserted sequence, such as a gene for drug resistance. Cell death, a drastic reduction of fitness, or preventing a plasmid&#39;s replication are potential results of deleting such DNA. Design of the length of the region between the DNA recognition site of the target and essential DNA enables a programmable delay in the outcome of the latter&#39;s removal. 
         [0046]      FIG. 5  provides a schematic showing that biomanufacturing efficiency is reduced when mutations in an industrial microorganism result in an increase in fitness, but a decrease in efficiency for synthesizing a desired product. Fixation of such mutations in the population can be eliminated by constraining the number of generations that can follow from a microorganism&#39;s replication. As shown, invertase and unidirectional recombination site pairs (e.g. Cre and LoxP) can be used to invert promoters while the microorganism flows from the feeder to manufacturing reactors; Thereby initiating both repeatable offset asymmetric cleavage and the metabolic pathway for manufacturing. Many other inducers and transcriptional control pairs can be used (e.g. small molecule and light-activated transcription factors). Inside the manufacturing reactor, eventual extension of the deletable region to essential DNA in an individual microorganism results in a drastic cost to its fitness, such that the microorganism cannot propagate its mutations. 
         [0047]      FIG. 6  shows that for site-specific mutations with nucleic acid templates (middle strand of  FIG. 6 ) in DRD-based genome engineering, the process of homology-directed repair (HDR) (also known as homologous recombination) competes with faster end joining processes that typically delete part of the recognition sequence in the repair of a DNA break. What&#39;s more, once the recognition sequence is removed, rate of HDR can be nearly negligible. As shown, repeatable cleavage asymmetrically offset from a DNA recognition sequence can preserve the recognition sequence to allow for HDR and end joining to compete again and again, until the occurrence of HDR. This technique may be most-valuable for multiplexed genome engineering for multiple loci. Alternative embodiments for genome engineering are covered in the following two figures. 
         [0048]      FIG. 7A  is a diagram of a system consisting of guide RNA, single-chain dCas9-FokI(ELD-S)-dFokI(KKR-S) and an oligonucleotide donor to achieve homologous recombination on a targeted position on the genome. Note, the ELD-S and KKR-S are a heterodimeric FokI pair, and dFokI denotes a variant consisting of a mutation that eliminates cleavage activity on one strand of the FokI dimer-DNA complex. Therefore, the single chain FokI(ELD-S)-dFokI(KKR-S) domain only induces a nick (or single-stranded break) in a DNA molecule. In the presence of an oligonucleotide donor, homologous recombination pathways result in a genome editing event templated by the donor strand that can also modify the recognition sequence to prevent further nicks. Such repair is described in Metzger et al.,  Nucleic Acids Research ; doi: 10.1093/nar/gkq826. If the nick instead results in non-homologous end joining, then the DNA recognition site is maintained to allow for further nicking events until achieving the desired genomic edits. 
         [0049]      FIG. 7B  is a diagram of a system similar to that of  FIG. 7A , but includes an additional single-chain nicking repeatable directed endonuclease (RDE). Orienting the pair such that offset nicks are interior to the DNA recognition domain enables a double-strand break to form when the resected ends of DNA meet. Again, the presence of an oligonucleotide donor allows for genome editing via homologous recombination.  FIG. 8A  is a diagram of a system similar to that of  FIG. 7B , but includes an additional pair of single-chain nicking RDEs and lacks an oligonucleotide donor. Simultaneous formation of single-stranded (or double-stranded) breaks within each pairs can result in large genomic excisions when the exterior ends from each break are joined via non-homologous end joining. 
         [0050]      FIG. 8B  is a diagram of a system consisting of two single-chain cleaving repeatable directed endonuclease (RDE). Orienting the pair such that offset double-strand breaks are exterior to the DNA recognition domain can result in large genomic excisions when the breaks occur simultaneously. Furthermore, both recognition domains are deleted by the excision. 
         [0051]      FIG. 9  provides additional preferred embodiments achieve DSBs and SSBs with homodimer-activated catalytic domains. Shown in the top left, the preferred embodiment of  FIG. 9  is generalized to encompass any combination of the aforementioned DRDs and homodimer-activated catalytic RE/HE domains for creating a DSB. As shown in the Bottom Left, one preferred embodiment links only one RE/HE domain to the DRD. The two embodiments illustrated on the right achieve single-strand breaks similar to the double-strand breaking embodiments on the right, but instead have one of the homodimer catalytic domains deactivated as described in Ramirez et al., 2012 , Nucleic Acids Research , pp. 1-9; doi:10.1093/nar/gks/179. 
         [0052]      FIG. 10  shows additional preferred embodiments to achieve DSBs and SSBs like those of  FIG. 9 , but instead with heterodimer-activated catalytic domains. A FokI based example of engineering such heterodimeric domains is described in Miller et al., 2007 , Nature Biotechnology,  25:778-785. The link between the DNA binding domain is shown linked to a catalytic domain on the opposite strand as illustrated in  FIG. 9  to reflect that the target site can be on either strand and the linker can be on either termini of the DNA binding domain. Since resection occurs in the 5′-3′ direction (Polo and Jackson, 2011 , Genes  &amp;  Development,  24: 409-433), deletions as a result of SSB can be made unidirectionally away from the recognition sequence. 
         [0053]      FIG. 11  shows additional preferred embodiments achieve DSBs and SSBs with catalytic domains that do not require dimerization for activity such as TevI (Beurdeley et al., 2013 , Nature Communications ; doi: 10/1038/ncomms2782) and I-SceI (Gabsalilow et al.,  Nucleic Acids Research,  2013, 41; doi: 10/1093/nar/gkt080) are dimeric and monomeric examples, respectively, of such catalytic domains. 
         [0054]      FIGS. 12 ,  13 , and  14  include peptide or nucleic acid sequences used for a preferred embodiment of the invention and two plasmid maps that can be used for such an embodiment. In the illustrated example guide RNA is expressed under the control of a pBAD promoter that is induced by the sugar arabinose. A deletion directly downstream of the recognition sequence growing towards the sgRNA is the result of multiple or continued exposures to arabinose. Said deletion no longer extends after removing the sgRNA—a component necessary for localizing the DRD-RE/HE complex to the recognition sequence. 
         [0055]    The ability for an RDE of the invention to repeatedly cleave a nucleic acid target can be determined using well-established techniques, such as next-generation sequencing and PCR-based fragment analysis. Adaptations of recently developed techniques, such as fluorescent in situ hybridization (Lee et al,  Science,  2014, 343; doi: 10.1126/science.1250212) and microfluidic DNA curtions (Redding and Greene,  Chemical Physical Letters,  2013; doi: 10.1016/j.cplett.2013.03.035), are alternative readouts compatible with the invention. 
         [0056]    The RDEs and methods of the invention are useful for example, i) for monitoring activation, exposure or synthesis of biomolecules as described in  FIGS. 2 and 12 ; ii) for sequential control of genetic pathways as described in  FIG. 3 ; iii) for programmable delayed cell death, loss of fitness or plasmid loss as described in  FIG. 4 ; iv) for containment of engineered organisms as described in  FIG. 4 ; v) for population control in biomanufacturing as described in  FIG. 5 ; and vi) for genome engineering as described in  FIG. 6 ,  FIG. 7A-7B , and  FIG. 8A-8B . 
         [0057]    The present invention also provides kits comprising nucleic acid molecules encoding the RDE described above and a substrate for the RDE. In another embodiment, the invention provides kits comprising the RDEs of the invention. Kits of the invention can be used for genomic editing using the methods described above. 
         [0058]    The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. 
         [0059]    While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive and that features from the various embodiments may be combined in whole or in part in accordance with the invention.