Composition and method for modifying genome

Abstract

To provide a composition and a method for modifying a genome DNA sequence.SOLUTION: A method for modifying a nucleotide sequence at a target site in a genome in an eukaryotic cell includes a step of introducing (i) DNA polynucleotide encoding DNA-targeted RNA or DNA-targeted RNA, and (ii) Cpf1 or Csm1 polypeptide, or polynucleotide encoding Cpf1 or Csm1 polypeptide into the eukaryotic cell, where a genome of the eukaryotic cell is a nucleus, plastid, or a mitochondrial genome.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to compositions and methods for genome sequence editing and modulation of gene expression at preselected locations.

[0002] References for sequence listings submitted as text files on EFS-WEB An official copy of this sequence listing has been submitted contemporaneously herewith as a text file via EFS-Web, with file name B88552_1060WO_0057_1_Seq_List.txt, file size 1.62 MB, in information interchange code (ASCII), created on February 14, 2017. The sequence listing filed via EFS-Web is a part of this specification and is hereby incorporated by reference in its entirety. [Background technology]

[0003] Modification of genomic DNA is of great importance for basic and applied research. Genome modification has the potential to elucidate and possibly cure the causes of diseases, and to provide desirable traits to cells and / or individuals containing the modification. Genome modification includes, for example, the modification of plants, animal, fungal, and / or prokaryotic genomes, etc. One of the areas in which genome modification is practiced is the field of plant genomic DNA modification.

[0004] Modification of plant genomic DNA is of great importance for both basic and applied plant research. Transgenic plants with stably modified genomic DNA can have new properties such as herbicide resistance, insect resistance, and / or accumulation of valuable proteins, including pharmaceutical proteins and industrial enzymes, endowed to them. The expression of a native plant gene can be altered in any way, either up-regulated or down-regulated (e.g., by changing the tissue in which the native plant gene is expressed), its expression can be completely disrupted, the DNA sequence can be altered (e.g., through point mutations, insertions, or deletions), or new non-native genes can be inserted into the plant genome to confer new traits on the plant.

[0005] The most common methods of modifying plant genomic DNA tend to modify DNA at random sites within the genome. Such methods include, for example, Agrobacterium-mediated plant transformation and biolistic transformation, also called particle bombardment. However, in many cases, it is desirable to modify genomic DNA at a predetermined target site within the plant genome of interest, for example, to avoid disruption of native plant genes or to insert a transgene cassette into a genomic locus that is known to provide robust gene expression. More recently, techniques for targeted modification of plant genomic DNA have become available. Such techniques rely on creating a double-stranded break (DSB) at the desired site. This DSB triggers the recruitment of the plant's natural DNA repair mechanisms against the DSB. The DNA repair mechanisms can be utilized to insert heterologous DNA at a predetermined site, to delete native plant genomic DNA, and to generate point mutations, insertions, or deletions at the desired site. Summary of the Invention

[0006] Compositions and methods are provided for modifying genomic DNA sequences. As used herein, genomic DNA refers to linear and / or chromosomal DNA, and / or to plasmids or other extrachromosomal DNA sequences present in a cell of interest. The methods generate double-strand breaks (DSBs) at predetermined target sites in the genomic DNA sequence, resulting in mutations, insertions, and / or deletions of DNA sequences at the target sites in the genome. The compositions include DNA constructs that include nucleotide sequences encoding Cpf1 or Csm1 proteins operably linked to a promoter operable in a cell of interest. The DNA constructs can be used to direct modification of genomic DNA at predetermined genomic loci. Methods of using these DNA constructs to modify genomic DNA sequences are described herein. Modified plants, plant cells, plant parts, and seeds are also included. Compositions and methods for regulating gene expression are also provided. The methods target proteins to predetermined sites in the genome to effect upregulation or downregulation of genes whose expression is regulated by the target location in the genome. The compositions contain nucleotide sequences encoding modified Cpf1 or Csm1 proteins with reduced or eliminated nuclease activity, optionally fused to a transcriptional activation or repression domain. Methods for using these DNA constructs to modify gene expression are described herein. [Brief description of the drawings]

[0007] [Figure 1] Figure 1 shows a schematic diagram of inserting a hygromycin resistance gene cassette into the rice CAO1 locus. The star indicates the site of the intended Cpf1-mediated double-strand break in wild-type DNA. The dashed line indicates the homology between the repair donor cassette and wild-type DNA. The small arrows indicate primer binding sites for the PCR reaction used to confirm the insertion at the intended genomic locus. 35STerm., CaMV35S terminator; hph, hygromycin resistance gene; ZmUbi, maize ubiquitin promoter. [Diagram 2]Figure 2 shows sequence data obtained from rice callus generated during experiment 1. Figure 2A shows the results of inserting the hph cassette at the CAO1 locus. The PAM sequence is boxed and the sequence targeted by the guide RNA is underlined. Ellipses indicate the presence of a large insertion, where full sequence data is not shown. Figures 2B, 2C, and 2D show data obtained from rice callus in experiment 01 (Table 7) where an FnCpf1-mediated deletion event occurred. In Figures 2B and 2C, the lanes show, from left to right, callus pieces #1-16 with molecular weight ladder lanes. Figure 2B shows PCR amplification of the FnCpf1 gene cassette, indicating the insertion of this cassette into the rice genome in callus pieces 1, 2, 4, 6, 7, and 15. Figure 2C shows the results of a T7EI assay with DNA extracted from the same callus pieces, along with a double band pattern in callus #15 indicating a possible insertion or deletion. Similar T7EI assay results were obtained on additional calli in repeats of experiment 01, resulting in the generation of callus pieces 01-20, 01-21, 01-30, and 01-31. Figure 2D shows an alignment of sequence data obtained from callus #15 (01-15) with sequence data obtained from callus pieces 01-20, 01-21, 01-30, and 01-31. The PAM sequence is boxed and the sequence targeted by the guide RNA is underlined. [Diagram 3]FIG. 3 shows sequence data from experiments 31, 46, 80, 81, 91, and 93, confirming Cpf1- and Csm1-mediated indels (insertions, deletions) at the rice CAO1 genomic locus. Figure 3A shows the alignment of the wild-type rice CAO1 locus with callus piece #21 (31-21) from experiment 31, callus piece #33 (80-33) from experiment 80, callus pieces #9, 30, and 46 from experiment 81 (81-09, 81-30, and 81-46, respectively), callus piece #47 (93-47) from experiment 93, callus piece #4 (91-04) from experiment 91, callus pieces #112 and 141 (97-112 and 97-141) from experiment 97, and callus pieces #4 and 11 (119-04 and 119-11) from experiment 119. Figure 3B shows the sequence data from callus pieces 46-38, 46-77, 46-86, 46-88, and 46-90 from experiment 46. In both 4A and 4B, the PAM site is boxed and the sequence targeted by the guide RNA is underlined. [Figure 4-1]FIG. 4 shows an overview of the unexpected recombination events recovered from runs 70 and 75. FIG. 4A shows a schematic diagram of the portion of the 131633 plasmid containing the 35S terminator and downstream arm homology regions that led to the recombination events recovered from run 70. The region of homology that appears to have mediated the unintended HDR event is underlined. FIG. 4B shows sequence data from callus piece 70-15. WT, wild type sequence; GE70, callus piece 70-15 sequence; 131633_upstream, upstream arm and 35STerm sequence from plasmid 131633; 131633_downstream, downstream arm sequence from plasmid 131633. FIG. 4C shows a schematic diagram of the portion of the 131633 plasmid containing the 35S terminator and downstream arm homology regions that led to the recombination events recovered from run 75. The region of homology that appears to have mediated the unintended HDR event is underlined. Figure 4D shows sequence data from callus pieces 75-46. WT, wild-type sequence; GE75, callus piece 75-46 sequence; 131633_upstream, upstream arm and 35STerm sequence from plasmid 131633; 131633_downstream, downstream arm sequence from plasmid 131633. 35STarm, CaMV35S terminator; hph, hygromycin phosphotransferase coding region; pZmUbi, maize ubiquitin promoter. PAM sites are boxed in Figures 4B and 4D. [Figure 4-2]FIG. 4 shows an overview of the unexpected recombination events recovered from runs 70 and 75. FIG. 4A shows a schematic diagram of the portion of the 131633 plasmid containing the 35S terminator and downstream arm homology regions that led to the recombination events recovered from run 70. The region of homology that appears to have mediated the unintended HDR event is underlined. FIG. 4B shows sequence data from callus piece 70-15. WT, wild type sequence; GE70, callus piece 70-15 sequence; 131633_upstream, upstream arm and 35STerm sequence from plasmid 131633; 131633_downstream, downstream arm sequence from plasmid 131633. FIG. 4C shows a schematic diagram of the portion of the 131633 plasmid containing the 35S terminator and downstream arm homology regions that led to the recombination events recovered from run 75. The region of homology that appears to have mediated the unintended HDR event is underlined. Figure 4D shows sequence data from callus pieces 75-46. WT, wild-type sequence; GE75, callus piece 75-46 sequence; 131633_upstream, upstream arm and 35STerm sequence from plasmid 131633; 131633_downstream, downstream arm sequence from plasmid 131633. 35STarm, CaMV35S terminator; hph, hygromycin phosphotransferase coding region; pZmUbi, maize ubiquitin promoter. PAM sites are boxed in Figures 4B and 4D. [Diagram 5] Figure 5 shows the sequence of the upstream region of callus piece #46-161 from experiment 46 (Table 7). The PAM site is boxed, indicating the expected mutation of this site in transformed rice callus, and the sequence data indicates successful insertion of vector 131633 at the rice CAO1 locus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Invention Provided herein are methods and compositions for controlling gene expression, including sequence targeting, such as genome perturbation or gene editing, involving CRISPR-Cpf or CRISPR-Csm systems and their components. In some embodiments, the CRISPR enzyme is a Cpf enzyme, such as a Cpf1 orthologue. In some embodiments, the CRISPR enzyme is a Csm enzyme, such as a Csm1 orthologue. The methods and compositions include nucleic acids for binding to target DNA sequences. This is advantageous, since nucleic acids are much easier and cheaper to produce, and specificity can be altered depending on the length of the stretch of homology required, for example, compared to peptides. For example, complex 3-D arrangements of multiple fingers are not required.

[0009] Also provided are nucleic acids encoding Cpf1 or Csm1 polypeptides, as well as methods of using Cpf1 or Csm1 polypeptides to modify chromosomal (i.e., genome) or organelle DNA sequences in host cells, including plant cells. Cpf1 polypeptides interact with specific guide RNAs (gRNAs), which direct Cpf1 or Csm1 endonuclease to specific target sites, where the Cpf1 or Csm1 endonuclease introduces double-strand breaks that can be repaired by DNA repair processes, such that the DNA sequence is modified. Because of the properties provided by the guide RNAs, Cpf1 or Csm1 polypeptides are universal and can be used with different guide RNAs to target different genomic sequences. Cpf1 and Csm1 endonucleases have certain advantages over Cas nucleases (e.g., Cas9) that have traditionally been used with CRISPR arrays. For example, Cpf1-bound CRISPR arrays are processed into mature crRNAs without the need for additional transactivating crRNAs (tracrRNAs). Also, the Cpf1-crRNA complex can cleave target DNAs that are preceded by short protospacer adjacent motifs (PAMs), which are often T-rich, as opposed to the G-rich PAMs that follow the target DNAs of many Cas9 systems. Furthermore, Cpf1 can introduce staggered DNA double-strand breaks with 4 or 5 nucleotide (nt) 5' overhangs. Without being bound by theory, the Csm1 protein processes its CRISPR arrays into mature crRNAs in a similar manner without the need for additional transactivating crRNAs (tracrRNAs), creating staggered cuts rather than blunt cuts. The methods disclosed herein can be employed to target and modify specific chromosomal sequences and / or to introduce exogenous sequences into target locations in plant cells or plant embryos. This method can also be employed to introduce sequences or modify regions within organelles (eg, chloroplasts and / or mitochondrial cells).Furthermore, the targeting is specific with limited off-target effects.

[0010] I. Cpf1 and Csm1 endonucleases Provided herein are Cpf1 and Csm1 endonucleases and fragments and variants thereof for use in genome modification, including plant genomes. As used herein, the term Cpf1 endonuclease or Cpf1 polypeptide refers to homologs and orthologs of the Cpf1 polypeptides disclosed in Zetsche et al. (2015) Cell 163:759-771, and to the Cpf1 polypeptides disclosed in US Patent Application Publication No. 2016 / 0208243, and fragments and variants thereof. Examples of Cpf1 polypeptides are set forth in SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-133, 135-146, 148-158, 161-173, and 231-236. As used herein, the term Csm1 endonuclease or Csm1 polypeptide refers to homologs and orthologs of SEQ ID NOs: 134, 147, 159, 160, and 230. Typically, Cpf1 and Csm1 endonucleases can act without tracrRNA and can introduce staggered DNA double-strand breaks. Generally, Cpf1 and Csm1 polypeptides contain at least one RNA recognition and / or RNA binding domain. The RNA recognition and / or RNA binding domain interacts with the guide RNA. Cpf1 and Csm1 polypeptides can also contain a nuclease domain (e.g., a DNase or RNase domain), a DNA binding domain, a helicase domain, an RNAse domain, a protein-protein interaction domain, a dimerization domain, and other domains. In certain embodiments, the Cpf1 or Csm1 polypeptide, or a polynucleotide encoding a Cpf1 or Csm1 polypeptide, comprises an RNA-binding portion that interacts with a DNA-targeting RNA and an active portion that exhibits site-specific enzymatic activity, such as a RuvC endonuclease domain.

[0011] The Cpf1 or Csm1 polypeptide may be a wild-type Cpf1 or Csm1 polypeptide, a modified Cpf1 or Csm1 polypeptide, or a fragment of a wild-type or modified Cpf1 or Csm1 polypeptide. The Cpf1 or Csm1 polypeptide may be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or alter other properties of the protein. For example, the nuclease (i.e., DNase, RNase) domain of the Cpf1 or Csm1 polypeptide may be modified, deleted, or inactivated. Alternatively, the Cpf1 or Csm1 polypeptide may be truncated to remove domains that are not essential for the function of the protein. In certain embodiments, the Cpf1 or Csm1 polypeptide forms homodimers or heterodimers.

[0012] In some embodiments, the Cpf1 or Csm1 polypeptide can be derived from a wild-type Cpf1 or Csm1 polypeptide or a fragment thereof. In other embodiments, the Cpf1 or Csm1 polypeptide can be derived from a modified Cpf1 or Csm1 polypeptide. For example, the amino acid sequence of a Cpf1 or Csm1 polypeptide can be modified to change one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, domains of the Cpf1 or Csm1 polypeptide that are not involved in RNA-guided cleavage can be removed from the protein such that the modified Cpf1 or Csm1 polypeptide is smaller than the wild-type Cpf1 or Csm1 polypeptide.

[0013] Generally, a Cpf1 or Csm1 polypeptide comprises at least one nuclease domain, but need not comprise an HNH domain as found in Cas9 proteins. For example, a Cpf1 or Csm1 polypeptide can comprise a RuvC-like nuclease domain. In some embodiments, a Cpf1 or Csm1 polypeptide can be modified to inactivate the nuclease domain such that it is no longer functional. In some embodiments where the nuclease domain is inactive, the Cpf1 or Csm1 polypeptide does not cleave double-stranded DNA. In certain embodiments, a mutated Cpf1 or Csm1 polypeptide comprises a mutation at a position corresponding to position 917 or 1006 of FnCpf1 (SEQ ID NO:3) or a position corresponding to position 701 or 922 of SmCsm1 (SEQ ID NO:160) when aligned for maximum identity that reduces or eliminates nuclease activity. For example, aspartate to alanine (D917A) and glutamate to alanine (E1006A) conversions in the RuvC-like domain completely inactivated the DNA cleavage activity of FnCpf1 (SEQ ID NO:3), while aspartate to alanine (D1255A) conversion significantly reduced the cleavage activity (Zetsche et al., (2015) Cell 163:759-711). The nuclease domain can be modified using well-known methods such as site-directed mutagenesis, PCR-mediated mutagenesis, and pre-gene synthesis, as well as other methods known in the art. Cpf1 or Csm1 proteins with inactivated nuclease domains (dCpf or dCsm1 proteins) can be used to regulate gene expression without modifying the DNA sequence. In certain embodiments, dCpf1 or dCsm1 proteins can be directed to specific regions of the genome, such as the promoter for a gene of interest, through a suitable gRNA. The dCpf1 or dCsm1 protein can bind to a desired region of DNA and can interfere with RNA polymerase binding to this region of DNA and / or the binding of transcription factors to this region of DNA.This technique may be used to up- or down-regulate the expression of one or more genes in a cell of interest. In certain other embodiments, the dCpf1 or dCsm1 protein may be fused to a repressor to further down-regulate the expression of genes whose expression is regulated by the interaction of RNA polymerases, transcription factors, or other transcriptional regulators with the region of chromosomal DNA targeted by the gRNA. In certain other embodiments, the dCpf1 or dCsm1 protein may be fused to an activation domain to effect the up-regulation of genes whose expression is regulated by the interaction of RNA polymerases, transcription factors, or other transcriptional regulators with the region of chromosomal DNA targeted by the gRNA.

[0014] The Cpf1 or Csm1 polypeptides disclosed herein further comprise at least one nuclear localization signal (NLS). Generally, an NLS comprises a stretch of basic amino acids. Localization signals are known in the art (see, for example, Lange et al., J. Biol. Chem. (2007) 282:5101-5105). The NLS can be located at the N-terminus, C-terminus, or at an internal position of the Cpf1 or Csm1 polypeptide. In some embodiments, the Cpf1 or Csm1 polypeptide can further comprise at least one cell-penetrating domain. The cell-penetrating domain can be located at the N-terminus, C-terminus, or at a single internal position.

[0015] The Cpf1 or Csm1 polypeptides disclosed herein can further include at least one plastid targeting signal peptide, at least one mitochondrial targeting signal peptide, or a single peptide that targets the Cpf1 or Csm1 polypeptide to both plastids and mitochondria. Plastid, mitochondrial, and dual targeting single peptide localization signals are known in the art (e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physiol dx.doi.org / 10.3389 / fphys.2015.00259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soll (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al. (2009) FEBS J 276:1187-1195; Silva-Filho (2003) Curr Opin Plant Biol (See, e.g., et al. (1998) Plant Biol 38:311-338; Peeters and Small (2001) Biochim Biophys Acta 1541:54-63; Murcha et al. (2014) J Exp Bot 65:6301-6335; Mackenzie (2005) Trends Cell Biol 15:548-554; Glaster et al. (1998) Plant Biol 38:311-338). The plastid, mitochondrial, or dual targeting single peptide can be located at the N-terminus, C-terminus, or internal position of the Cpf1 or Csm1 polypeptide.

[0016] In yet other embodiments, the Cpf1 or Csm1 polypeptide can also include at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In some embodiments, the marker domain can be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed). Monomeric, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent protein (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), or any other suitable fluorescent protein. In other embodiments, the marker domain can be a purification tag and / or an epitope tag.Non-limiting examples of tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity (TAP) tags, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag1, Softag3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, biotin carboxyl carrier protein (BCCP), and calmodulin.

[0017] In some embodiments, the Cpf1 or Csm1 polypeptide may be part of a protein-RNA complex that includes a guide RNA. The guide RNA interacts with the Cpf1 or Csm1 polypeptide and directs the Cpf1 or Csm1 polypeptide to a specific target site, where the 5' end of the guide RNA can base pair with a specific protospacer sequence of a nucleotide sequence of interest in the plant genome, which may be part of the nuclear, plastid, and / or mitochondrial genome. As used herein, the term "DNA-targeting RNA" refers to a guide RNA that interacts with a Cpf1 or Csm1 polypeptide and a target site of a nucleotide sequence of interest in the genome of a plant cell. A DNA-targeting RNA, or a DNA polynucleotide that encodes a DNA-targeting RNA, may include a first segment that includes a nucleotide sequence that is complementary to a sequence in the target DNA and a second segment that interacts with the Cpf1 or Csm1 polypeptide.

[0018] Polynucleotides encoding the Cpf1 or Csm1 polypeptides disclosed herein can be used to isolate corresponding sequences from other prokaryotes or eukaryotes. Thus, methods such as PCR, cross-breeding, and the like can be used to identify such sequences based on sequence identity or identity to the sequences described herein. Sequences isolated based on their sequence identity to the entire Cpf1 or Csm1 sequences described herein or to variants and fragments thereof are included in the present invention. Such sequences include sequences that are orthologs of the disclosed Cpf1 or Csm1 sequences. "Ortholog" is intended to mean genes that are derived from a common ancestral gene and are found in different species as a result of speciation. Genes found in different species are considered orthologs if their nucleotide sequences and / or their encoded protein sequences share at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity. The function of orthologs is often highly conserved between species. Thus, isolated polynucleotides that encode a polypeptide having Cpf1 or Csm1 endonuclease activity and share at least about 75% or more sequence identity to the sequences disclosed herein are included in the present invention. As used herein, Cpf1 or Csm1 endonuclease activity refers to CRISPR endonuclease activity, where a guide RNA (gRNA) combined with a Cpf1 or Csm1 polypeptide binds the Cpf1-gRNA or Csm1-gRNA complex to a predetermined nucleotide sequence that is complementary to the RNA, and where the Cpf1 or Csm1 activity can introduce a double-stranded break at or near the site targeted by the guide RNA. In some embodiments, the double-stranded break can be a staggered DNA double-stranded break.As described herein, a "staggered DNA double-stranded break" results in a double-stranded break with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotide overhangs on the 3' or 5' ends after cleavage. In certain embodiments, the Cpf1 or Csm1 polypeptide introduces a staggered DNA double-stranded break with a 4 or 5-nt 5' overhang. The double-stranded break can occur at or near the sequence to which the DNA-targeting RNA (e.g., guide RNA) sequence is targeted.

[0019] Fragments and variants of Cpf1 or Csm1 polypeptides and the Cpf1 or Csm1 amino acid sequences encoded thereby are included herein. By "fragment" is intended a portion of a polypeptide or a portion of an amino acid sequence. By "variants" is intended to mean substantially similar sequences. In the case of polynucleotides, variants include polynucleotides having 5' and / or 3' deletions (i.e., truncations), deletions and / or additions of one or more nucleotides at one or more internal sites in a naturally occurring polynucleotide, and / or one or more substitutions at one or more sites in a naturally occurring polynucleotide. As used herein, a "native" polynucleotide or polypeptide includes a naturally occurring nucleotide sequence or amino acid sequence, respectively. Generally, a variant of a particular polynucleotide of the present invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the particular polynucleotide as determined by sequence alignment programs and parameters described herein.

[0020] A "variant" amino acid or protein is intended to mean an amino acid or protein derived from a native amino acid or protein by the deletion of one or more amino acids at the N-terminus and / or C-terminus of the native amino acid (so-called truncation), by the deletion and / or addition of one or more amino acids at one or more internal sites of the native protein, or by the substitution of one or more amino acids at one or more sites of the native protein. The variant proteins encompassed by the present invention are biologically active, i.e., retain the desired biological activity of the native protein. A biologically active variant of a native polypeptide will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of the native sequence, as determined by sequence alignment programs and parameters described herein. A biologically active variant of a protein of the present invention may differ from the protein by as few as 1-15 amino acid residues, as few as 1-10, e.g., 6-10, as few as 5, as few as 4, 3, 2, or even as few as one amino acid residue.

[0021] Mutant sequences can also be identified by analysis of existing databases of sequenced genomes. Thus, corresponding sequences can be identified and used in the methods of the invention.

[0022] Methods of alignment of sequences for comparison are well known in the art. Thus, the percentage of sequence identity between two sequences can be determined using a mathematical algorithm. Examples of such mathematical algorithms include, but are not limited to, the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the local alignment methodology of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. 90:5873-5877.

[0023] Computer implementations of these mathematical algorithms can be employed to compare sequences to determine sequence identity. Such implementations include, but are not limited to, CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); GAP, BESTFIT, BLAST, FASTA, and TFASTA in the ALIGN program (Version 2.0) and GCG Wisconsin Genetics Software Package Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs are performed using default parameters. The CLUSTAL program is described in detail in Higgins et al. (1988) Gene 73:237-244; Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Mayers and Miller (1988) supra. When comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap extension penalty of 4 can be employed with the ALIGN program. The BLAST program of Altschul et al. (1990) J. Mol. Biol. 215:403 is based on the algorithm of Karlin and Altschul (1990), supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleotide sequences encoding the proteins of the invention. BLAST searches can be performed with the BLASTX program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins or polypeptides of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST (BLAST 2.0) can be employed as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997), supra. When employing BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs can be used (e.g., nucleotide sequences for BLASTN, proteins for BLASTX). See the website at www.ncbi.nlm.nih.gov. Alignments can also be performed manually by inspection.

[0024] Nucleic acid molecules encoding Cpf1 and Csm1 polypeptides, or fragments or variants thereof, can be codon optimized for expression in a plant of interest or in other cells or organisms of interest. A "codon-optimized gene" is a gene with a codon usage designed to mimic the preferred codon usage of the host cell. A nucleic acid molecule can be fully or partially codon-optimized. Since any of the amino acids (except methionine and tryptophan) are coded for by many codons, the sequence of a nucleic acid molecule can be altered without changing the amino acid coded for. Codon optimization is when one or more codons are altered at the nucleic acid level such that the amino acid is not changed but expression in a particular host organism is increased. Given that suitable information for a wide range of organisms is available in the art, one of ordinary skill in the art will recognize codon tables and other references (see, e.g., Zhang et al. (1991) Gene 105:61-72; Murray et al. (1989) Nucl. Acids Res. 17:477-508). Methodologies for optimizing nucleotide sequences for expression in plants are described, for example, in U.S. Patent No. 6,015,891 and references cited therein. Examples of codon-optimized polynucleotides for expression in plants are set forth in SEQ ID NOs: 5, 8, 11, 14, 17, 19, 22, 25, and 174-206.

[0025] II. Fusion Proteins Provided herein is a fusion protein comprising a Cpf1 or Csm1 polypeptide, or a fragment or variant thereof, and an effector domain. The Cpf1 or Csm1 polypeptide is directed by a guide RNA to a target site where the effector domain can modify or act on a nucleic acid sequence. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. The fusion protein can further comprise at least one additional domain selected from a nuclear localization signal, a plastid signal peptide, a mitochondrial signal peptide, a signal peptide capable of transporting a protein to multiple intracellular locations, a cell penetrating domain, or a marker domain, any of which can be located at the N-terminus, the C-terminus, or an internal location of the fusion protein. The Cpf1 or Csm1 polypeptide can be located at the N-terminus, the C-terminus, or an internal location of the fusion protein. The Cpf1 or Csm1 polypeptide can be directly fused to the effector domain or fused to a linker. In certain embodiments, the linker sequence fusing the Cpf1 or Csm1 polypeptide to the effector domain is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 amino acids in length. For example, the linker is 1-5, 1-10, 1-20, 1-50, 2-3, 3-10, 3-20, 5-20, or 10-50 amino acids in length.

[0026] In some embodiments, the Cpf1 or Csm1 polypeptide of the fusion protein may be derived from a wild-type Cpf1 or Csm1 protein. The Cpf1- or Csm1-derived protein may be an engineered variant or fragment. In some embodiments, the Cpf1 or Csm1 polypeptide may be engineered to include a nuclease domain (e.g., a RuvC-like domain) with reduced or eliminated nuclease activity. For example, the Cpf1- or Csm1-derived polypeptide may be engineered such that the nuclease domain is deleted or modified so that it is no longer functional (i.e., has no nuclease activity). In particular, when aligned for maximum identity, the Cpf1 or Csm1 polypeptide includes a mutation at a position corresponding to positions 917 or 1006 of FnCpf1 (SEQ ID NO:3) or positions 701 or 922 of SmCsm1 (SEQ ID NO:160). For example, aspartate to alanine (D917A) and glutamate to alanine (E1006A) conversions in the RuvC-like domain completely inactivated the DNA cleavage activity of FnCpf1, while an aspartate to alanine (D1255A) conversion significantly reduced the cleavage activity (Zetsche et al., (2015) Cell 163:759-711). Examples of Cpf1 polypeptides with mutations in the RuvC domain are set forth in SEQ ID NOs: 26-41 and 63-70. The nuclease domain can be modified using other methods known in the art as well, such as by one or more deletion, insertion, and / or substitution mutations, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis. In an exemplary embodiment, the Cpf1 or Csm1 polypeptide of the fusion protein is modified by mutating the RuvC-like domain such that the Cpf1 or Csm1 polypeptide does not have nuclease activity.

[0027] The fusion protein comprises an effector domain located at the N-terminus, C-terminus, or at an internal position of the fusion protein. In some embodiments, the effector domain is a cleavage domain. As used herein, "cleavage domain" refers to a domain that cleaves DNA. The cleavage domain can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing nucleases. See, e.g., New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). See also, Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) can be used as the source of the cleavage domain.

[0028] In some embodiments, the cleavage domain may be derived from a type II-S endonuclease. Type II-S endonucleases cleave DNA at sites typically several base pairs away from the recognition site, and as such have separable recognition and cleavage domains. These enzymes are generally monomers that transiently associate to form dimers and cleave each strand of DNA at staggered positions. Non-limiting examples of suitable II-S endonucleases include Bfil, BpmI, BsaI, BsgI, BsmBI, BsmI, BspMI, FokI, MbolI, and SapI.

[0029] In certain embodiments, type II-S cleavage is modified to promote dimerization of two different cleavage domains, each of which is bound to a Cpf1 or Csm1 polypeptide or fragment thereof. In embodiments in which the effector domain is a cleavage domain, the Cpf1 or Csm1 polypeptide can be modified to eliminate endonuclease activity as discussed herein. For example, a Cpf1 or Csm1 polypeptide can be modified by mutating the RuvC-like domain such that the polypeptide no longer exhibits endonuclease activity.

[0030] In other embodiments, the effector domain of the fusion protein may be an epigenetic modification domain. In general, epigenetic modification domains change histone and / or chromosome structure without changing DNA sequence. Changes in histone and / or chromatin structure can lead to changes in gene expression. Examples of epigenetic modifications include, but are not limited to, acetylation or methylation of lysine residues in histone proteins and methylation of cytosine residues in DNA. Examples of suitable epigenetic modification domains include, but are not limited to, histone acetyltransferase domains, histone deacetylase domains, histone methyltransferer domains, histone demethylase domains, DNA methyltransferer domains, and DNA demethylase domains.

[0031] In embodiments where the effector domain is a histone acetyltransferase (HAT) domain, the HAT domain may be derived from EP300 (i.e., E1A binding protein p300), CREBBP (i.e., CREB binding protein), CDY1, CDY2, CDYL1, CLOCK, ELP3, ESA1, GCN5 (KAT2A), HAT1, KAT2B, KAT5, MYST1, MYST2, MYST3, MYST4, NCOA1, NCOA2, NCOA3, NCOAT, P / CAF, Tip60, TAFII250, or TF3C4. In embodiments where the effector domain is an epigenetic modification domain, the Cpf1 or Csm1 polypeptide may be modified as discussed herein to delete its endonuclease activity. For example, a Cpf1 or Csm1 polypeptide can be modified by mutating the RuvC-like domain such that the polypeptide no longer exhibits endonuclease activity.

[0032] In some embodiments, the effector domain of the fusion protein may be a transcription activation domain. Generally, a transcription activation domain interacts with a transcription control element and / or a transcription regulatory protein (i.e., transcription factor, RNA polymerase, etc.) to increase and / or activate the transcription of one or more genes. In some embodiments, the transcription activation domain includes, but is not limited to, herpes simplex virus VP16 activation domain, VP64 (which is a tetrameric derivative of VP16), NFκB p65 activation domain, p53 activation domains 1 and 2, CREB (cAMP response element binding protein) activation domain, E2A activation domain, and NFAT (nuclear factor of activated T cells) activation domain. In other embodiments, the transcription activation domain may be Gal4, Gcn4, MLL, Rtg3, Gln3, Oaf1, Pip2, Pdr1, Pdr3, Pho4, and Leu3. The transcription activation domain may be wild-type or a modified version of the original transcription activation domain. In some embodiments, the effector domain of the fusion protein is a VP16 or VP64 transcription activation domain. In embodiments in which the effector domain is a transcription activation domain, the Cpf1 or Csm1 polypeptide can be modified to delete its endonuclease activity as discussed herein. For example, the Cpf1 or Csm1 polypeptide can be modified by mutating the RuvC-like domain so that the polypeptide no longer has endonuclease activity.

[0033] In yet other embodiments, the effector domain of the fusion protein can be a transcriptional repressor domain. Generally, a transcriptional repressor domain interacts with a transcriptional control element and / or a transcriptional regulatory protein (i.e., a transcription factor, an RNA polymerase, etc.) to reduce and / or terminate the transcription of one or more genes. Examples of suitable transcriptional repressor domains include, but are not limited to, an inducible cAMP early repressor (ICER) domain, a Kruppel-associated box A (KRAB-A) repressor domain, a YY1 glycine-rich repressor domain, an Sp1-like repressor, an E(sp1) repressor, an I.κ.B repressor, and MeCP2. In embodiments where the effector domain is a transcriptional repressor domain, the Cpf1 or Csm1 polypeptide can be modified as discussed herein to delete its endonuclease activity. For example, a Cpf1 or Csm1 polypeptide can be modified by mutating the RuvC-like domain such that the polypeptide no longer has nuclease activity.

[0034] In some embodiments, the fusion protein comprises at least one additional domain. Examples of suitable additional domains include, but are not limited to, nuclear localization signals, cell penetration or translocation domains, and marker domains.

[0035] When the effector domain of the fusion protein is a cleavage domain, a dimer containing at least one fusion protein can be formed. The dimer can be a homodimer or a heterodimer. In some embodiments, the heterodimer contains two different fusion proteins. In other embodiments, the heterodimer contains one fusion protein and an additional protein.

[0036] The dimer may be a homodimer in which the two fusion protein monomers are identical to each other in terms of primary amino acid sequence. In one embodiment in which the dimer is a homodimer, the Cpf1 or Csm1 polypeptide can be modified to lack endonuclease activity. In an embodiment in which the Cpf1 or Csm1 polypeptide is modified to lack endonuclease activity, each fusion protein monomer can comprise an identical Cpf1 or Csm1 polypeptide and an identical cleavage domain. The cleavage domain can be any cleavage domain, including any of the exemplary cleavage domains provided herein. In such an embodiment, a particular guide RNA will target the fusion protein monomers to different, but adjacent, sites such that upon dimerization, the nuclease domains of the two monomers can create a double-stranded break in the target DNA.

[0037] The dimer may also be a heterodimer of two different fusion proteins. For example, the Cpf1 or Csm1 polypeptide of each fusion protein may be derived from a different Cpf1 or Csm1 polypeptide, or may be derived from an orthologous Cpf1 or Csm1 polypeptide from a different bacterial species. For example, each fusion protein may comprise a Cpf1 or Csm1 polypeptide derived from a different bacterial species. In these embodiments, each fusion protein will recognize a different target site (i.e., specified by a protospacer and / or PAM sequence). For example, a guide RNA may position the heterodimer at a different but adjacent site such that its nuclease domain produces an effective double-strand break in the target DNA.

[0038] Alternatively, the two fusion proteins of the heterodimer may have different effector domains. In embodiments where the effector domain is a cleavage domain, each fusion protein may contain a different modified cleavage domain. In these embodiments, the Cpf1 or Csm1 polypeptides are modified to delete their endonuclease activity. The two fusion proteins forming the heterodimer may differ in both the Cpf1 or Csm1 polypeptide domain and the effector domain.

[0039] In any of the above embodiments, the homodimer or heterodimer may include a nuclear localization signal (NLS), a plastid signal peptide, a mitochondrial signal peptide, a signal peptide capable of transporting the protein to multiple subcellular locations, a cell penetrating translocation domain, and a marker domain, as detailed above. In any of the above embodiments, one or both of the Cpf1 or Csm1 polypeptides may be modified to have deleted or modified endonuclease activity.

[0040] The heterodimer may further comprise one fusion protein and an additional protein. For example, the additional protein may be a nuclease. In one embodiment, the nuclease is a zinc finger nuclease. The zinc finger nuclease comprises a zinc finger DNA binding domain and a cleavage domain. The zinc fingers recognize and bind three (3) nucleotides. The zinc finger DNA binding domain may comprise about 3 to about 7 zinc fingers. The zinc finger DNA binding domain may be derived from a naturally occurring protein or may be engineered. See, e.g., Beerli et al. (2002) Nat. Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nat. Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; Zhang et al. (2000) J. Biol. Chem. 275(43):33850-33860; Doyon et al. (2008) Nat. Biotechnol. 26:702-708; and Santiago et al. (2008) Proc. Natl. Acad. Sci. See, USA 105:5809-5814. The cleavage domain of the zinc finger nuclease can be any cleavage domain described in detail herein. In some embodiments, the zinc finger nuclease can further comprise at least one additional domain selected from a nuclear localization signal, a plastid signal peptide, a mitochondrial signal peptide, a signal peptide capable of transporting the protein to multiple intracellular locations, a cell penetration or translocation domain, all of which are described in detail herein.

[0041] In certain embodiments, any of the fusion proteins detailed above or a dimer containing at least one fusion protein may be part of a protein-RNA complex containing at least one guide RNA that interacts with the Cpf1 or Csm1 polypeptide of the fusion protein and targets the fusion protein to a target site, where the 5' end of the guide RNA base pairs with a specific protospacer sequence.

[0042] III. Nucleic Acids Encoding Cpf1 or Csm1 Polypeptides or Fusion Proteins Nucleic acids are provided that encode any of the Cpf1 and Csm1 polypeptides or fusion proteins described herein. The nucleic acids may be RNA or DNA. Examples of polynucleotides encoding Cpf1 polypeptides are set forth in SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 21, 22, 24, 25, and 174-184, 187-192, 194-201, and 203-206. Examples of polynucleotides encoding Csm1 polypeptides are set forth in SEQ ID NOs: 185, 186, 193, and 202. In one embodiment, the nucleic acid encoding the Cpf1 or Csm1 polypeptide or fusion protein is mRNA. The mRNA may be 5' capped and / or 3' polyadenylated. In another embodiment, the nucleic acid encoding the Cpf1 or Csm1 polypeptide or fusion protein is DNA. The DNA may be present in a vector.

[0043] A nucleic acid encoding a Cpf1 or Csm1 polypeptide or fusion protein can be codon optimized for efficient translation into a protein in a plant cell of interest. Programs for codon optimization are available in the art (e.g., OPTIMIZER, genomes.urv.es / OPTIMIZER; OptimumGene.TM., GenScript, Inc., www.genscript.com / codon_opt.html).

[0044] In some embodiments, the DNA encoding the Cpf1 or Csm1 polypeptide or fusion protein may be operably linked to at least one promoter sequence. The DNA coding sequence may be operably linked to a promoter control sequence for expression in a host plant of interest. In some embodiments, the host cell is a plant cell. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operably linked linkage between a promoter and a plant coding region of interest (e.g., a Cpf1 or Csm1 polypeptide or a guide RNA) is a functional linkage that allows expression of the linked region of interest. The operably linked elements may be contiguous or discontinuous. When used to refer to the linkage of two protein coding regions, "operably linked" is intended to mean that the coding regions are in the same reading frame.

[0045] The promoter sequence may be constitutive, regulated, developmental stage specific, or tissue specific. It is recognized that different applications may be enhanced by using different promoters in the nucleic acid molecule to regulate the timing, location, and / or extent of expression of the Cpf1 or Csm1 polypeptide and / or guide RNA. Such nucleic acid molecules may also optionally include promoter regulatory regions (e.g., inducible, equivocal, environmentally or developmentally regulated, or cell or tissue specific / selective expression), transcription initiation sites, ribosome binding sites, RNA processing signals, transcription termination sites, and / or polyadenylation signals.

[0046] In some embodiments, the nucleic acid molecules provided herein can be combined with constitutive, tissue-preferred, developmentally preferred, developmentally preferred, or other promoters for expression in plants. Examples of constitutive promoters functional in plant cells include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'-promoter from the T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and transcription initiation regions from various plant genes known in the art. If a low expression level is desired, a weak promoter can be used. Weak constitutive promoters include, for example, the nuclear promoter of the Rsyn7 promoter (WO 99 / 43838 and U.S. Pat. No. 6,072,050), the nuclear 35S CaMV promoter, and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also U.S. Patent No. 6,177,611, which is incorporated herein by reference.

[0047] Inducible promoters include the Adh1 promoter, which is induced by hypoxia or cold stress, the Hsp70 promoter, which is induced by heat stress, and the PPDK and pep carboxylase promoters, both of which are induced by light. Also useful are chemically inducible promoters, such as the safener-induced In2-2 promoter (U.S. Pat. No. 5,364,780), the estrogen-induced ERE promoter, and the Axig1 promoter, which is auxin-inducible and tapetum-specific but also active in callus (International Application PCT / US01 / 22169).

[0048] Examples of promoters under developmental control in plants include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, fruits, seeds, or flowers. A "tissue-specific" promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of a gene, tissue-specific expression is the result of several interactions at the genetic level of regulation. Thus, promoters from homologous or closely related plant species are preferably used to achieve efficient and reliable expression of transgenes in specific tissues. In some embodiments, the expression comprises a tissue-preferred promoter. A "tissue-preferred" promoter is a promoter that preferentially initiates transcription, but does not necessarily initiate transcription entirely or only in a specific tissue.

[0049] In some embodiments, the nucleic acid molecule encoding the Cpf1 or Csm1 polypeptide and / or guide RNA comprises a cell type specific promoter. A "cell type specific promoter" is a promoter that drives expression primarily in a certain cell type in one or more organs. Some examples of plant cells in which a cell type specific promoter that is functional in plants may be primarily active include, for example, BETL cells, root vascular cells, leaves, stem cells, and stem cells. The nucleic acid molecule can also comprise a cell type preferred promoter. A "cell type preferred" promoter is a promoter that drives transcription primarily, but not necessarily exclusively, in a certain cell type in one or more tissues. Some examples of plant cells in which a cell type preferred promoter that is functional in plants may be primarily active include, for example, BETL cells, root vascular cells, leaves, stem cells, and stem cells. The nucleic acid molecules described herein can also comprise a species preferred promoter. In some embodiments, the species preferred promoter has expression in the embryo sac, early embryo, early endosperm, aleurone, and / or basal germ transfer cell layer (BETL).

[0050] Examples of seed-preferred promoters include, but are not limited to, the 27 kD gamma zein promoter and the waxy promoter, Boronat, A. et al. (1986) Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res. 18(21):6426; and Kloesgen, RB et al. (1986) Mol. Gen. Genet. 203:237-244. Promoters that express in the embryo, pericarp, and endosperm are described in U.S. Patent No. 6,225,529 and International Application PCT Publication No. 00 / 12733, the disclosures of each of which are incorporated herein by reference in their entirety.

[0051] Promoters capable of promoting gene expression in a plant seed-preferential manner, with expression in the embryo sac, early embryo, early endosperm, aleurone, and / or basal germ transfer cell layer (BETL), can be used in the compositions and methods disclosed herein. Such promoters include, but are not limited to, Zea mays early endosperm 5 gene, Zea mays early endosperm 1 gene, Zea mays early endosperm 2 gene, GRMZM2G124663, GRMZM2G006585, GRMZM2G120008, GRMZM2G157806, GRMZM2G176390, GRMZM2G472234, GRMZM2G138727, Zea mays CLAVATA1, Zea mays MRP1, Oryza sativa PR602, Oryza sativa PR9a, Zea mays BET1, Zea mays BETL-2, Zea mays BETL-3, Zea mays BETL-4, Zea mays BETL-9, Zea mays BETL-10, Zea mays MEG1, Zea mays TCCR1, Zea mays TCCR2 ... mays ASP1, Oryza sativa ASP1, Triticum durum PR60, Triticum durum PR91, Triticum durum GL7, AT3G10590, AT4G18870, AT4G21080, AT5G23650, AT3G05860, AT5G42910, AT2G26320, AT3G03260, AT5G26630, AtlPT4, AtlPT8, AtLEC2, and LFAH12.Further such promoters are disclosed in U.S. Pat. Nos. 7,803,990, 8,049,000, 7,745,697, 7,119,251, 7,964,770, 7,847,160, 7,700,836, U.S. Patent Application Publication Nos. 20100313301, 20090049571, 20090089897, and 20100281569. International Publication Nos. WO 20100281570, WO 20120066795, WO 20040003427; WO 1999 / 050427, WO 2010 / 129999, WO 2009 / 094704, WO 2010 / 019996 and WO 2010 / 147825, each of which is incorporated herein by reference in its entirety for all purposes. Functional variants or functional fragments of the promoters described herein can also be operably linked to the nucleic acids disclosed herein.

[0052] Chemically regulated promoters can be used to regulate the expression of genes through the application of exogenous chemical regulators. Depending on the purpose, the promoter can be a chemically inducible promoter, where application of a chemical induces gene expression, or a chemically repressible promoter, where application of a chemical represses gene expression. Chemically inducible promoters known in the art include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemically regulated promoters of interest are steroid-responsive promoters (see, e.g., glucocorticoid-inducible promoters described in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0053] Tissue-preferred promoters can be used to target enhanced expression of an expression construct in a specific tissue. In some embodiments, the tissue-preferred promoter is active in plant tissue. Tissue-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol 112(2):525-535; Canevascini et al. (1996) Plant Physiol 112(2):513-524; Yamamoto et al. (1994) Plant Cell See Physiol 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified for weak expression, if necessary.

[0054] Leaf-preferred promoters are known in the art.See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.In addition, the promoters of Cab and rubisco can also be used. See, e.g., Simpson et al. (1958) EMBO J 4:2723-2729 and Timko et al. (1988) Nature 318:57-58.

[0055] Root-preferred promoters are well known and can be selected from many available in the literature, or can be isolated de novo from a variety of compatible species. See, e.g., Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific regulatory elements in the GRP1.8 gene of French bean; Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding a cytosolic glutamine synthetase (GS) that is expressed in soybean roots and nodules). Also see Bogusz et al. (1990) Plant Cell 2(7):633-641, which describes two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing non-legume Parasponia andersonii and the related non-nitrogen-fixing non-legume Trema tomentosa. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases, root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe the promoters of the highly expressed roIC and roID root-inducible genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They conclude that enhancers and tissue-preferential DNA determinants are dissociated in their promoters.Teeri et al. (1989) used genes fused to laZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is particularly active in the epidermis of the root tip, and that the TR2' gene is root-specific in intact plants and can be stimulated by wounding leaf tissue in a combination of particularly desirable properties for use with insecticidal or larvicidal genes (see EMBO J. 8(2):343-350). The TR1' gene fused to nptII (neomycin phosphotransferase II) showed similar properties. Further root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and the roIB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179. The phaseolin gene (Murai et al. (1983) Science 23:476-482 and Sengopta-Gopalen et al. (1988) PNAS 82:3320-3324). The promoter sequences may be wild-type or may be modified for more efficient or effective expression.

[0056] The nucleic acid sequence encoding the Cpf1 or Csm1 polypeptide or fusion protein can be operably linked to a promoter sequence recognized by a phage RNA polymerase for in vitro mRNA synthesis. In such embodiments, the in vitro transcribed RNA can be purified for use in the genome modification methods described herein. For example, the promoter sequence is a T7, T3, or SP6 promoter sequence or a variant of a T7, T3, or SP6 promoter sequence. In some embodiments, the sequence encoding the Cpf1 or Csm1 polypeptide or fusion protein can be operably linked to a promoter sequence for expression of an in vitro Cpf1 or Csm1 polypeptide or fusion protein in a plant cell. In such embodiments, the expressed protein can be purified for use in the genome modification methods described herein.

[0057] In certain embodiments, the DNA encoding the Cpf1 or Csm1 polypeptide or fusion protein can also be linked to a polyadenylation signal (e.g., an SV40 polyA signal or other signal functional in plants) and / or at least one transcription termination sequence. Additionally, the sequence encoding the Cpf1 or Csm1 polypeptide or fusion protein can be linked to a sequence encoding at least one localization signal, at least one plastid signal peptide, at least one mitochondrial signal peptide, at least one signal peptide capable of transporting the protein to multiple subcellular locations, at least one cell penetration domain, and / or at least one marker domain, as described herein.

[0058] The DNA encoding the Cpf1 or Csm1 polypeptide or fusion protein may be present in a vector. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial / minichromosomes, transposons, and virus vectors (e.g., lentiviral vectors, adeno-associated virus vectors, etc.). In one embodiment, the DNA encoding the Cpf1 or Csm1 polypeptide or fusion protein is present in a plasmid vector. Examples of suitable plasmid vectors include, but are not limited to, pUC, pBR322, pET, pCAMBIA, and variants thereof. The vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selection marker sequences (e.g., antibiotic resistance genes), origins of replication, etc. Additional information can be found in Current Protocols in Molecular, Ausubel et al., John Wiley & Sons, New York, 2003, or Molecular Cloning: A Laboratory Manual, Sambrook & Russell, Cold Spring Harbor, NY, 3rd ed., 2001.

[0059] In some embodiments, the expression vector comprising a sequence encoding a Cpf1 or Csm1 polypeptide or fusion protein can further comprise a sequence encoding a guide RNA. The sequence encoding the guide RNA can be operably linked to at least one transcription control sequence for expression of the guide RNA in a plant or plant cell of interest. For example, the DNA encoding the guide RNA can be operably linked to a promoter sequence recognized by RNA polymerase III (PolIII). Examples of suitable PolIII promoters include, but are not limited to, mammalian U6, U3, H1, and 7SL RNA promoters and rice U6 and U3 promoters.

[0060] IV. Methods for Modifying Nucleotide Sequences in Plant Genomes A method for modifying a nucleotide sequence in a plant cell, plant organelle, or plant embryo is proposed herein. The method includes introducing into the plant cell, plant organelle, or plant embryo a DNA-targeting RNA, the DNA-targeting RNA, or a DNA polynucleotide encoding the DNA-targeting RNA, the DNA-targeting RNA comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence in a target DNA and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide, the Cpf1 or Csm1 polypeptide, or a polynucleotide encoding the Cpf1 or Csm1 polypeptide, the Cpf1 or Csm1 polypeptide comprising (a) an RNA-binding portion that interacts with the DNA-targeting RNA and (b) an activity portion that exhibits site-specific enzymatic activity, into the plant cell. The plant cell or plant embryo can then be cultured under conditions in which the Cpf1 or Csm1 polypeptide is expressed and cleaves the nucleotide sequence. The system described herein can be used to culture the plant cell or plant embryo ... 2+ Note that the addition of ions such as arginine, arginine, methylglutaminase ...

[0061] In some embodiments, the method can include introducing a Cpf1 or Csm1 polypeptide (or encoding nucleic acid) and an RNA (or encoding DNA) into a plant cell, organelle, or embryo, where the Cpf1 or Csm1 polypeptide introduces a double-stranded break in a target nucleotide sequence of a plant chromosomal DNA. In embodiments in which no donor polynucleotide is present, the double-stranded break in the nucleotide sequence can be repaired by a non-homologous end joining (NHEJ) repair process. Because NHEJ is error-prone, a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof can occur when the break is repaired. In this way, the target nucleotide sequence can be modified or inactivated. For example, a single nucleotide change (SNP) can result in an increase in an altered protein product, or a shift in the reading frame of a coding sequence can inactivate or "knock out" the sequence so that no protein product is made. In embodiments where any donor polynucleotide is present, the donor sequence in the donor polynucleotide can be replaced or integrated into the nucleotide sequence at the target site during repair of the double break. For example, in embodiments where the donor sequence has substantial sequence identity with and is adjacent to the upstream and downstream sequences of the target site in the nucleotide sequence of the plant, the donor sequence can be replaced or integrated into the nucleotide sequence at the target site during repair mediated by a homology-directed repair process. Alternatively, in embodiments where the donor sequence is adjacent to a compatible overhang (or a compatible overhang is generated in situ by a Cpf1 or Csm1 polypeptide), the donor sequence can be directly joined to the cleaved nucleotide sequence by a non-homologous repair process during repair of the double break. The replacement or integration of the donor sequence into the nucleotide sequence will modify the target nucleotide sequence of the plant or introduce an exogenous sequence into the nucleotide sequence of the plant cell, plant organelle, or plant embryo.

[0062] The methods disclosed herein can further include introducing two Cpf1 or Csm1 polypeptides (or encoding nucleic acids) and two guide RNAs (or encoding DNAs) into a plant cell, organelle, or embryo, where the Cpf1 or Csm1 polypeptides introduce a double-stranded break in a nucleotide sequence of the nuclear and / or organelle chromosomal DNA. The two breaks can occur within a few base pairs, within tens of base pairs, or can be separated by thousands of base pairs. In embodiments where no donor nucleotide is present, the resulting double-stranded break can be repaired by a non-homologous repair process such that during repair of the break, the sequence between the two cleavage sites is lost and / or at least one nucleotide is deleted, at least one nucleotide is inserted, at least one nucleotide is substituted, or a combination thereof can occur. In embodiments where an optional donor nucleotide is present, during repair of the double-stranded break by either a homology-based repair process (e.g., in embodiments where the donor sequence has substantial sequence identity with, and is flanked by, upstream and downstream sequences, respectively, of the target site in the plant's nucleotide sequence) or a non-homologous repair process (e.g., in embodiments where the donor sequence is flanked by compatibility overhangs), the donor sequence in the donor polynucleotide may be exchanged with or incorporated into the plant's nucleotide sequence.

[0063] By "altering" or "modulating" the expression level of a gene, it is intended that the expression of the gene is up-regulated or down-regulated. It has been recognized that in some instances, increasing the expression level of one or more genes encoding proteins in photosynthesis, i.e., up-regulating expression, increases plant growth and yield. Similarly, in some instances, decreasing the expression level of one or more genes encoding proteins in photosynthesis, i.e., down-regulating expression, may decrease plant growth and yield. Thus, the present invention includes up-regulating or down-regulating the expression level of one or more genes encoding proteins involved in photosynthesis by using the Cpf1 or Csm1 polypeptides disclosed herein. Additionally, the present methods include up-regulating at least one gene encoding a protein involved in photosynthesis and down-regulating at least one gene encoding a protein involved in photosynthesis in a plant of interest. By modulating the level and / or activity of at least one gene encoding a protein involved in photosynthesis in a transformed plant, it is intended that the level and / or activity be increased or decreased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more compared to a naturally occurring control plant, plant part, or cell that does not have the introduced sequence of the present invention.

[0064] Plant cells have nuclear, plastid, and mitochondrial genomes. The compositions and methods of the present invention can be used to modify sequences of the nuclear, plastid, and / or mitochondrial genomes or can be used to modulate the nuclear, plastid, and / or mitochondrial genomes. Thus, by "chromosome" or "chromosomal," we mean nuclear, plastid, and mitochondrial genomic DNA. When applied to plant cells, "genome" includes not only chromosomal DNA found in the nucleus, but also organelle DNA found in subcellular components of the cell (e.g., mitochondria or plastids). Any nucleotide sequence of interest in a plant cell, organelle, or embryo can be modified by using the methods disclosed herein. In certain embodiments, the methods disclosed herein can be used to modify nucleotide sequences encoding agriculturally important traits such as plant hormones, plant defense proteins, nutrient transport proteins, biologically relevant proteins, desirable input traits, desirable output traits, stress resistance genes, disease / pathogen resistance genes, male sterility genes, developmental genes, regulatory genes, genes involved in photosynthesis, DNA repair genes, transcriptional regulatory genes, or any other polynucleotides and / or polypeptides of interest. Agronomically important traits such as oil, starch and protein content can also be modified. Modifications include increasing oleic acid, saturated and unsaturated oil content, increasing lysine and sulfur values, providing essential amino acids, and modifying starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389, which are incorporated herein by reference. Another example is lysine and / or sulfur-rich seeds encoded by soybean 2S albumin, described in U.S. Pat. No. 5,850,016, and barley-derived chymotrypsin inhibitors, described in Williamson et al. (1987) Eur. J. Biochem. 165:99-106, the disclosures of which are incorporated herein by reference.

[0065] Using the methods disclosed herein, a derivative of a coding sequence can be made to increase the value of a preselected amino acid in the encoded polypeptide. For example, the gene encoding the Barley Lysine-rich (BHL) polypeptide is derived from the barley chymotrypsin inhibitor of U.S. Patent Application Serial No. 08 / 740682, filed November 1, 1996, and WO 98 / 20133, the disclosures of which are incorporated herein by reference. Other proteins include methionine-rich proteins from sunflower seeds (Lilley et al. (1989) Proceedings of the world Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American oil Chemists Society, Champain, Illinois) pp. 497-502; incorporated herein by reference); corn (Pedersen et al. (1986) J. Biol. Chem. 261:6279 and Kirihara et al. (1988) Gene 71:359; both of which are incorporated herein by reference); and rice (Musumura et al. (1989) Plant Mol. Biol. 12:123; incorporated herein by reference). Other agronomically important genes encode latex, floury2 growth factors, seed storage factors, and transcription factors.

[0066] The methods disclosed herein can be used to modify herbicide tolerance traits including genes coding for tolerance to herbicides that act to inhibit the action of acetolactate synthase (ALS), particularly sulfonylurea-type herbicides (e.g., acetolactate synthase (ALS) genes containing mutations leading to such tolerance, particularly S4 and / or Hra mutations), genes coding for tolerance to herbicides that act to inhibit the action of glutamine synthase (ALS), such as phosphinothricin or BASTA (e.g., bar gene); glyphosate (e.g., EPSPS gene and GAT gene; see, e.g., U.S. Publication No. 20040082770 and WO 03 / 092360); or other such genes known in the art. The bar gene codes for tolerance to the herbicide BASTA, the nptII gene codes for tolerance to the antibiotics kanamycin and geneticin, and the ALS gene mutant codes for tolerance to the herbicide chlorsulfuron. Further herbicide tolerance traits are described, for example, in U.S. Patent Application No. 2016 / 0208243, incorporated herein by reference.

[0067] Sterility genes can also be modified to provide an alternative physical detasseling. Examples of genes used in such a manner include male tissue preference genes and genes with male sterility phenotypes, such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those that code for compounds that are toxic to either male or female gametophyte development. Additional sterility traits are described, for example, in U.S. Patent Application Publication No. 2016 / 0208243, which is incorporated herein by reference.

[0068] Grain quality can be altered by modifying genes that code for traits such as oil value and type, saturated and unsaturated, quality and quantity of essential amino acids, cellulose value, etc. In corn, modified hordothionin proteins are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0069] Commercial traits can also be modified by modifying genes or by providing expression of proteins, for example to increase starch for ethanol production. Another important commercial application of modified plants is the production of polymers and bioplastics, as described in U.S. Pat. No. 5,602,321. Genes such as β-ketothiolase, PHBase (polyhydrobutyrate synthase) and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) promote the expression of polyhydroxyalkanoase (PHA).

[0070] Exogenous products include plant enzymes and those from other sources, including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, etc. The value of proteins can be increased, especially engineered proteins with improved amino acid distribution and improved nutritional value in plants. This is accomplished by expression of such proteins with enhanced amino acid content.

[0071] The methods disclosed herein can also be used to insert heterologous genes and / or modify native plant gene expression to achieve desired plant traits, including, for example, disease resistance, herbicide tolerance, drought tolerance, salt tolerance, insect resistance, parasitic weed resistance, improved plant nutritional value, increased feed digestibility, increased grain yield, cytoplasmic male sterility, altered fruit ripening, increased shelf life of plants or plant parts, reduced allergen production, and increased or reduced lignin content. Genes that can confer these desirable traits are described in U.S. Patent Application Publication No. 2016 / 0208243, which is incorporated herein by reference.

[0072] (a) a Cpf1 or Csm1 polypeptide The methods disclosed herein include introducing at least one Cpf1 or Csm1 polypeptide or a nucleic acid encoding at least one Cpf1 or Csm1 polypeptide into a plant cell, plant organelle, or plant embryo, as described herein. In some embodiments, the Cpf1 or Csm1 polypeptide can be introduced into the plant cell, organelle, or embryo as an isolated protein. In such embodiments, the Cpf1 or Csm1 polypeptide can further comprise at least one cell-penetrating domain that facilitates uptake of the protein into the cell. In some embodiments, the Cpf1 or Csm1 polypeptide can be introduced into the plant cell, organelle, or embryo as a ribonucleoprotein complexed with a guide RNA. In other embodiments, the Cpf1 or Csm1 polypeptide can be introduced into the plant cell, organelle, or embryo as an mRNA. In yet other embodiments, the Cpf1 or Csm1 polypeptide can be introduced into the plant cell, organelle, or embryo as a DNA molecule. Generally, the sequences encoding the Cpf1 or Csm1 polypeptides or fusion proteins described herein are operably linked to a promoter sequence that functions in the plant cell, organelle, or embryo of interest. The DNA sequence may be linear or the DNA sequence may be part of a vector. In yet another embodiment, the Cpf1 or Csm1 polypeptides or fusion proteins may be introduced into the plant cell, organelle, or embryo as an RNA-protein complex comprising a guide RNA or a fusion protein and a guide RNA.

[0073] In some embodiments, an mRNA encoding a Cpf1 or Csm1 polypeptide may be targeted to an organelle (e.g., plastids or mitochondria). In some embodiments, an mRNA encoding one or more guide RNAs may be targeted to an organelle (e.g., plastids or mitochondria). In some embodiments, an mRNA encoding a Cpf1 or Csm1 polypeptide and one or more guide RNAs may be targeted to an organelle (e.g., plastids or mitochondria). Methods for targeting mRNA to an organelle are known in the art (see, e.g., U.S. Patent Application No. 2011 / 0296551; U.S. Patent Application No. 2011 / 0321187; Gomes and Pallas (2010) PLos One 5:e12269) and are incorporated herein by reference.

[0074] In some embodiments, the DNA encoding the Cpf1 or Csm1 polypeptide may further comprise a sequence encoding a guide RNA. In general, each sequence encoding the Cpf1 or Csm1 polypeptide and the guide RNA is operably linked to one or more suitable promoter control sequences that allow expression of the Cpf1 or Csm1 polypeptide and the guide RNA, respectively, in a plant cell, organelle, or embryo. The DNA sequence encoding the Cpf1 or Csm1 polypeptide and the guide RNA may further comprise additional expression control, regulation, and / or processing sequences. The DNA sequence encoding the Cpf1 or Csm1 polypeptide and the guide RNA may be linear or part of a vector.

[0075] (b) Guide RNA The methods described herein may also include introducing at least a guide RNA or a DNA encoding at least a guide RNA into a plant cell, organelle, or embryo. The guide RNA interacts with the Cpf1 or Csm1 polypeptide, directing the Cpf1 or Csm1 polypeptide to its specific target site, where the 5' end of the bases of the guide RNA pairs with a specific protospacer sequence in the plant nucleotide sequence. The guide RNA may include three regions: a first region that is complementary to a target site in the target chromosomal sequence, a second region that forms a stem-loop structure, and a third region that remains essentially single-stranded. The first region of each guide RNA is different such that each guide RNA directs the Cpf1 or Csm1 polypeptide to a specific target site. The second and third regions of each guide RNA may be the same in all guide RNAs.

[0076] A region of the guide RNA is complementary to a sequence (i.e., a protospacer sequence) at a target site in the plant genome, including plastid or mitochondrial 1 sequences as well as nuclear chromosomal sequences, such that the first region of the guide RNA can base pair with the target site. In various embodiments, the first region of the guide RNA can comprise from about 8 nucleotides to about 30 or more nucleotides. For example, the base-paired region between the guide RNA and the target site in the nucleotide sequence is about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 22, about 23, about 24, about 25, about 27, about 30, or more than 30 nucleotides in length. In exemplary embodiments, the first region of the guide RNA is about 23, 24, or 25 nucleotides in length. The guide RNA can also include a second region forming a second structure. In some embodiments, the second structure comprises a stem or a hairpin. The length of the stem can vary. For example, the stem may range in length from about 6 to about 10, about 15, about 20, or about 25 base pairs. The stem may include one or more bulges of 1 to 10 nucleotides. Thus, the total length of the second region may range in length from about 16 to about 25 nucleotides. In one embodiment, the loop is about 5 nucleotides long and the stem comprises about 10 base pairs.

[0077] The guide RNA can also include a third region that remains essentially single stranded. Thus, the third region has no complementarity to any nucleotide sequence in the cell of interest, and has no complementarity to the remainder of the guide RNA. The length of the third region can vary, and generally the third region is about 4 or more nucleotides long. For example, the length of the third region is within the range of about 5 to about 60 nucleotides long. The total length of the second and third regions (also called universal or scaffold regions) of the guide RNA is within the range of about 30 to about 120 nucleotides long. In one embodiment, the total length of the second and third regions is within the range of about 40 to about 45 nucleotides long.

[0078] In some embodiments, the guide RNA comprises a single molecule that includes all three regions. In other embodiments, the guide RNA comprises two separate molecules. The first RNA molecule may comprise the first region of the guide RNA and half of the stem of the second region of the guide RNA. The second RNA molecule may comprise the other half of the stem of the second region of the guide RNA and the third region of the guide RNA. Thus, in this embodiment, the first and second RNA molecules each comprise a sequence of nucleotides that are complementary to each other. For example, in one embodiment, the first and second RNA molecules each comprise a sequence (about 6 to about 25 nucleotides) that base pairs with the remaining sequence to form a functional guide RNA. In certain embodiments, the guide RNA is a single molecule (i.e., crRNA) that interacts with target sites in chromosomes and Cpf1 polypeptides without the need for a second guide RNA (i.e., tracrRNA).

[0079] In some embodiments, the guide RNA can be introduced into the plant cell, organelle, or embryo as an RNA molecule. The RNA molecule can be in vitro transcribed. Alternatively, the RNA molecule can be chemically synthesized. In other embodiments, the guide RNA can be introduced into the plant cell, organelle, or embryo as a DNA molecule. In such cases, the DNA encoding the guide RNA can be operably linked to a promoter control sequence for expression of the guide RNA in the plant cell, organelle, or embryo of interest. For example, the RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (PolIII). In an exemplary embodiment, the RNA coding sequence is bound to a plant-specific promoter.

[0080] The DNA encoding the guide RNA may be linear or circular. In some embodiments, the DNA encoding the guide RNA may be part of a vector. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial / minichromosomes, transposons, and viral vectors. In an exemplary embodiment, the DNA encoding the Cpf1 or Csm1 polypeptide is present in a plasmid vector. Suitable plasmid vectors include, but are not limited to, pUC, pBR322, pET, pBluescript, pCAMBIA, and variants thereof. The vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selection marker sequences (e.g., antibiotic resistance genes), replicator sequences, etc.

[0081] In embodiments in which both a Cpf1 or Csm1 polypeptide and a guide RNA are introduced into a plant cell, organelle, or embryo as DNA molecules, each can be part of a separate molecule (e.g., a second vector containing the Cpf1 or Csm1 polypeptide or fusion protein coding sequence and the guide RNA coding sequence) or both can be part of the same molecule (e.g., one vector containing the coding (and regulatory) sequences for both the Cpf1 or Csm1 polypeptide or fusion protein coding sequence and the guide RNA).

[0082] (c) Target site The Cpf1 or Csm1 polypeptide, together with the guide RNA, is directed to a target site in a plant or plant embryo, including a plant chromosomal sequence, a plant cell, a plant organelle (e.g., a plastid or a mitochondrion), where the Cpf1 or Csm1 polypeptide introduces a double-stranded break in the chromosomal sequence. The target site has no sequence restrictions other than that the sequence immediately precedes (upstream of) a consensus sequence. This consensus sequence is also known as a protospacer adjacent motif (PAM). Examples of PAM sequences include, but are not limited to, TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN, where N is defined as any nucleotide. It is known in the art that PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (Karvelis et al. (2015) Genome Biol 16:253). Thus, modulating the concentration of Cpf1 or Csm1 protein delivered to a cell or in vitro system of interest represents a way to modify the PAM site or sites associated with the Cpf1 or Csm1 enzyme. Modulation of Cpf1 or Csm1 protein concentration in a system of interest can be achieved, for example, by modifying the promoter used to express the gene encoding Cpf1 or encoding Csm1, by modifying the concentration of ribonucleoprotein delivered to the cell or in vitro system, or by adding or removing introns that may play a role in modulating gene expression levels. As detailed herein, the first region of the guide RNA is complementary to the protospacer of the target sequence. Typically, the guide RNA is about 19 to 21 nucleotides in length.

[0083] The target site may be in the coding region of a gene, in an intron of a gene, in a regulatory region of a gene, in a non-coding region between genes, etc. The gene may be a protein-coding gene or an RNA-coding gene. The gene may be any gene of interest as described herein.

[0084] (d) donor polynucleotide In some embodiments, the methods disclosed herein further include introducing at least one donor polynucleotide into the plant cell, organelle, or embryo. The donor polynucleotide comprises at least one donor sequence. In some aspects, the donor sequence of the donor polynucleotide corresponds to an endogenous or native plant genomic sequence found in the cell nucleus or in the organelle (e.g., plastid or mitochondrion) of interest. For example, the donor sequence can be essentially identical to a portion of the chromosomal sequence at or near the target site, which contains at least one nucleotide change. Thus, the donor sequence can include a modified version of the wild-type sequence at the target site, such that upon integration or replacement with the native sequence, the sequence at the target site contains at least one nucleotide change. For example, the change can be an insertion of one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more nucleotides, or a combination thereof. As a result of integration of the modified sequence, the plant, plant cell, or plant embryo can produce a modified gene product from the target chromosomal sequence.

[0085] Alternatively, the donor sequence of the donor polynucleotide may correspond to an exogenous sequence. As used herein, an "exogenous" sequence refers to a sequence that is not native to a plant cell, organelle, or embryo, or that is in a different location than its natural location in the genome of the cell, organelle, or embryo. For example, an exogenous sequence can include a protein coding sequence that can be operably linked to an exogenous promoter, such that upon integration into the genome, the plant cell or organelle can express the protein encoded by the integrated sequence. For example, the donor sequence can be any gene of interest, such as one that encodes an agronomically important trait, as described elsewhere herein. Alternatively, the exogenous sequence can be integrated into a nuclear, plastid, and / or mitochondrial 1 chromosomal sequence such that its expression is regulated by an exogenous promoter control sequence. In other iterations, the exogenous sequence can be a transcription control sequence, another expression control sequence, or an RNA coding sequence. The integration of an exogenous sequence into a chromosomal sequence is referred to as a "knock-in." Donor sequences can vary in length from a few nucleotides to hundreds to hundreds of thousands of nucleotides.

[0086] In some embodiments, the donor sequence of the donor polynucleotide is flanked by upstream and downstream sequences that have substantial sequence identity to sequences located upstream and downstream, respectively, of the target site in a plant nuclear, plastid, and / or mitochondrial 1 chromosome sequence. Due to these sequence similarities, the upstream and downstream sequences of the donor polynucleotide allow for homologous recombination between the donor polynucleotide and the target sequence such that the donor sequence is integrated (or replaced) into the target plant sequence.

[0087] As used herein, an upstream sequence refers to a nucleic acid sequence that shares substantial sequence identity with a chromosomal sequence upstream of a target site. Similarly, a downstream sequence refers to a nucleic acid sequence that shares substantial sequence identity with a chromosomal sequence downstream of a target site. As used herein, the phrase "substantially sequence identity" refers to a sequence that has at least about 75% sequence identity. Thus, the upstream and downstream sequences in a donor polynucleotide can have about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the upstream and downstream sequences of a target site. In exemplary embodiments, the upstream and downstream sequences of the donor polynucleotide can have about 95% or 100% identity with the nucleotide sequences upstream and downstream of the target site. In one embodiment, the upstream sequence shares substantial sequence identity with a nucleotide sequence located immediately upstream of the target site (i.e., adjacent to the target site). In another embodiment, the upstream sequence shares substantial sequence identity with a nucleotide sequence located within about one hundred (100) nucleotides upstream of the target site. Thus, for example, the upstream sequence can share substantial sequence identity with a nucleotide sequence located about 1 to about 20, about 21 to 40, about 41 to 60, about 61 to 80, or about 81 to 100 nucleotides upstream of the target site. In one embodiment, the upstream sequence shares substantial sequence identity with a nucleotide sequence located immediately downstream of the target site (i.e., adjacent to the target site). In another embodiment, the downstream sequence shares substantial sequence identity with a nucleotide sequence located within about one hundred (100) nucleotides downstream of the target site. Thus, for example, the downstream sequence can share substantial sequence identity with a nucleotide sequence located about 1 to about 20, about 21 to 40, about 41 to 60, about 61 to 80, or about 81 to 100 nucleotides downstream of the target site.

[0088] The upstream or downstream sequences can each range from about 20 nucleotides to about 5000 nucleotides in length. In some embodiments, the upstream or downstream sequences can comprise 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 nucleotides. In exemplary embodiments, the upstream or downstream sequence may be in the range of about 50 to about 1500 nucleotides.

[0089] The donor polynucleotide, which includes upstream and downstream sequences with similar sequences to the target nucleotide sequence, can be linear or circular. In embodiments where the donor polynucleotide is circular, the donor polynucleotide can be part of a vector. For example, the vector can be a plasmid vector.

[0090] In some embodiments, the donor polynucleotide may further comprise at least one target cleavage site recognized by a Cpf1 or Csm1 polypeptide. The target cleavage site added to the donor polynucleotide may be located upstream or downstream or both upstream and downstream of the donor sequence. For example, the donor sequence may be adjacent to the target cleavage site, such that upon cleavage by the Cpf1 or Csm1 polypeptide, the donor sequence is adjacent to an overhang that is compatible with the nucleotide sequence generated upon cleavage by the Cpf1 or Csm1 polypeptide. Thus, the donor sequence may be ligated to the cleaved nucleotide sequence during repair of the double-strand break by a non-homologous repair process. In general, the donor polynucleotide containing the target cleavage site may be circular (e.g., may be part of a plasmid vector).

[0091] The donor polynucleotide may be a linear molecule containing a short donor sequence with any short overhang that is compatible with the overhang generated by the Cpf1 or Csm1 polypeptide. In such an embodiment, the donor sequence may be directly ligated to the cleaved chromosomal sequence during repair of the double-strand break. In some instances, the donor sequence may be less than about 1000, less than about 500, less than about 250, or less than about 100 nucleotides. In some instances, the donor polynucleotide may be a linear molecule containing a short donor sequence with blunt ends. In other iterations, the donor polynucleotide may be a linear molecule containing a short donor sequence with a 5' and / or 3' overhang. The overhang may comprise 1, 2, 3, 4, or 5 nucleotides.

[0092] In some embodiments, the donor polynucleotide will be DNA. The DNA may be single-stranded or double-stranded and / or linear or circular. The donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear fragment of DNA, naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. In some embodiments, the donor polynucleotide comprising the donor sequence may be part of a plasmid vector. In any of these situations, the donor polynucleotide comprising the donor sequence may further comprise at least one additional sequence.

[0093] (e) Introduction into plant cells The Cpf1 or Csm1 polypeptide (or encoding nucleic acid), guide RNA (or encoding DNA), and any donor polynucleotides can be introduced into plant cells, organelles, or embryos by various means. Transformation protocols and protocols for introducing polypeptide or polynucleotide sequences into plants vary depending on the type of plant or plant cell targeted for transformation, i.e., monocotyledonous or dicotyledonous. Suitable methods for introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Biggs et al. (1986) Proc. Natl. Acad. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and lectin conversion (WO 00 / 28058). Further references include Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27p:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Patents 5,240,855; 5,322,783; and 5,324,646; Klein et al. (1988) Plant Physiol 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Keppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christon and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize with Agrobacterium tumefaciens), all of which are incorporated herein by reference. Site-specific genome editing in plant cells by gene gun delivery of a ribonucleoprotein containing a nuclease and a suitable guide RNA has been demonstrated (Svitashev et al. (2016) Nat Commun doi:10.1038 / ncomms13274); these methods are incorporated herein by reference. "Stable transformation" is intended to mean that a nucleotide construct introduced into a plant can be integrated into the genome of the plant and inherited by its progeny. The nucleotide construct may be integrated into the nuclear, plastid, or mitochondrial genome of the plant. Methods for plastid transformation are known in the art (see, e.g., Chloroplast Biotechnology: Methods and Protocols (2014) Pal Maliga, ed. and U.S. Patent Application No. 2011 / 0321187), and methods for plant mitochondrial transformation have been disclosed in the art (see, e.g., U.S. Patent Application No. 2011 / 0296551), which are incorporated herein by reference.

[0094] The transformed cells can be grown (i.e., cultured) in plants according to conventional methods. See, e.g., McCormick et al. (1986) Plant Cell Reports 5:81-84. Thus, the present invention provides transformed seeds (also referred to as "transgenic seeds") having the nucleic acid modification stably integrated into their genome.

[0095] "Introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell means "transfection" or "transformation" or "transduction" and also includes the incorporation of a nucleic acid fragment into a plant cell, where the nucleic acid fragment may be integrated into the genome of the cell (e.g., a nuclear chromosome, a plasmid, a plastid chromosome, or a mitochondrial chromosome), converted into an autonomous replicon, or transiently expressed (e.g., as transfected mRNA).

[0096] The present invention may be employed for the transformation of any plant species, including, but not limited to, monocotyledonous and dicotyledonous plants (ie, monocotyledons and dicotyledons, respectively). Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica (e.g., B. napus, B. rapa, B. juncea), particularly those useful as a source of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), foxtail millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), mengka (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musas pp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaer), papaya (Carica papaya), cashew nut (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), oil palm (Elaeis guineensis), poplar (Populus spp.), eucalyptus (Eucalyptus spp.), oats (Avena sativa), barley (Hordeum vulgare), vegetables, ornamental plants, and conifers.

[0097] The Cpf1 or Csm1 polypeptide (or encoding nucleic acid), guide RNA (or DNA encoding the guide RNA) and any donor polynucleotide can be introduced simultaneously or sequentially into a plant cell, organelle, or embryo. The ratio of Cpf1 polypeptide (or encoding nucleic acid) to guide RNA (encoding DNA) will generally be approximately stoichiometric, allowing the two components to form an RNA-protein complex with the target DNA. In one embodiment, the DNA encoding the Cpf1 or Csm1 polypeptide and the DNA encoding the guide RNA are delivered together in a plasmid vector.

[0098] The compositions and methods disclosed herein can be used to alter the expression of a gene of interest in a plant, such as a gene involved in photosynthesis. Thus, the expression of a gene encoding a protein involved in photosynthesis may be altered compared to a control plant. A "subject plant or plant cell" is a plant or plant cell that contains a genetic alteration, such as a mutation, carried out on a gene of interest, or that is derived from a plant or cell so altered. A "control" or "control plant" or "control plant cell" provides a reference point for measuring phenotypic changes in the subject plant or plant cell. Thus, expression levels may be higher or lower than those in a control plant by the methods of the present invention.

[0099] A control plant or plant cell can be, for example, (a) a wild-type plant or cell, i.e., a plant or plant cell of the same genotype as the starting material for the genetic modification resulting in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material, but that has been transformed with a null construct (i.e., a construct that does not affect the trait of interest, such as a construct containing a marker gene); (c) a plant or plant cell that is a non-transformed segregant among the progeny of the subject plant or cell; (d) a plant or plant cell that is genetically identical to the subject plant or cell, but that has not been exposed to a condition or stimulus that induces expression of the gene of interest; or (e) the subject plant or cell itself, under conditions in which the gene of interest is not expressed.

[0100] Although the invention is described in terms of transformed plants, it should be recognized that the transformed organisms of the invention include intact plant cells in plants, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant mass, and plants or parts of plants such as embryos, pollen, eggs, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, husks, stems, roots, root tips, anthers, etc. By grain, it is intended to mean mature seeds produced by commercial growers for purposes other than cultivating or regenerating the seed. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention if these parts contain the introduced polynucleotide.

[0101] (f) Use of fusion proteins to modify or regulate the expression of plant sequences. The methods disclosed herein further include modifying a nucleotide sequence or modulating expression of a nucleotide sequence in a plant cell, plant organelle, or plant embryo, the methods comprising introducing into a plant cell or plant embryo at least one fusion protein or a nucleic acid encoding at least one fusion protein, the fusion protein comprising a Cpf1 or Csm1 polypeptide or a fragment or variant thereof and an effector domain, and (b) at least one guide RNA or a DNA encoding a guide RNA, the guide RNA directing the Cpf1 or Csm1 polypeptide of the fusion protein to a target site in a chromosomal sequence, and the effector domain of the fusion protein modulates a chromosomal sequence or modulates expression of a chromosomal sequence.

[0102] Fusion proteins comprising a Cpf1 or Csm1 polypeptide or a fragment or variant thereof and an effector domain are described herein. In general, the fusion proteins disclosed herein may further comprise at least one nuclear localization signal, a plastid signal peptide, a mitochondrial signal peptide, or a signal peptide capable of transporting the protein to multiple subcellular locations. Nucleic acids encoding the fusion proteins are described herein. In some embodiments, the fusion protein is introduced into a cell or embryo as an isolated fusion protein, which may further comprise a cell-penetrating domain. Furthermore, the isolated fusion protein may be part of a protein-RNA complex that includes a guide RNA. In other embodiments, the fusion protein may be introduced into a cell or embryo as an RNA molecule, which may be capped and / or polyadenylated. In yet other embodiments, the fusion protein may be introduced into a cell or embryo as a DNA molecule. For example, the fusion protein and the guide RNA may be introduced into a cell or embryo as separate DNA molecules or as part of the same DNA molecule. Such a DNA molecule may be a plasmid vector.

[0103] In some embodiments, this involves introducing at least one donor polynucleotide into a cell, organelle, or embryo, as described elsewhere herein. Means for introducing molecules into a plant cell, organelle, or plant embryo, as well as means for culturing a cell (including a cell that contains an organelle) or embryo are described herein.

[0104] In some embodiments where the effector domain of the fusion protein is a cleavage domain, the method can include introducing a fusion protein (or a nucleic acid encoding a fusion protein) and two guide RNAs (or DNA encoding two guide RNAs) into a plant cell, organelle, or embryo. The two RNAs direct the fusion protein to two different target sites in a chromosomal sequence, where the fusion protein dimerizes (e.g., forms a homodimer) so that the two cleavage domains can introduce a double-stranded break in the chromosomal sequence. In embodiments where no donor polynucleotide is present, the double-stranded break in the chromosomal sequence can be repaired by a non-homologous end joining (NHEJ) repair process. Because NHEJ is error-prone, a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof can occur during repair of the break. Thus, the targeted chromosomal sequence can be modified or inactivated. For example, a single nucleotide (SNP) may result in an increase in an altered protein product, or a shift in the reading frame of a coding sequence may inactivate or "knock out" the sequence so that no protein product is made. In embodiments where any donor polynucleotide is present, the donor sequence in the donor polynucleotide may be exchanged or integrated into the chromosomal sequence at the target site during repair of the double break. For example, in embodiments where the donor sequence has substantial sequence identity with and is adjacent to the upstream and downstream sequences of the target site in the chromosomal sequence, respectively, the donor sequence may be exchanged or integrated into the chromosomal sequence at the target site during repair mediated by a homology-directed repair process. Alternatively, in embodiments where the donor sequence is adjacent to a compatible overhang (or where the compatible overhang is generated in situ by a Cpf1 or Csm1 polypeptide), the donor sequence may be directly ligated to the cleaved chromosomal sequence by a non-homologous repair process during repair of the double break.Exchange or integration of the donor sequence with a chromosomal sequence will modify the target chromosomal sequence or introduce an exogenous sequence into the target chromosomal sequence of a plant cell, organelle, or embryo.

[0105] In other embodiments where the effector domain of the fusion protein is a cleavage domain, the method can introduce two different fusion proteins (or nucleic acids encoding two different fusion proteins) and two guide RNAs (or DNA encoding two guide RNAs) into a plant cell, organelle, or embryo. The fusion proteins are different as described elsewhere herein. Each RNA directs the fusion protein to a specific target site in the chromosomal sequence where the fusion proteins can dimerize (e.g., form heterodimers) such that the two cleavage domains can introduce a double-stranded break in the chromosomal sequence. In embodiments where no donor polynucleotide is present, the resulting double-stranded break can be repaired by a non-homologous repair process such that a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof can occur during repair of the break. In embodiments in which an optional donor polynucleotide is present, the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence during repair of the double-stranded break by either a homology-based repair process (e.g., in embodiments in which the donor sequence has substantial sequence identity with, and is flanked by, upstream and downstream sequences, respectively, of the target site in the chromosomal sequence) or a non-homologous repair process (e.g., in embodiments in which the donor sequence is flanked by compatible overhangs).

[0106] In some embodiments, where the effector domain of the fusion protein is a transcription activation domain or a transcription repressor domain, the method can include introducing a fusion protein (or a nucleic acid encoding a fusion protein) and a guide RNA (or a guide RNA) into a plant cell, organelle, or embryo. The guide RNA targets the fusion protein to a specific chromosomal sequence, where the transcription activation domain or the transcription repressor domain activates or represses, respectively, the expression of one or more genes located near the targeted chromosomal sequence. That is, transcription can be affected for genes that are close to the targeted chromosomal sequence or for genes that are located far from the targeted chromosomal sequence. It is known in the art that gene transcription can be regulated by sequences located thousands of bases away from the transcription start site or on another chromosome. (Harmston and Lenhard (2013) Nucleic Acids Res 41:7185-7199).

[0107] In another embodiment, where the effector domain of the fusion protein is an epigenetic modification domain, the method can include introducing a fusion protein (or a nucleic acid encoding a fusion protein) and a guide RNA (or a DNA encoding a guide RNA) into a plant cell, organelle, or embryo. The guide RNA targets the fusion protein to a specific chromosomal sequence, where the epigenetic modification domain modifies the structure of the targeted chromosomal sequence. Epigenetic modifications include protein acetylation, methylation, and / or nucleotide methylation. In some instances, the structural modification of the chromosomal sequence leads to a change in the expression of the chromosomal sequence.

[0108] V. Plants and Plant Cells Containing Genetic Modifications Provided are plant cells, plant organelles, and plant embryos that contain at least one nucleotide sequence modified by employing the Cpf1 or Csm1 polypeptide-mediated or fusion protein-mediated processes described herein. Also provided are plant cells, organelles, and plant embryos that contain at least one DNA or RNA molecule encoding a Cpf1 or Csm1 polypeptide or fusion protein directed to a chromosomal sequence or fusion protein of interest, at least one guide RNA, and optionally one or more donor polynucleotides. The genetically modified plants disclosed herein can be heterozygous for the modified nucleotide sequence or homozygous for the modified nucleotide sequence. The plant cells that contain one or more genetic modifications in the organelle DNA can be heteroplasmic or homoplasmic.

[0109] The modified chromosomal sequence of the plant, plant organelle, or plant cell may be modified or contain an integrated sequence to be inactivated, to have its expression up-regulated or down-regulated, or to produce a modified protein. The modified chromosomal sequence may be inactivated such that the sequence is not transcribed and / or a functional protein product is not produced. Thus, a genetically modified plant containing an inactivated chromosomal sequence may be termed a "knockout" or a "conditional knockout". The inactivated chromosomal sequence may contain a deletion mutation (i.e., deletion of one or more nucleotides), an insertion mutation (i.e., insertion of one or more nucleotides), a nonsense mutation (i.e., substitution of a single nucleotide for another nucleotide such that one or more nucleotide stop codons are introduced). As a result of the mutation, the targeted chromosomal sequence is inactivated and no functional protein is produced. The inactivated chromosomal sequence does not contain an exogenously introduced sequence. Also included herein are genetically modified plants in which 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomal sequences are inactivated.

[0110] The modified chromosomal sequence can also be altered to encode a mutant protein product. For example, a genetically modified plant containing a modified chromosomal sequence can contain targeted point mutations or other mutations such that a modified protein product is produced. In one embodiment, the chromosomal sequence can be modified such that at least one nucleotide is changed and the expressed protein product contains one altered amino acid residue (missense mutation). In another embodiment, the chromosomal sequence can be modified to contain more than one missense mutation such that more than one amino acid is changed. In addition, the chromosomal sequence can be modified to have three nucleotide deletions or insertions such that the expressed protein contains a single amino acid deletion or insertion. The modified or mutant protein can have modified properties or activities compared to the wild-type protein, such as modified substrate specificity, modified enzymatic activity, modified reaction rate, etc.

[0111] In some embodiments, the genetically modified plant may include a nucleotide sequence integrated into at least one chromosome. A genetically modified plant including an integrated sequence may be termed a "knock-in" or a "conditional knock-in". The integrated sequence may, for example, code for an orthologous protein, an endogenous protein, or a combination of both. In one embodiment, a sequence encoding an orthologous protein or an endogenous protein may be integrated into a nuclear or organelle chromosome sequence encoding a protein such that the chromosome sequence is inactivated, but the exogenous sequence is expressed. In such a case, the sequence encoding an orthologous protein or an endogenous protein may be operably linked to a promoter control sequence. Alternatively, the sequence encoding an orthologous protein or an endogenous protein may be integrated into a nuclear or organelle chromosome sequence without affecting the expression of the chromosome sequence. For example, the protein coding sequence may be integrated into a "safe harbor" locus. The present disclosure also includes genetically modified plants having 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sequences integrated into the genome, including sequences encoding proteins. As disclosed herein, any gene of interest can be introduced and integrated into the chromosomal sequence of the plant nucleus or organelle. In certain embodiments, a gene that increases plant growth or production is integrated into the chromosome.

[0112] The integrated sequence on the chromosome encoding the protein can encode a wild type of the protein of interest, or can encode a protein that includes at least one modification such that a modified version of the protein is produced. For example, the integrated sequence on the chromosome encoding a protein associated with a disease or disorder can include at least one modification such that the modified version of the protein produced causes or enhances the associated disorder. Alternatively, the integrated sequence on the chromosome encoding a protein associated with a disease or disorder can include at least one modification such that the modified version of the protein produced protects the plant from developing the associated disease or disorder.

[0113] In some embodiments, the genetically modified plant can include at least one modified chromosomal sequence encoding a protein such that the expression pattern of the protein is modified. For example, the regulatory regions controlling the expression of the protein, such as promoters or transcriptional gene binding sites, can be modified such that the protein is overexpressed or the tissue specific or temporal expression of the protein is modified or a combination thereof. Alternatively, the expression pattern of the protein can be modified using a conditional knockout system. Examples of conditional knockout systems include, but are not limited to, the Cre-lox recombination system. The Cre-lox recombination system contains the enzyme Cre recombinase, a site-specific DNA recombinase that can catalyze the recombination of nucleic acid sequences between specific sites (lox sites) in a nucleic acid molecule. Methods of using this system to produce temporal and tissue specific expression are known in the art.

[0114] VI. METHODS FOR MODIFYING NUCLEOTIDE SEQUENCES IN NON-PLANT EUKARYOTIC GENOMES AND NON-PLANT EUKARYOTIC CELLS CONTAINING GENETIC MODIFICATIONS A method for modifying a nucleotide sequence in a non-plant eukaryotic cell or non-plant eukaryotic organelle is provided herein. The method includes introducing a DNA-targeting RNA, comprising (a) a first segment comprising a nucleotide sequence complementary to a sequence in a target DNA and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide, a DNA polynucleotide encoding the DNA-targeting RNA, into the target cell or organelle, and also introducing a Cpf1 or Csm1 polypeptide, comprising (a) an RNA-binding portion that interacts with the DNA-targeting RNA and (b) an activity portion that exhibits site-specific enzymatic activity, the Cpf1 or Csm1 polypeptide, or a polynucleotide encoding the Cpf1 or Csm1 polypeptide into the target cell or organelle. The target cell or organelle can then be cultured under conditions in which the chimeric nuclease polypeptide is expressed and cleaves the nucleotide sequence. The system described herein can be used in combination with exogenous Mg 2+ Note that the addition of cations, ions, or other ions is not required. Finally, a non-plant eukaryotic cell or organelle can be selected that contains the modified nucleotide sequence.

[0115] In some embodiments, the method comprises introducing a Cpf1 or Csm1 polypeptide (or encoding nucleic acid) and a guide RNA (or encoding DNA) into a non-plant eukaryotic cell or organelle, where the Cpf1 or Csm1 polypeptide introduces a double-stranded break in a target nucleotide sequence in chromosomal DNA of the nucleus or organelle. In some embodiments, the method can comprise introducing a Cpf1 or Csm1 polypeptide (or encoding nucleic acid) and at least one guide RNA (or encoding DNA) into a non-plant eukaryotic cell or organelle, where the Cpf1 or Csm1 polypeptide introduces one or more double-stranded breaks (i.e., 2, 3, or more than 3 double-stranded breaks) in a target nucleotide sequence in chromosomal DNA of the nucleus or organelle. In embodiments in which no donor polynucleotide is present, the double-stranded break in the nucleotide sequence can be repaired by a non-homologous end joining (NHEJ) repair process. Since NHEJ is error-prone, deletion of at least one nucleotide, insertion of at least one nucleotide, substitution of at least one nucleotide, or a combination thereof may occur during repair of the break. Thus, the targeted nucleotide sequence may be altered or inactivated. For example, a single nucleotide (SNP) may result in an increase in an altered protein product, or a shift in the reading frame of the coding sequence may inactivate or "knock out" the sequence so that no protein product is made. In embodiments in which any donor polynucleotide is present, the donor sequence in the donor polynucleotide may be replaced or incorporated into the nucleotide sequence at the target site during repair of the double break. For example, in embodiments in which the donor sequence has substantial sequence identity with and is adjacent to the upstream and downstream sequences of the target site in the nucleotide sequence of a non-plant eukaryotic cell or organelle, the donor sequence may be replaced or incorporated into the nucleotide sequence at the target site during repair mediated by a homology-directed repair process.Alternatively, in embodiments where the donor sequence is adjacent to a compatibility overhang (or where a compatibility overhang is generated in situ by a Cpf1 or Csm1 polypeptide), the donor sequence can be directly joined to the cleaved nucleotide sequence by a non-homologous repair process during repair of the double break. Exchange or incorporation of the donor sequence into the nucleotide sequence will modify the target nucleotide sequence or introduce an exogenous sequence into the target nucleotide sequence in the non-plant eukaryotic cell or organelle.

[0116] In some embodiments, double strand breaks caused by the action of Cpf1 or Csm1 nuclease are repaired by a method such that DNA is deleted from the chromosome of the non-plant eukaryotic cell or organelle, hi some embodiments, a single base, a few bases (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases), or large segments of DNA (i.e., 10 or more, 50 or more, 100 or more, or 500 or more bases) are deleted from the chromosome of the non-plant eukaryotic cell or organelle.

[0117] In some embodiments, expression of non-plant eukaryotic genes can be altered as a result of double-strand breaks caused by Cpf1 or Csm1 nuclease. In some embodiments, expression of non-plant eukaryotic genes can be altered by mutant Cpf1 or Csm1 enzymes that contain a mutation that renders Cpf1 or Csm1 nuclease unable to create double-strand breaks. In some preferred embodiments, mutant Cpf1 or Csm1 nucleases that contain a mutation that renders Cpf1 or Csm1 nuclease unable to create double-strand breaks can be fused to a transcriptional activation or inactivation domain.

[0118] In some embodiments, eukaryotic cells having mutations in nuclear and / or organelle chromosomal DNA caused by the action of Cpf1 or Csm1 nucleases are cultured to produce eukaryotic organisms. In some embodiments, eukaryotic cells having altered gene expression as a result of one or more Cpf1 or Csm1 nucleases or one or more mutant Cpf1 or Csm1 nucleases are cultured to produce eukaryotic organisms. Methods of culturing non-plant eukaryotic cells to produce eukaryotic organisms are known in the art, for example in U.S. Patent Application Publication Nos. 2016 / 0208243 and 2016 / 0138008, and are incorporated herein by reference.

[0119] The present invention can be employed for the transformation of eukaryotic species, whether such as, but not limited to, animals (including, but not limited to, mammals, insects, fish, birds, and reptiles), fungi, amoebas, and yeast.

[0120] Methods for the introduction of nuclease proteins, DNA or RNA molecules encoding nuclease proteins, guide RNAs or DNA molecules encoding guide RNAs, and any donor sequence DNA molecules into non-plant eukaryotic cells or organelles are known in the art, for example in US Patent Application No. 2016 / 0208243, and are incorporated herein by reference. Exemplary genetic modifications to non-plant eukaryotic cells or organelles that may be of special value in industrial applications are also known in the art, for example in US Patent Application No. 2016 / 0208243, and are incorporated herein by reference.

[0121] VII. METHODS FOR MODIFYING NUCLEOTIDE SEQUENCES IN PROKARYOTIC GENOMES AND PROKARYOTIC CELLS CONTAINING GENETIC MODIFICATIONS Provided herein is a method for modifying a nucleotide sequence in a prokaryotic (e.g., bacterial or archaeal) cell. The method includes introducing into the target cell a DNA-targeting RNA or a DNA polynucleotide encoding the DNA-targeting RNA, where the DNA-targeting RNA comprises (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide, and also includes introducing into the target cell a Cpf1 or Csm1 polypeptide or a polynucleotide encoding a Cpf1 or Csm1 polypeptide, where the Cpf1 or Csm1 polypeptide comprises (a) an RNA-binding portion that interacts with the DNA-targeting RNA and (b) an activity portion that exhibits site-specific enzymatic activity. The target cell is then cultured under conditions in which the Cpf1 or Csm1 polypeptide is expressed and cleaves the nucleotide sequence. The system described herein can be used to culture the target cell using exogenous Mg 2+ It should be noted that the addition of ions such as arginine, ...

[0122] In some embodiments, the method can include introducing a Cpf1 or Csm1 polypeptide (or encoding nucleic acid) and a guide RNA (or encoding DNA) into a prokaryotic cell, where the Cpf1 or Csm1 polypeptide introduces a double-stranded break in a target nucleotide sequence of the prokaryotic cell DNA. In some embodiments, the method can include introducing a Cpf1 or Csm1 polypeptide (or encoding nucleic acid) and at least one guide RNA (or encoding DNA) into a prokaryotic cell, where the Cpf1 or Csm1 polypeptide introduces more than one double-stranded break (i.e., 2, 3, or more than 3 double-stranded breaks) in a target nucleotide sequence of the prokaryotic cell DNA. In embodiments in which no donor polynucleotide is present, the double-stranded break in the nucleotide sequence can be repaired by a non-homologous end joining (NHEJ) repair process. Since NHEJ is error-prone, deletion of at least one nucleotide, insertion of at least one nucleotide, substitution of at least one nucleotide, or a combination thereof may occur during repair of the break. Thus, the targeted nucleotide sequence may be altered or inactivated. For example, a single nucleotide (SNP) may result in an increase in an altered protein product, or a shift in the reading frame of the coding sequence may inactivate or "knock out" the sequence so that no protein product is made. In embodiments where any donor polynucleotide is present, the donor sequence in the donor polynucleotide may be replaced or incorporated into the nucleotide sequence at the target site during repair of the double break. For example, in embodiments where the donor sequence has substantial sequence identity with and is adjacent to the upstream and downstream sequences of the target site in the nucleotide sequence of the prokaryotic cell, the donor sequence may be replaced or incorporated into the nucleotide sequence at the target site during repair mediated by a homology-directed repair process.Alternatively, in embodiments where the donor sequence is adjacent to a compatibility overhang (or where a compatibility overhang is generated in situ by a Cpf1 or Csm1 polypeptide), the donor sequence can be directly joined to the cleaved nucleotide sequence by a non-homologous repair process during repair of the double break. Exchange or incorporation of the donor sequence with a nucleotide sequence will modify the target nucleotide sequence or introduce an exogenous sequence into the target nucleotide sequence of the prokaryotic cell DNA.

[0123] In some embodiments, double strand breaks caused by the action of Cpf1 or Csm1 nuclease are repaired by a method such that DNA is deleted from the prokaryotic cell DNA, hi some embodiments, a single base, a few bases (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases), or large segments of DNA (i.e., 10 or more, 50 or more, 100 or more, or 500 or more bases) are deleted from the prokaryotic cell DNA.

[0124] In some embodiments, expression of a prokaryotic gene may be altered as a result of a double-stranded break caused by a Cpf1 or Csm1 nuclease. In some embodiments, expression of a prokaryotic gene may be altered by a mutant Cpf1 or Csm1 nuclease that includes a mutation that renders the Cpf1 or Csm1 nuclease unable to create a double-stranded break. In some preferred embodiments, a mutant Cpf1 or Csm1 nuclease that includes a mutation that renders the Cpf1 or Csm1 nuclease unable to create a double-stranded break may be fused to a transcriptional activation or inactivation domain.

[0125] The present invention is directed to, but not limited to, Cyanobacteria, the genus Corynebacterium, the genus Bifidobacterium, the genus Mycobacterium, the genus Streptomyces, the genus Thermobifida, the genus Chlamydia, the genus Prochlorococcus, the genus Synechococcus, the genus Thermosynechococcus, the genus Thermo ... The genus Rmosynechococcus, Thermus, Bacillus, Clostridium, Geobacillus, Lactobacillus, Listeria, Staphylococcus, Streptococcus, Fusobacterium, Agrobacterium ), Bradyrhizobium spp., Ehrlichia spp., Mesorhizobium spp., Nitrobacter spp., Rickettsia spp., Urbachia spp. (genus Wolbachia), genus Zymomonas, genus Burkholderia, genus Neisseria, genus Ralstonia, genus Acinetobacter, genus Acinetobacter, - Erwinia, Escherichia, Haemophilus, Legionella, Pasteurella, Pseudomonas, Psychrobacter, Salmonella, Shewanella, Shigella, Vibrio, Xanthomonas,It can be used for transformation of any prokaryotic species, including those of the genera Xylella, Yersinia, Campylobacter, Desulfovibrio, Helicobacter, Geobacter, Leptospira, Treponema, Mycoplasma, and Thermotoga.

[0126] Methods for introducing nuclease proteins, DNA or RNA encoding nuclease proteins, guide RNA or DNA encoding guide RNA, and any donor sequence DNA molecules into prokaryotic cells or organelles are known in the art, for example, in U.S. Patent Application No. 2016 / 0208243, and are incorporated herein by reference. Exemplary genetic modifications to prokaryotic cells that may be of special value in industrial applications are also known in the art, for example, in U.S. Patent Application No. 2016 / 0208243, and are incorporated herein by reference.

[0127] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0128] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0129] Embodiments of the present invention include the following.

[0130] 1. 1. A method for modifying a nucleotide sequence at a target site in the genome of a eukaryotic cell, comprising: introducing into said eukaryotic cell: (i) a DNA-targeting RNA or a DNA polynucleotide encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of a target DNA, and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide; (ii) a Cpf1 or Csm1 polypeptide, or a polynucleotide encoding a Cpf1 or Csm1 polypeptide, wherein the Cpf1 or Csm1 polypeptide comprises (a) an RNA-binding portion that interacts with a DNA-targeting RNA and (b) an active portion that exhibits site-specific enzymatic activity.

[0131] 2. 1. A method for modifying a nucleotide sequence at a target site in the genome of a prokaryotic cell, comprising: introducing into the prokaryotic cell: (i) a DNA-targeting RNA or a DNA polynucleotide encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of a target DNA, and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide; (ii) a Cpf1 or Csm1 polypeptide, or a polynucleotide encoding a Cpf1 or Csm1 polypeptide, wherein the Cpf1 or Csm1 polypeptide comprises (a) an RNA-binding portion that interacts with a DNA-targeting RNA and (b) an active portion that exhibits site-specific enzymatic activity.

[0132] 3. 1. A method for modifying a nucleotide sequence at a target site in the genome of a plant cell, comprising: 1. A method comprising the steps of introducing into a plant cell: (i) a DNA-targeting RNA, or a DNA polynucleotide encoding a DNA-targeting RNA, where the DNA-targeting RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA, and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide; (ii) a Cpf1 or Csm1 polypeptide, or a polynucleotide encoding a Cpf1 or Csm1 polypeptide, wherein the Cpf1 or Csm1 polypeptide comprises (a) an RNA-binding portion that interacts with a DNA-targeting RNA and (b) an active portion that exhibits site-specific enzymatic activity.

[0133] 4. The method of any one of embodiments 1 to 3, further comprising the steps of growing a plant under conditions in which a Cpf1 or Csm1 polypeptide is expressed and cleaves the nucleotide sequence at the target site to produce a modified nucleotide sequence, and selecting a plant containing the modified nucleotide sequence.

[0134] 5. The method of any one of embodiments 1-4, wherein cleaving the nucleotide sequence of the target site comprises making a double-stranded break in a sequence at or near the targeted DNA-targeting RNA sequence.

[0135] 6. The method of embodiment 5, wherein the double-stranded break is a staggered double-stranded break.

[0136] 7. The method of embodiment 6, wherein the staggered double-stranded break creates a 5'-end overhang of 3 to 6 nucleotides.

[0137] 8. The method of any one of embodiments 1-7, wherein the DNA-targeting RNA is a guide RNA (gRNA).

[0138] 9. The method of any one of embodiments 1 to 8, wherein the modified nucleotide sequence comprises an insertion of heterologous DNA into the genome of the cell, a deletion of a nucleotide sequence from the genome of the cell, or a mutation of at least one nucleotide in the genome of the cell.

[0139] 10. The method of any one of embodiments 1-9, wherein the Cpf1 or Csm1 polypeptide is selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236.

[0140] 11. The method of any one of embodiments 1-10, wherein the polynucleotide encoding the Cpf1 or Csm1 polypeptide is selected from the group consisting of SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 21, 22, 24, 25, and 174-206.

[0141] 12. The method of any one of embodiments 1-11, wherein the Cpf1 or Csm1 polypeptide has at least 80% identity to one or more polypeptide sequences selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236.

[0142] 13. The method of any one of embodiments 1-12, wherein the Cpf1 or Csm1 polypeptide has at least 70% identity to one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 21, 22, 24, 25, and 174-206.

[0143] 14. The method of any one of embodiments 1 to 13, wherein the Cpf1 or Csm1 polypeptide forms a homodimer or a heterodimer.

[0144] 15. The method of any one of embodiments 1-14, wherein the plant cell is from a monocotyledonous plant.

[0145] 16. The method of any one of embodiments 1-14, wherein the plant cell is from a dicotyledonous plant.

[0146] 17. The method of any one of embodiments 1-16, wherein expression of the Cpf1 or Csm1 polypeptide is under the control of an inducible or constitutive promoter.

[0147] 18. The method of any one of embodiments 1-17, wherein expression of the Cpf1 or Csm1 polypeptide is under the control of a cell-type-specific or developmentally preferred promoter.

[0148] 19. The method of any one of embodiments 1-18, wherein the PAM sequence comprises 5'-TTN, where N can be any nucleotide.

[0149] 20. The method of any one of embodiments 1-19, wherein the nucleotide sequence at the target site in the genome of the cell encodes an SBPase, FBPase, FBP aldolase, AGPase large subunit, AGPase small subunit, sucrose phosphate synthase, starch synthase, PEP carboxylase, pyruvate phosphate dikinase, transketolase, Rubisco small subunit, or Rubisco activation protein, or encodes a transcription factor that regulates expression of one or more genes encoding SBPase, FBPase, FBP aldolase, AGPase large subunit, AGPase small subunit, sucrose phosphate synthase, starch synthase, PEP carboxylase, pyruvate phosphate dikinase, transketolase, Rubisco small subunit, or Rubisco activation protein.

[0150] 21. The method of any one of embodiments 1 to 20, further comprising contacting the target site with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into target DNA.

[0151] 22. The method of any one of embodiments 1-21, wherein the target DNA is modified such that nucleotides within the target DNA are deleted.

[0152] 23. The method of any one of embodiments 1-22, wherein the polynucleotide encoding a Cpf1 or Csm1 polypeptide is codon optimized for expression in a plant cell.

[0153] 24. The method of any one of embodiments 1 to 23, wherein expression of the nucleotide is increased or decreased.

[0154] 25. The method of any one of embodiments 1 to 24, wherein the polynucleotide encoding a Cpf1 or Csm1 polypeptide is operably linked to a promoter that is constitutive, cell-specific, inducible, or activated by alternative splicing of a suicide exon.

[0155] 26. The method of any one of embodiments 1-25, wherein the Cpf1 or Csm1 polypeptide comprises one or more mutations that reduce or eliminate nuclease activity of the Cpf1 or Csm1 polypeptide.

[0156] 27. The method of embodiment 26, wherein the mutated Cpf1 or Csm1 polypeptide comprises a mutation at a position corresponding to position 917 or 1006 of FnCpf1 (sequence number 3) or at a position corresponding to position 701 or 922 of SmCsm1 (sequence number 160) when aligned for maximum identity, or wherein the mutated Cpf1 or Csm1 polypeptide comprises a mutation at position 917 or 1006 of FnCpf1 (sequence number 3) or at position 701 or 922 of SmCsm1 (sequence number 160) when aligned for maximum identity.

[0157] 28. The method of embodiment 27, wherein the mutation at a position corresponding to position 917 or 1006 of FnCpf1 (sequence number 3) is D917A and E1006A, respectively, or the mutation at a position corresponding to position 701 or 922 of SmCsm1 (sequence number 160) is D701A and E922A, respectively.

[0158] 29. The method of any one of embodiments 26-28, wherein the mutated Cpf1 or Csm1 polypeptide comprises an amino acid sequence set forth in the group consisting of SEQ ID NOs: 26-41 and 63-70.

[0159] 30. The method of any one of embodiments 26-29, wherein the mutated Cpf1 or Csm1 polypeptide is fused to a transcription activation domain.

[0160] 31. The method of embodiment 30, wherein the mutated Cpf1 or Csm1 polypeptide is fused directly to a transcription activation domain or fused to a transcription activation domain by a linker.

[0161] 32. The method of any one of embodiments 26-29, wherein the mutated Cpf1 or Csm1 polypeptide is fused to a transcriptional repressor domain.

[0162] 33. The method of embodiment 32, wherein the mutated Cpf1 or Csm1 polypeptide is fused to the transcriptional repressor domain by a transcriptional linker.

[0163] 34. The method of any one of embodiments 1 to 33, wherein the Cpf1 or Csm1 polypeptide further comprises a nuclear localization signal.

[0164] 35. The method of embodiment 34, wherein the nuclear localization signal comprises SEQ ID NO:1 or is encoded by SEQ ID NO:2.

[0165] 36. The method of any one of embodiments 1-33, wherein the Cpf1 or Csm1 polypeptide further comprises a chloroplast signal peptide.

[0166] 37. The method of any one of embodiments 1-33, wherein the Cpf1 or Csm1 polypeptide further comprises a mitochondrial signal peptide.

[0167] 38. The method of any one of embodiments 1-33, wherein the Cpf1 or Csm1 polypeptide further comprises a signal peptide that targets the Cpf1 or Csm1 polypeptide to multiple intracellular locations.

[0168] 39. A nucleic acid molecule comprising a polynucleotide sequence encoding a Cpf1 or Csm1 polypeptide, wherein the polynucleotide sequence is codon-optimized for expression in a plant cell.

[0169] 40. A nucleic acid molecule comprising a polynucleotide sequence encoding a Cpf1 or Csm1 polypeptide, wherein the polynucleotide sequence is codon-optimized for expression in a eukaryotic cell.

[0170] 41. A nucleic acid molecule comprising a polynucleotide sequence encoding a Cpf1 or Csm1 polypeptide, wherein the polynucleotide sequence is codon-optimized for expression in a prokaryotic cell that is not a natural host for said Cpf1 or Csm1 polypeptide.

[0171] 42. The polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 21, 22, 24, 25, and 174-206, or a fragment or variant thereof; or the polynucleotide sequence encodes a Cpf1 or Csm1 polypeptide selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236; 42. The nucleic acid molecule of any one of embodiments 39 to 41, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide is operably linked to a promoter that is heterologous to the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide.

[0172] 43. The nucleic acid molecule of any one of embodiments 39-41, wherein the variant polynucleotide sequence has at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 21, 22, 24, 25, and 174-206, or wherein the polynucleotide sequence encodes a Cpf1 or Csm1 polypeptide having at least 80% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236, and wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide is operably linked to a promoter that is heterologous to the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide.

[0173] 44. Any one of the nucleic acid molecules of 39 to 41, wherein the Cpf1 or Csm1 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236, or a fragment or variant thereof.

[0174] 45. The nucleic acid molecule of embodiment 44, wherein the variant polypeptide sequence has at least 70% sequence identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236.

[0175] 46. ​​The nucleic acid molecule of any one of embodiments 39 to 45, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide is operably linked to a promoter that is active in a plant cell.

[0176] 47. The nucleic acid molecule of any one of embodiments 39 to 45, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide is operably linked to a promoter active in a eukaryotic cell.

[0177] 48. The nucleic acid molecule of any one of embodiments 39 to 45, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide is operably linked to a promoter active in a prokaryotic cell.

[0178] 49. The nucleic acid molecule of any one of embodiments 39 to 45, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide is operably linked to a constitutive promoter, an inducible promoter, a cell-specific promoter, or a developmentally preferred promoter.

[0179] 50. The nucleic acid molecule of any one of embodiments 39 to 45, wherein the nucleic acid molecule encodes a fusion protein comprising the Cpf1 or Csm1 polypeptide and an effector domain.

[0180] 51. The nucleic acid molecule of embodiment 50, wherein the effector domain is selected from the group consisting of a transcriptional activator, a transcriptional repressor, a nuclear localization signal, and a cell penetration signal.

[0181] 52. The nucleic acid molecule of embodiment 51, wherein the Cpf1 or Csm1 polypeptide is mutated to reduce or eliminate nuclease activity.

[0182] 53. The nucleic acid molecule of embodiment 52, wherein the mutated Cpf1 or Csm1 polypeptide comprises a mutation at a position corresponding to position 917 or 1006 of FnCpf1 (sequence number 3) or a position corresponding to position 701 or 922 of SmCsm1 (sequence number 160) when aligned for maximum identity, or wherein the mutated Cpf1 or Csm1 polypeptide comprises a mutation at position 917 or 1006 of FnCpf1 (sequence number 3) or a position 701 or 922 of SmCsm1 (sequence number 160) when aligned for maximum identity.

[0183] 54. The nucleic acid molecule of any one of embodiments 50 to 53, wherein the Cpf1 or Csm1 polypeptide is fused to the effector domain via a linker.

[0184] 55. The nucleic acid molecule of any one of embodiments 39 to 54, wherein the Cpf1 or Csm1 polypeptide forms a dimer.

[0185] 56. A fusion protein encoded by any one of the nucleic acid molecules of embodiments 50 to 55.

[0186] 57. A Cpf1 or Csm1 polypeptide encoded by the nucleic acid molecule of any one of embodiments 39 to 45.

[0187] 58. A Cpf1 or Csm1 polypeptide mutated to reduce or eliminate nuclease activity.

[0188] 59. The Cpf1 or Csm1 polypeptide of embodiment 58, wherein the mutated Cpf1 or Csm1 polypeptide comprises a mutation at a position corresponding to position 917 or 1006 of FnCpf1 (sequence number 3) or a position corresponding to position 701 or 922 of SmCsm1 (sequence number 160) when aligned for maximum identity, or wherein the mutated Cpf1 or Csm1 polypeptide comprises a mutation at position 917 or 1006 of FnCpf1 (sequence number 3) or at position 701 or 922 of SmCsm1 (sequence number 160) when aligned for maximum identity.

[0189] 60. A plant cell, a eukaryotic cell, or a prokaryotic cell comprising a nucleic acid molecule of any one of embodiments 39 to 55.

[0190] 61. A plant cell, a eukaryotic cell, or a prokaryotic cell comprising the fusion protein or polypeptide of any one of embodiments 56 to 59.

[0191] 62. A plant cell produced by the method of any one of embodiments 1 and 3-38.

[0192] 63. A plant comprising a nucleic acid molecule produced by the method of any one of embodiments 39 to 55.

[0193] 64. A plant comprising the fusion protein or polypeptide of any one of embodiments 56 to 59.

[0194] 65. A plant produced by the method of any one of embodiments 1 and 3-38.

[0195] 66. A seed of the plant of any one of embodiments 63 to 65.

[0196] 67. The method of any one of embodiments 1 and 3-38, wherein the modified nucleotide sequence comprises an insertion of a polynucleotide encoding a protein that confers antibiotic or herbicide resistance to the transformed cell.

[0197] 68. The method of embodiment 67, wherein the polynucleotide encoding a protein that confers antibiotic or herbicide resistance to the transformed cell comprises SEQ ID NO: 76 or encodes a protein comprising SEQ ID NO: 77.

[0198] 69. The method of any one of embodiments 3 to 38, wherein the target site in the genome of the plant cell comprises SEQ ID NO:71 or shares at least 80% identity with a portion or fragment of SEQ ID NO:71.

[0199] 70. The method of any one of embodiments 1 to 38, wherein the DNA polynucleotide encoding the DNA-targeting RNA comprises SEQ ID NO:73, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:95.

[0200] 71. The nucleic acid molecule of any one of embodiments 39 to 55, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide further comprises a polynucleotide sequence encoding a nuclear localization signal.

[0201] 72. The nucleic acid molecule of embodiment 71, wherein the nuclear localization signal comprises SEQ ID NO:1 or is encoded by SEQ ID NO:2.

[0202] 73. The nucleic acid molecule of any one of embodiments 39 to 55, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide further comprises a polynucleotide sequence encoding a chloroplast signal peptide.

[0203] 74. The nucleic acid molecule of any one of embodiments 39 to 55, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide further comprises a polynucleotide sequence encoding a mitochondrial signal peptide.

[0204] 75. The nucleic acid molecule of any one of embodiments 39 to 55, wherein the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide further comprises a polynucleotide sequence encoding a signal peptide that targets the Cpf1 or Csm1 polypeptide to multiple intracellular locations.

[0205] 76. The fusion protein of embodiment 56, wherein the fusion protein further comprises a nuclear localization signal, a chloroplast signal peptide, a mitochondrial signal peptide, or a signal peptide that targets the Cpf1 or Csm1 polypeptide to multiple subcellular locations.

[0206] 77. The Cpf1 or Csm1 polypeptide of any one of embodiments 57 to 59, wherein the Cpf1 or Csm1 polypeptide further comprises a nuclear localization signal, a chloroplast signal peptide, a mitochondrial signal peptide, or a signal peptide that targets the Cpf1 or Csm1 polypeptide to multiple subcellular locations.

[0207] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0208] Example 1 Cloning of the cpf1 construct The constructs containing Cpf-1 (construct nos. 131306-131311 and 131313) are summarized in Table 1. Briefly, the cpf1 gene was synthesized de novo by GenScript (Piscataway, NJ) and augmented by PCR to add an N-terminal SV40 nuclear localization tag (SEQ ID NO:2) in frame with the cpf1 coding sequence of interest as well as restriction enzyme sites for cloning. Using the appropriate restriction enzyme sites, each cpf1 gene was cloned downstream of the 2x35 promoter (SEQ ID NO:43).

[0209] Guide RNAs directed to the region of DNA linking the junction of the promoter and the 5' end of the GFP coding region were synthesized as complete cassettes by Integrated DNA Technologies (Coralville, IA). Each cassette contained a U3 promoter (SEQ ID NO: 42) operably linked to an appropriate gRNA (SEQ ID NO: 47-53) that was operably linked to a U3 terminator (SEQ ID NO: 44). While each gRNA was directed to the same region of the promoter and GFP gene, each gRNA was designed to ensure that it contained the appropriate scaffold to interact correctly with its respective Cpf1 enzyme.

[0210] The constructs were assembled and cloned into a modified pSB11 vector backbone containing the hptII gene capable of conferring hygromycin B resistance to plants. The hptII gene was located downstream from the maize ubiquitin promoter and 5'UTR (pZmUbi; SEQ ID NO: 46).

[0211] [Table 1]

[0212] Example 2 Agrobacterium-mediated transformation of rice Rice (Oryza sativa cv. Kitaake) callus was infected with Agrobacterium cells containing a superbinary plasmid containing green fluorescent protein (GFP; SEQ ID NO:55 encoding SEQ ID NO:56) operably linked to a constitutive promoter. Three infected calli that showed high levels of GFP-derived fluorescence based on visual inspection were selected and divided into multiple fragments. These fragments were allowed to grow on selective medium. After the callus pieces were allowed to recover and grow larger, these calli were reinfected with Agrobacterium cells containing the genes encoding the Cpf1 enzyme and its respective guide RNAs (gRNAs). Following infection with the cpf1-containing vector, they were grown on selective medium containing hygromycin b. Callus fragments that were putatively expressing functional Cpf1 protein were selected by visually inspecting the callus fragments for areas that were no longer visibly luminescent. This loss of luminescence was likely due to continued Cpf1-mediated revision of the GFP-coding sequence, resulting in a non-functional GFP gene. For example, rice callus was first transformed with a GFP construct and then with the 131307 construct because it contains a gene encoding the Cpf1 protein from Acidaminococcus sp. BV3L6 (SEQ ID NO:8, encoding SEQ ID NO:6) leading to portions of the callus showing a clear loss of GFP-derived fluorescence. These rice callus portions containing cell clusters that do not show GFP-derived fluorescence are prioritized due to their deeper molecular characterization.

[0213] Example 3 T7EI Assay The T7 endonuclease I (T7EI) assay has been used to identify samples with insertions and / or deletions at desired locations and to evaluate the efficiency of target genome encoding enzymes. The protocol for the assay has been modified from Shan et al. (2014) Nature Protocols 9:2395-2410. The basis of the assay is that T7EI recognizes and disrupts mismatched DNA. Briefly, a PCR reaction is performed to amplify the DNA region containing the DNA sequence targeted by the gRNA. As both modified and unmodified DNA are expected to be present in the sample, a mixture of PCR products is obtained. The PCR products are melted and allowed to reanneal. If unmodified PCR products reanneal with modified PCR products, it will lead to DNA mismatches. These DNA mismatches are degraded by T7EI and can be identified by a gel-based assay. DNA is extracted from emerged rice calli that show a loss of GFP-derived fluorescence. PCR is performed with the DNA as template, using primers designed to amplify the DNA region spanning the junction between the promoter and the GFP open reading frame. The PCR products are melted, reannealed, and then digested with T7EI (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The resulting DNA is electrophoresed on a 2% agarose gel. In those samples in which Cpf1 has created an insertion or deletion at the desired location, the first band is digested to produce two smaller bands.

[0214] Example 4 Sequencing of DNA from rice callus DNA extracted from rice calli that have emerged to contain modified genomic DNA as a result of accumulation of functional Cpf1 enzyme, based on loss of trend based on visual inspection and / or results of T7EI assays, is selected for sequence-based assays. DNA is extracted from the appropriate rice callus portions and primers are used to PCR amplify the GFP coding sequence from this DNA. The resulting PCR product is cloned into a plasmid that is then transfected into E. coli cells. These plasmids are recovered and Sanger sequencing is used for DND assays to identify insertions, deletions and / or point mutations in the GFP-encoding DNA.

[0215] Example 5 Use of inactivated Cpf1 protein to regulate gene expression The RuvC-like domain of Cpf1 has been shown to mediate DNA cleavage (Zetsche et al. (2015) Cell 163:759-771), with specific residues from Francisella tularensis subsp. novicida U112 (i.e., D917 and E1006) that, when mutated from natural amino acids to alanine, completely inactivate the DNA cleavage activity identified in the Cpf1 enzyme. Amino acid-based alignments using Clustal W multiple alignments (Thompson et al. (1994) Nucleic Acid Research 22:4673-4680) of the eight Cpf1 enzymes surveyed here were performed to identify corresponding amino acid residues in other enzymes. These amino acid residues are listed in Table 2. The amino acid sequences of the inactivated Cpf1 proteins corresponding to point mutations at each of the amino acid residues listed in Table 2 are found in SEQ ID NOs: 26-41. For each cpf1 protein in Table 2, the amino acid sequence of a double mutant inactivated Cpf1 protein containing mutations at both listed residues is found in SEQ ID NOs:63-70.

[0216] [Table 2]

[0217] Suitable primers are designed to perform Quickchanger PCR (Agilent Technologies, Santa Clara, CA) to generate genes encoding inactivated Cpf1 sequences listed in SEQ ID NOs: 26-41 and to generate genes encoding inactivated Cpf1 sequences listed in SEQ ID NOs: 63-70. PCR is performed to generate genes encoding fusion proteins containing inactivated Cpf1 fused to a gene expression activation or repression domain, such as an EDLL or TAL activation domain or an SRDX repressor domain, with an SV40 localization signal (SEQ ID NO: 2, encoding SEQ ID NO: 1) fused in frame with the gene at the 5' end. A guide RNA (gRNA) is designed to allow the RNA to interact with the inactivated Cpf1 protein and to guide the inactivated Cpf1 protein to a desired location in the plant genome. A cassette containing a gRNA of interest operably linked to a promoter operable in plant cells and containing a gene encoding a Cpf1 fusion protein fused to an activation and / or repression domain is cloned into a vector suitable for plant transformation. This vector is transformed into a plant cell, leading to the production of a gRNA and a Cpf1 fusion protein in the plant cell. The fusion protein, which contains an inactivated Cpf1 protein and an activator or repressor domain, results in the modulation of the expression of adjacent genes in the plant genome.

[0218] Example 6 Editing of Selected Genomic Loci in Maize (Zea mays) One or more gRNAs are designed to anneal to a desired site in the maize genome and interact with one or more Cpf1 or Csm1 proteins. These gRNAs are cloned into a vector such that they are operably linked to a promoter operable in a plant cell (gRNA cassette). One or more genes encoding a Cpf1 or Csm1 protein are cloned into a vector such that they are operably linked to a promoter operable in a plant cell (cpf1 cassette or csm1 cassette). The gRNA cassette and the cpf1 cassette or csm1 cassette are each cloned into a vector suitable for plant transformation, which vector is then transformed into an Agrobacterium cell. These cells are contacted with maize tissue suitable for transformation. Following incubation with the Agrobacterium cells, the maize cells are cultured in a tissue culture medium suitable for whole plant regeneration. Maize plants are regenerated from the cells contacted with the Agrobacterium cells harboring the vector containing the cpf1 or csm1 cassette and the gRNA cassette. After regeneration of the sugar locust plants, plant tissue is harvested and DNA is extracted from the tissue. If appropriate, T7EI assays and / or sequencing assays are performed to analyze whether a DNA sequence change has occurred at the desired genomic location.

[0219] Alternatively, particle bombardment is used to introduce the cpf1 or csm1 cassette and the gRNA cassette into corn cells. The vector containing the cpf1 or csm1 cassette and the gRNA cassette is coated onto gold or titanium beads, which are then used to bombard corn tissue suitable for regeneration. Following bombardment, the corn tissue is transferred to tissue culture medium for regeneration of corn plants. Following regeneration of corn plants, the plant tissue is harvested and DNA is extracted from the tissue. T7EI assays and / or sequence assays are performed as needed to determine whether the DNA sequence change has occurred at the desired genomic location.

[0220] Example 7 Editing of defined genomic loci in Setaria viridis One or more gRNAs are designed to anneal to a desired site in the foxtail genome and to interact with one or more Cpf1 or Csm1 proteins. These RNAs are cloned into a vector so that they are operably linked to a promoter operable in a plant cell (the "gRNA cassette"). One or more genes encoding Cpf1 or Csm1 proteins are cloned into a vector so that they are operably linked to a promoter operable in a plant cell (the "cpf1 cassette" or "csm1 cassette"). The gRNA cassette and the cpf1 cassette or csm1 cassette are each cloned into a vector suitable for plant transformation, which is then transformed into Agrobacterium cells. These cells are contacted with foxtail tissue suitable for transformation. Following incubation with the Agrobacterium cells, the foxtail cells are cultured on tissue culture medium suitable for regeneration of intact plants. Setaria plants are regenerated from cells inoculated with Agrobacterium cells harboring a vector containing the cpf1 or csm1 cassette and the gRNA cassette. Following regeneration of Setaria plants, plant tissue is harvested and DNA is extracted from the tissue. T7EI and / or sequence assays are performed, as needed, to determine whether the DNA sequence change has occurred at the desired genomic location.

[0221] Alternatively, particle bombardment is used to introduce the cpf1 or csm1 cassette and the gRNA cassette into foxtail cells. The vectors containing the cpf1 or csm1 cassette and the gRNA cassette are coated onto gold or titanium beads, which are then used to bombard foxtail tissue suitable for regeneration. Following bombardment, the foxtail tissue is transferred to tissue culture medium for regeneration of intact plants. Following plant regeneration, the plant tissue is harvested and DNA is extracted from the tissue. T7EI assays and / or sequence assays are performed as necessary to determine whether the DNA sequence change has occurred at the desired genomic location.

[0222] Example 8. Deletion of DNA from a defined genomic locus A first gRNA is designed to anneal to a first desired site in the genome of a plant of interest and to interact with one or more Cpf1 or Csm1 proteins. A second gRNA is designed to anneal to a first desired site in the genome of a plant of interest and to interact with one or more Cpf1 or Csm1 proteins. Each of these RNAs is operably linked to a promoter operable in a plant cell and then cloned into a vector suitable for plant transformation. One or more genes encoding Cpf1 or Csm1 proteins are cloned into a vector so as to be operably linked to a promoter operable in a plant cell ("cpf1 cassette" or "csm1 cassette"). The cpf1 cassette or csm1 cassette and the gRNA cassette are cloned into a single plant transformation vector and then the vector is transformed into an Agrobacterium cell. These cells are contacted with a plant tissue suitable for transformation. Following incubation with the Agrobacterium cells, the plant cells are cultured on tissue culture medium suitable for regenerating intact plants. Alternatively, the vector containing the cpf1 or csm1 cassette and the gRNA cassette is coated on gold or titanium beads suitable for bombardment of plant cells. The bombarded cells are then transferred to tissue culture medium suitable for regenerating intact plants. The gRNA-Cpf1 or gRNA-Csm1 complex performs a double-strand break at the desired genomic locus, and in some cases, DNA repair mechanisms allow the DNA to be repaired to delete the native DNA sequence located between the two targeted genomic loci. Plants are regenerated from cells contacted with Agrobacterium cells harboring a vector containing the cpf1 or csm1 cassette and the gRNA cassette, or from cells bombarded with beads coated with this vector. Following plant regeneration, the plant tissue is harvested and DNA is extracted from the tissue. T7EI assays and / or sequence assays are performed, as appropriate, to determine whether DNA has been deleted from the desired genomic location.

[0223] Example 9 DNA insertion at a defined genomic locus A gRNA is designed to anneal with a first desired site in the genome of a plant of interest and to interact with one or more Cpf1 or Csm1 proteins. The gRNA is operably linked to a promoter operable in a plant cell and then cloned into a vector suitable for plant transformation. One or more genes encoding Cpf1 or Csm1 proteins are cloned into a vector operably linked to a promoter operable in a plant cell ("cpf1 cassette" or "csm1 cassette"). The cpf1 cassette or csm1 cassette and the gRNA cassette are cloned into a single plant transformation vector, which is then transformed into Agrobacterium cells. These cells are contacted with plant tissue suitable for transformation. At the same time, donor DNA is introduced into these same plant cells. The donor DNA contains a DNA molecule to be inserted into a desired location in the plant root, flanked by upstream and downstream flanking regions. The upstream flanking region is homologous to a region of genomic DNA upstream of the genomic locus targeted by the gRNA, and the downstream flanking region is homologous to a region of genomic DNA downstream of the genomic locus targeted by the gRNA. The upstream and downstream flanking regions mediate DNA insertion into the desired site of the plant genome. Following this incubation with Agrobacterium cells and introduction of donor DNA, the plant cells are cultured on a tissue culture medium suitable for regenerating intact plants. Plants are regenerated from cells contacted with Agrobacterium cells harboring a vector that contained the cpf1 or csm1 cassette and the gRNA cassette. Following plant regeneration, plant tissue is harvested and DNA is extracted from the tissue. T7EI and / or sequence assays are performed as necessary to determine whether DNA has been deleted from the desired genomic location.

[0224] Example 10. Biolistic DNA insertion at the rice CAO1 locus Vectors were designed with cpf1 or csm1 cassettes for biolistic insertion of DNA into a given genomic locus. These vectors contained a 2X35S promoter (SEQ ID NO: 43) upstream of the cpf1 or csm1 ORF and a 35S polyA terminator sequence (SEQ ID NO: 54) downstream of the cpf1 or csm1 ORF. Table 3 summarizes these cpf1 or csm1 vectors.

[0225] [Table 3-1] [Table 3-2] [Table 3-3]

[0226] In addition to the cpf1 and csm1 vectors listed in Table 3, vectors carrying gRNA cassettes were designed such that the gRNA anneals to a region of the CAO1 locus in the rice (Oryza sativa) genome (SEQ ID NO: 71) and can interact with the appropriate Cpf1 or Csm1 protein. In these vectors, the gRNA is operably linked to the rice U6 promoter (SEQ ID NO: 72) and terminator (SEQ ID NO: 74). Table 4 summarizes these gRNA vectors.

[0227] [Table 4]

[0228] To facilitate the insertion of the hygromycin gene cassette into the rice CAO1 genomic locus, a repair donor cassette was designed with approximately 1000 base pairs of homology upstream and downstream of the site of the intended double-stranded break caused by the action of Cpf1 or Csm1 enzymes in combination with a gRNA targeting this locus. Figure 1 is a schematic diagram of the CAO1 genomic locus and the homology arms used to induce homologous recombination and insertion of the hygromycin gene cassette in the CAO1 genomic locus. The hygromycin gene cassette inserted into the rice CAO1 genomic locus contained a maize ubiquitin promoter (SEQ ID NO: 46) driving the expression of a hygromycin resistance gene (SEQ ID NO: 76, encoding SEQ ID NO: 77) flanked at the 3' end by a cauliflower mosaic virus 35SpolyA sequence (SEQ ID NO: 54). Table 5 is a schematic diagram of the modified donor cassette vector constructed for hygromycin insertion into the rice CAO1 genomic locus.

[0229] [Table 5]

[0230] Particle bombardment was employed to introduce the cpf1 or csm1 cassette, the gRNA-containing plasmid, and the repair donor cassette into rice cells. For bombardment, 2 mg of 0.6 μm gold particles were weighed and transferred to a sterile 1.5 mL test tube. 500 mL of 100% ethanol was added and the tube was sonicated for 10-15 seconds. Following centrifugation, the ethanol was removed. Next, 1 mL of sterile double-distilled water was added to the test tube containing the gold beads. The bead pellet was briefly vortexed and then reconstituted by centrifugation, after which the water was removed from the tube. DNA was coated onto the beads in a sterile laminar flow hood. Table 6 shows the amount of DNA added to the beads. The cpf1 or csm1 cassette, the gRNA-containing plasmid, and the repair donor cassette-containing plasmid were added to the beads, and sterile double-distilled water was added to a total volume of 50 μL. To this was added 20 μL of spermidine (1 M) followed by 50 μL of CaCl2 (2.5 M). The gold particles were allowed to pellet by gravity for several minutes and then centrifuged to pellet. The supernatant was removed and 800 μL of 100% ethanol was added. Following a brief sonication, the gold particles were allowed to pellet by gravity for 3-5 minutes and then the tube was centrifuged to pellet. The supernatant was removed and 30 μL of 100% ethanol was added to the tube. The DNA-coated gold particles were resuspended in this ethanol by vortexing and 10 μL of the resuspended gold particles were added to each of three macrocarriers (Bio-Rad, Hercules, CA). The macrocarriers were allowed to air dry for 5-10 minutes in a laminar flow hood to allow the ethanol to evaporate.

[0231] [Table 6]

[0232] Rice callus tissue was used for bombardment. Prior to bombardment, rice callus was kept in callus induction medium (CIM; 3.99 g / L N6 salts and vitamins, 0.3 g / L casein hydrolysate, 30 g / L sucrose, 2.8 g / L L-proline, 2 mg / L 24-D8 g / L agar, adjusted to pH 5.8) at 28 degrees for 4-7 days. Prior to particle bombardment, approximately 80-100 callus pieces, each 0.2-0.3 cm in size and weighing 1-1.5 g in total, were arranged in the center of a Petri dish containing permeable solid medium (CIM supplemented with 0.4 M sorbitol and 0.4 M mannitol) for 4 h of permeable pretreatment. For bombardment, macrocarriers containing DNA-coated gold particles were assembled into a macrocarrier holder. The burst plate (1100 psi), stopping screen, and microcarrier holder were assembled according to the manufacturer's instructions. The plate containing the rice callus to be bombarded was placed 6 cm below the stopping screen, and the callus pieces were bombarded after the vacuum chamber reached 25-28 in. Hg. After bombardment, the callus was left on the infiltration medium for 16-20 hours, after which the callus pieces were transferred to selection medium (CIM supplemented with 50 mg / L hygromycin and 100 mg / L timentin). The plates were transferred to an incubator and kept at 28°C in the dark to initiate recovery of transformed cells. Every two weeks, the callus was subcultured on fresh selection medium. After approximately 5-6 weeks on selection medium, hygromycin-resistant callus pieces began to appear. Individual hygromycin-resistant callus pieces were transferred to new selection plates and allowed to divide and grow, generating enough tissue to be harvested for molecular analysis. Table 7 summarizes the DNA vector combinations used for these rice bombardment experiments.

[0233] [Table 7-1] [Table 7-2]

[0234] Individual hygromycin-resistant callus pieces from each experiment were transferred to new plates and then grown until large enough to be harvested. A small amount of tissue was harvested from each individual hygromycin-resistant rice callus piece, and DNA was extracted from these tissue samples for PCR and DNA sequence analysis. For experiments using repair donor plasmids 131760 or 131632, PCR was performed on these DNA extracts using primers with sequences SEQ ID NOs: 78 and 79 designed to amplify a DNA region spanning from the ZmUbi promoter into the region of the rice genome outside the downstream repair donor arm, as shown diagrammatically in Figure 1. For those experiments using repair donor plasmid 131633, primers with sequences SEQ ID NOs: 102 and 103 were used to amplify a DNA region spanning from the CaMV35S terminator into the region of the rice genome outside the upstream repair donor arm, as shown diagrammatically in Figure 1. These PCR reactions did not produce amplicons from wild-type rice DNA or from the repair donor plasmid, thus suggesting an insertion event at the rice CAO1 locus. Table 8 summarizes the number of hygromycin-resistant callus pieces generated from each experiment shown in Table 7 as well as the number of PCR-positive callus pieces that were presumed to have contained an insertion event. The number of callus pieces used in each bombardment experiment was estimated by survey of 10 plates with 159±11.1 callus pieces per plate, based on weight.

[0235] [Table 8]

[0236] Further PCR analysis was performed on the PCR-positive callus pieces listed in Table 8 to amplify across the junction between both homology arms and the rice genome. Primers with sequences SEQ ID NOs: 96 and 97 were used to amplify the upstream region for these experiments using the repair donor plasmids 131760 or 131632. The locations of these primer binding sites are shown diagrammatically in Figure 1.

[0237] Sanger sequencing of PCR amplicons generated using the above primer pairs to amplify the downstream region of the insertion event showed that the expected sequence was present in the transformed rice calli, confirming the insertion of the hygromycin gene cassette at the expected genomic locus mediated by a double-stranded break created by the Cpf1 or Csm1 enzyme. Sanger sequencing of PCR amplicons generated using the above primer pairs to amplify the upstream region of the insertion event also showed that the expected sequence was present in the transformed rice calli, confirming the insertion of the hygromycin gene cassette at the expected genomic locus mediated by a double-stranded break created by the Cpf1 or Csm1 enzyme. Importantly, a deletion of five base pairs (GCCTT) from the rice genome sequence was predicted to occur at the upstream insertion site following Cpf1-mediated DSB formation, and this deletion was confirmed from the sequence data, thus proving that the observed insertion event was mediated by the action of Cpf1. Figure 2A shows an alignment summarizing the sequence data that confirmed the insertion event at the rice CAO1 locus targeted in experiment 1 (see Table 7).

[0238] PCR products used to confirm the presence of targeted insertions at the rice CAO1 locus were sequenced as targeted in experiments 5 and 7 (see Table 7). Primers with sequences SEQ ID NOs: 104 and 105 were used to amplify the downstream region of these insertion events. These PCR products were sequenced and the expected sequences were observed for insertion events mediated by DSB production by FnCpf1 (experiment 5) and MbCpf1 (experiment 7). The hph strand set was inserted into the CAO1 locus at the targeted site with no base changes in the downstream arm.

[0239] Experiment 70 (Table 7) resulted in the insertion of the 35S terminator present in plasmid 131633 at the intended insertion site in the rice CAO1 genomic locus, rather than the insertion of the entire hph cassette. Sequence analysis showed that the 35S terminator contains an 11 base pair region that shares 10 bases with the downstream arm (Figure 4A). It appears that this region in the 35S terminator mediated an unintended homologous recombination event with the downstream arm in rice callus piece no. 70-15, while plasmid 131633 mediated an intended recombination event between this plasmid and the sequence upstream of the locus in the rice CAO1 gene targeted by the guide RNA and Cpf1 enzyme, resulting in the insertion sequence shown in Figure 4B. The resulting insertion led to a 179 base pair deletion and a 133 base pair insertion in the rice CAO1 locus. While the insertion event uncovered in experiment 70 contained only a portion of the 35S terminator rather than the entire hph cassette as intended for insertion, the event was found to be at the intended site in the CAO1 locus targeted by the Prevotella bryantii Cpf1 enzyme (encoded by SEQ ID NO:179, SEQ ID NO:138), indicating that this Cpf1 enzyme is effective in generating the intended DSB at the CAO1 locus.

[0240] Experiment 75 (Table 7) resulted in the insertion of a portion of the 35S terminator present in plasmid 131633 at the intended insertion site in the rice CAO1 genomic locus, rather than the insertion of the entire hph cassette. Sequence analysis showed that the 35S terminator contained a 12 base pair region that shared 8 bases with the downstream arm (Figure 4C). It appears that this region in the 35S terminator mediated an unintended homologous recombination event with the downstream arm in rice callus piece no. 75-46, while the upstream arm in plasmid 131633 mediated an intended recombination event between this plasmid and the sequence upstream of the locus in the rice CAO1 gene targeted by the guide RNA and Cpf1 enzyme, resulting in the insertion sequence shown in Figure 4D. The resulting insertion led to a 47 base pair deletion and a 24 base pair insertion in the rice CAO1 locus. While the insertion event uncovered in experiment 75 contained only a portion of the 35S terminator rather than the entire hph cassette as intended for insertion, the event was found to be at the intended site in the CAO1 locus targeted by the Proteocatella sphenisci Cpf1 enzyme (encoded by SEQ ID NO:191, SEQ ID NO:142), indicating that this Cpf1 enzyme is effective in generating the intended DSB at the CAO1 locus.

[0241] Experiment 46 (Table 7) resulted in an insertion mediated by the Lachnospiraceae bacterium ND2006 Cpf1 enzyme (SEQ ID NO:18, encoded by SEQ ID NO:19) at the intended insertion site in the rice CAO1 genomic locus. PCR analysis of the region of the intended site in the CAO1 locus resulted in the amplification of a band diagnostic of the insertion in callus piece no. 46-161. This genomic region was subjected to sequence analysis to confirm the presence of the intended DNA insertion at the rice CAO1 locus. Figure 5 shows the results of this sequence analysis with the expected insertion from the 131633 vector present in the rice DNA at the expected site. The mutated PAM site (TTTC>TAGC) present in the 131633 vector was also detected in rice DNA from callus piece no. 46-161, further supporting HDR-mediated insertion of the 131633 vector insert at the rice CAO1 gene as mediated by site-specific DSB introduction by Lachnospiraceae bacteriumND2006Cpf1 enzyme.

[0242] Experiment 58 (Table 7) was the result of an insertion mediated by Anaerovibrio sp. RM50Cpf1 enzyme (SEQ ID NO:143, encoded by SEQ ID NO:176) at the intended insertion site in the rice CAO1 genomic locus. PCR analysis of the region of the intended insertion site in the CAO1 locus resulted in the amplification of a band diagnostic of the insertion in callus piece no. 58-169. This genomic region was subjected to sequence analysis to confirm the presence of the intended DNA insertion at the rice CAO1 locus.

[0243] Example 11 Cpf1-mediated genomic DNA modification at the rice CAO1 locus Rice callus was bombarded with gold beads coated with cpf1 and gRNA vectors as described above. Rice callus bombarded for experiment 1 (Table 7) was placed on infiltration medium for 16-20 hours following bombardment, and then rice callus was transferred to selective medium (CIM supplemented with 50 mg / L hygromycin and 100 mg / L timentin). Plates were transferred to an incubator and kept at 28°C in the dark to allow transformation cells to begin to recover. Callus was subcultured on fresh selective medium every two weeks. After approximately 5-6 weeks on selective medium, hygromycin-resistant callus pieces began to appear. Individual hygromycin-resistant callus pieces were transferred to new selective plates and allowed to divide and grow, generating enough tissue to be harvested for molecular analysis.

[0244] DNA was extracted from 16 hygromycin-resistant callus pieces generated in experiment 01 (Table 7) and PCR was performed using primers with sequences of SEQ ID NO: 100 and 101 to confirm the presence of the cpf1 cassette. This PCR reaction showed that DNA extracted from rice calli numbers 1, 2, 4, 6, 7, and 15 produced the expected 853 base pair aplicon, consistent with the insertion of the cpf1 cassette in the rice genome (Figure 2B). PCR was also performed with DNA extracted from hygromycin-resistant callus pieces using primers with sequences of SEQ ID NO: 98 and 99 to amplify the region of the rice CAO genomic locus targeted by gRAN in vector 131608. This PCR reaction produced a 595 base pair aplicon when wild-type rice DNA was used as template. Following the PCR reaction with SEQ ID NO: 98 and 99 as primers, T7 endonuclease analysis was performed on the resulting PCR products to check for small insertions and / or deletions at this locus. DNA from callus piece no. 15 showed a banding pattern consistent with a small insertion or deletion (Figure 2C). PCR products generated from reactions using primers SEQ ID NOs: 98 and 99 were cloned into E. coli cells using the pGEM® system (Promega, Madison, WI) according to the manufacturer's instructions. DNA was extracted from eight single E. coli colonies for sequencing. Five of the eight colonies showed the same seven base pair deletion at the predicted site of the Cpf1-mediated double-strand break at the CAO1 locus (Figure 2D). Without being limited by theory, a possible explanation for this deletion is that the deletion was generated following repair of the double-strand break caused by FnCpf1 by rice cell DNA repair machinery at the CAO1 locus.

[0245] Experiment 01 (Table 7) was repeated with additional rice callus pieces to confirm the reproducibility of the results initially obtained. Repeating experiment 01 led to the identification of four additional callus pieces that showed positive for indel generation based on the results of the T7EI analysis. DNA was extracted from these callus pieces for sequence analysis. PCR was performed to amplify the region of the rice genome surrounding the targeted rice site of the CAO1 locus, and Sanger sequencing was performed. The sequencing results confirmed the results of the T7EI analysis. Figure 2D shows the sequence data obtained. These four callus pieces showed a variety of deletion sizes ranging from 3 base pairs to 75 base pair deletions, all located at the predicted site targeted by FnCpf1 (encoded by SEQ ID NO: 5, SEQ ID NO: 3).

[0246] In experiments 31 and 46 (Table 7), the ability of LbCpf1 (SEQ ID NO:18, encoded by SEQ ID NO:19) to cause DSBs at two different sites in the rice CAO1 gene was tested. Experiment 31 used plasmid 132033 as the gRNA source, while experiment 46 used plasmid 132054 as the gRNA source. Following bombardment of rice callus with the plasmids used in these experiments, DNA was extracted from hygromycin-resistant rice callus pieces and subjected to T7EI assays. Following PCR amplification of the rice CAO1 genomic locus, the T7EI assay identified one callus piece from experiment 31 and five callus pieces from experiment 46 that appeared to contain indels at the expected site. PCR products from these rice callus pieces were analyzed by Sanger sequencing to identify sequences present at the CAO1 locus in these callus pieces. Figure 3 shows the results of Sanger sequencing analysis, confirming the presence of indels at the predicted locations in the rice CAO1 locus. Figure 3A shows the results from experiment 31, and Figure 3B shows the results from experiment 46. As Figure 3A shows, callus piece 31-21 showed a 56 base pair deletion along with a 10 base pair insertion. Callus from experiment 46 (data shown in Figure 3B) showed deletion sizes ranging from 3 to 15 base pairs. It should be noted that callus pieces 46-38 and 46-77 showed two different indels, indicating multiple indel generation events in independent cells within these callus pieces. All of the indels from these experiments were located at the predicted site of the CAO1 locus targeted by the respective guide RNAs, indicating faithful generation of DSBs at this site by the LbCfp1 enzyme.

[0247] Experiment 80 (Table 7) tested the ability of the Moraxella caprae Cpf1 enzyme (SEQ ID NO:133, encoded by SEQ ID NO:175) to cause a DSB at the rice CAO1 locus. Following bombardment of rice callus with the plasmid used in this experiment, DNA was extracted from hygromycin-resistant rice callus pieces and subjected to a T7EI assay. Following PCR amplification of the rice CAO1 genomic locus, the T7EI assay identified one callus piece from the experiment containing an indel at the expected site. The PCR product from this rice callus piece was analyzed by Sanger sequencing to identify the sequence present at the CAO1 locus in this callus piece. Figure 3A shows the results of these sequence assays, along with the eight base pair deletion at the expected site in the CAO1 locus targeted by each guide RNA in callus piece no. 80-33, demonstrating faithful generation of a DSB at this site by the Moraxella caprae Cpf1 enzyme.

[0248] Experiment 91 (Table 7) tested the ability of the Lachnospiraceae bacterium COE1 Cpf1 enzyme (SEQ ID NO:125, encoded by SEQ ID NO:189) to cause a DSB at the rice CAO1 locus. Following bombardment of rice callus with the plasmid used in this experiment, DNA was extracted from hygromycin-resistant rice callus pieces and subjected to a T7EI assay. Following PCR amplification of the rice CAO1 genomic locus, the T7EI assay identified one callus piece from the experiment containing an indel at the expected site. The PCR product from this rice callus piece was analyzed by Sanger sequencing to identify the sequence present at the CAO1 locus in this callus piece. Figure 3A shows the results of these sequence assays, along with a nine base pair deletion in callus piece number 91-4 at the expected site at the CAO1 locus targeted by each guide RNA, demonstrating faithful generation of a DSB at this site by the Lachnospiraceae bacterium COE1 Cpf1 enzyme.

[0249] Experiment 119 (Table 7) tested the ability of the Eubacterium coprostanoligenes Cpf1 enzyme (SEQ ID NO:173, encoded by SEQ ID NO:205) to cause DSBs at the rice CAO1 locus. Following bombardment of rice callus with the plasmid used in this experiment, DNA was extracted from hygromycin-resistant rice callus pieces and subjected to a T7EI assay. Following PCR amplification of the rice CAO1 genomic locus, the T7EI assay identified two callus pieces from the experiment that contained indels at the expected sites. PCR products from the rice callus pieces were analyzed by Sanger sequencing to identify sequences present at the CAO1 locus in the callus pieces. Figure 3A shows the results of these sequence assays, along with an eight base pair deletion in both callus piece numbers 119-4 and 119-11 at the predicted site in the CAO1 locus targeted by each guide RNA, demonstrating faithful generation of a DSB at this site by the Eubacterium coprostanoligenes Cpf1 enzyme.

[0250] Example 12 Regeneration of rice plants with an insertion at the CAO1 locus Rice calluses transformed with the hph cassette targeting the CAO1 locus by FnCpf1-mediated DSB in experiment 1 (see Tables 7 and 8) were grown in tissue culture medium to generate shoots. These shoots were then transferred to rooting medium and the rooted plants were transferred to soil for cultivation in the greenhouse. The rooted plants appeared phenotypically normal in soil. DNA was extracted from the rooted plants for PCR analysis. PCR amplification of the upstream and downstream arms confirmed that the hph cassette was present at the rice CAO1 genomic locus.

[0251] The T0 generation rice plants generated in experiment 1 with the hph cassette insertion in CAO1 were cultivated and self-pollinated to generate T1 generation seeds. The seeds were planted and the resulting T1 generation plants were genotyped to identify homozygous, hemizygous, and null plants. The T1 plants separated as expected, with approximately 25% of the T1 plants in the hemizygous state, 25% being null segregants, and 50% being heterozygous due to the hph insertion. The homozygous plants were observed phenotypically with the expected yellow leaf phenotype associated with knockout of the CAO1 gene (Lee et al. (2005) Plant Mol Biol 57:805-818).

[0252] T0 generation plants were regenerated from GE0046 callus pieces nos. 33, 40, 62, and 90, which tested positive for indels through T7EI assays and (for callus piece no. 90) sequence verification (Figure 3B). Regenerated plants derived from callus pieces 46-33, 40, 62, and 90 were positive for the presence of indels at the CAO1 locus based on T7EI assays using DNA extracted from regenerated plant tissues. Plants were also regenerated from GE0046 callus pieces 46-96 and 46-161, which were previously shown to have an insertion of the hygromycin marker at the CAO1 locus. Plants derived from callus pieces 46-96 and 46-161 were all positive for the insertion as detected by PCR screening. Sequence data obtained from DNA extracted from the two plants regenerated from callus piece no. 46-90 showed the same eight base pair deletion detected in the callus (Figure 3B), indicating that the deletion was stable throughout the regeneration process. Sequence data obtained from DNA extracted from plants derived from callus pieces no. 46-40 and no. 46-62 showed deletions of 8-, 9-, 10-, and 11 base pairs (data not shown).

[0253] Example 13 Identification of a putative new class of Cpf1-like proteins Examination of the phylogenetic tree of putative Cpf1 proteins (Zetsche et al. (2015) Cell 163:759-771, data not shown), together with sequence analysis of Cpf1 and Cpf1-like proteins identified through BLAST searches, revealed a small group of proteins that appear to be related to Cpf1 proteins but have significantly different sequences compared to known Cpf1 proteins. Two of these proteins are found in Smithella genus SCADs and Microgenomates, so this putative new class of proteins is likely to be related to Csm1 ( S mithella and M Derived from icrogenomates C These Csm1 proteins were named RISPR-associated proteins (RISPR-associated proteins). Like Cpf1 proteins, these Csm1 proteins contain RuvCI, RuvCII, and RuvCIII domains, but importantly, the amino acid sequences of these domains are often significantly different from the Cpf1 protein amino acid sequence, especially that of the RuvCIII domain. Furthermore, compared to Cpf1 proteins, the spacing of RuVCI-RuVCII and RuVCII-RuVCIII in Csm1 proteins is significantly altered.

[0254] Alignment of the Smithella genus SCADC Csm1 protein (SmCsm1; SEQ ID NO: 160) with known Cpf1 proteins using the BLASTP algorithm default parameters (blast.ncbi.nlm.nih.gov / Blast.cgi) shows little apparent sequence identity between these proteins. It is particularly clear that the RuvCI domain in the SmCsm1 protein appears to be present and well aligned with the corresponding sequence in the Cpf1 protein, while the RuvCII and RuvCIII (Shmakov et al. (2016) Mol Cell 60:385-397), which are well conserved in the Cpf1 protein, appear to be initially absent in the putative Csm1 protein. Further analysis employing HHPred (toolkit.tuebingen.mpg.de / hhpred; Soding et al. (2006) Nucleic Acids Res 34:W374-W378) revealed putative RuvCII and RuvCIII domains in this SmCsm1 protein. Table 9 shows the putative RuvCII-corresponding domains of several Cpf1 and putative Csm1 proteins and a representative C2c1 protein, with each sequence listing the amino acid residue that corresponds to the listed RuvCII sequence. The putative active residues are underlined for each protein listed.

[0255] [Table 9]

[0256] Table 10 shows the putative RuvCIII domains of several Cpf1 and putative Csm1 proteins along with a representative C2c1 protein, with each sequence listing the amino acid residues that correspond to the listed RuvCIII sequence. The putative active residues are underlined for each protein listed.

[0257] [Table 10]

[0258] As shown in Tables 9 and 10, the RuvCII and RuvCIII domains identified by HHPred for the putative Csm1 proteins (SEQ ID NOs: 134, 147, 159, 160, and 230) are significantly different from those found in the Cpf1 proteins (representative sequences shown above, SEQ ID NOs: 6 and 18). In particular, the ANGAY motif following the active residues in the RuvCIII domain is highly conserved among Cpf1 proteins (Shmakov et al. (2016) Mol Cell 60:385-397, data not shown), but is absent or altered in most of these Csm1 proteins. Analysis of the RuvCII and RuvCIII domains of the Csm1, Cpf1, and C2c1 proteins (Shmakov et al. (2016) Mol Cell 60:385-397) suggests that the Csm1 protein appears to be intermediate between the Cpf1 and C2c1 proteins, as the Csm1 RuvCII sequence is similar to that found in the Cpf1 protein, while the Csm1 RuvCIII sequence is similar to that found in the C2c1 protein. The RuvCIII domain of the Csm1 protein contains a DXXAA motif that is largely captured in the C2c1 protein sequence.

[0259] While Csm1 protein shares some sequence similarity with C2c1 protein, these genomic contexts indicate that Csm1 protein functions like Cpf1 protein in many ways. Specifically, C2c1 protein requires both crRNA and tracrRNA in situations where tracrRNA is partially complementary to the crRNA sequence. The genomic locus containing ORF encoding Csm1 from Smithella genus SCADC (SEQ ID NO:238) contains an array of CRISPRs with Cpf1-like direct repeats after Csm1 ORF, Cas4 ORF, Cas1 ORF, and Cas2 ORF. This is consistent with the genomic organization seen in genomes encoding Cpf1 (Shmakov et al. (2017) Nat Rev Microbiol doi:10.1038 / nrmicro.2016.184). In contrast, C2c1 genomic organization tends to contain fused Cas1 / Cas4 ORFs. Furthermore, C2c1-containing genomic loci tend to encode both crRNA arrays and tracrRNAs with partial complementarity to the crRNA direct repeats. Examination of Smithella SCADC genomic loci that contain Csm1-encoding ORFs and integrated crRNA sequences did not find any trarRNA-like sequences, strongly suggesting that tracrRNA is not required to generate double-stranded breaks.

[0260] Recently, a new class of nucleases called CasX proteins has been reported (Burstein et al. (2016) Nature http: / / dx.doi.org10.1038 / nature20159). The CasX protein from Deltaproteobacteria (SEQ ID NO: 239) is described as a protein of approximately 980 amino acids found in the region encoding Cas1, Cas4, and Cas2 proteins, as well as in the CRISPR repeat region and the genomic region containing the tracrRNA. The report describing CasX concludes that the tracrRNA is required for endonuclease function, in clear contrast to the Csm1 protein, which does not require the tracrRNA. BLASTP alignment of SmCsm1 (SEQ ID NO: 160) with Deltaproteobacterial CasX (SEQ ID NO: 239) showed a very poor alignment (data not shown). HHRred analysis of this CasX protein was employed to identify the putative RuvCI, RuvCII, and RuvCIII domains and their respective active site residues.

[0261] In addition to the altered amino acid sequences of the predicted RuvCI, RuvCII, and RuvCIII domains of the Csm1 protein compared to the Cpf1 protein, the protein architecture is significantly altered such that the spacing between these domains is significantly different from that between the Csm1 and Cpf1 proteins. Table 11 shows a comparison of the spacing between active residues in the RuVC subdomains of known Cpf1 proteins (AsCpf1 and LbCpf1; SEQ ID NOs: 6 and 18) compared to the spacing in these predicted Csm1 proteins (SEQ ID NOs: 134, 147, 159, 160, and 230), the Deltaproteobacterial CasX protein (SEQ ID NO: 239), and the representative C2c1 protein (SEQ ID NO: 237). The data in Table 11 clearly show that the Cpf1, C2c1, and Csm1 proteins have characteristic RuvC domain spacing, with a CasX RuvCI-RuvCII spacing similar to Cpf1 and a RuvCII-RuvCIII spacing similar to Csm1 / C2c1. Although the RuvCI, RuvCII, and RuvCIII spacings in the C2c1 and Csm1 proteins are similar, the distinct RuvCIII sequence in the Csm1 system and the lack of tracrRNA support the classification of the Csm1 nuclease as separate from the C2c1 nuclease.

[0262] [Table 11]

[0263] It should be noted that, in many cases, with the different RuvCII and RuvCIII amino acid sequences in the Csm1 protein compared to the Cpf1 protein and the altered sequences of these domains, HHPred analysis did not find any Csm1 sequence corresponding to the amino acid residue D1225 in FnCpf1 (SEQ ID NO:3) (D1234 in AsCpf1 (SEQ ID NO:6) and D1148 in LbCpf1 (SEQ ID NO:18)). Mutational analysis of FnCpf1 D1225 showed that mutation of this residue very significantly reduced the catalytic activity of this nuclease, suggesting that the enzymatic function of this residue is very important for the Cpf1 enzyme (Zetsche et al. (2015) Cell 163:759-771).

[0264] In addition to the altered amino acid sequence of the putative RuvC domain in the Csm1 protein compared to the Cpf1 protein, HHPred analysis of the Csm1 protein shows no match with the Cpf1 protein at their N-terminus, in contrast to HHPred analysis based on the known Cpf1 protein. HHPred analysis using the FnCpf1 amino acid sequence (SEQ ID NO:3) resulted in only two matches with AsCpf1 (SEQ ID NO:6) and LbCpf1 (SEQ ID NO:18) at 100% probability over the entire FnCpf1 amino acid sequence. In contrast, HHPred analysis using SmCsm1 (SEQ ID NO:160) found only matches with the Cpf1 protein covering the region from amino acids 391-1017 and 1003-1064 of SmCsm1. Amino acids 1003-1030 find matches to a variety of proteins, including possibly lysine biosynthetic proteins, amino acid carrier proteins, transcription initiation factors, 50S and 30S ribosomal proteins, and DNA-directed RNA polymerases. No matches to the first 390 amino acids of Csm1 are found in this HHPred analysis. Similar HHPred analyses with additional Csm1 proteins (SEQ ID NOs: 134, 147, 159, 160, and 230) also fail to find matches to the N-termini of these Csm1 proteins, further supporting the conclusion that these proteins share some similarity with the Cpf1 protein but are not the Cpf1 protein itself.

[0265] Example 14 Functional characterization of Csm1 Given the distinct properties of the Csm1 proteins compared to the Cpf1 proteins, we sought to confirm that these proteins could generate DSBs in vivo. While the amino acid sequences of the Csm1 proteins are significantly different from those of the Cpf1 proteins, genomic analysis of the source organisms of these Csm1 proteins revealed CRISPR arrays (data not shown), suggesting that these proteins are indeed functional.

[0266] In experiment 81 (Table 7), the ability of the Smithella sp. SCADC Csm1 enzyme (encoded by SEQ ID NO:185, SEQ ID NO:160) to effect DSB at the rice CAO1 locus was tested. Following bombardment of rice callus with the plasmids used in this experiment, DNA was extracted from hygromycin-resistant rice callus pieces and subjected to T7EI assays. Following PCR amplification of the rice CAO1 genomic locus, T7EI analysis identified three callus pieces from this experiment that contained indels at the expected sites. PCR products from these rice calli were analyzed by Sanger sequencing to identify sequences at the CAO1 locus in this callus. Figure 3A shows the results of these sequence assays for an 8 base pair deletion in callus piece no. 81-46, an identical 8 base pair deletion in callus piece no. 81-30, and a 12 base pair deletion in callus piece no. 81-9 at the predicted site in the CAO1 locus targeted by each guide RNA, demonstrating faithful DSB generation at this site by the Smithella SCADC Csm1 enzyme.

[0267] In experiment 93 (Table 7), the ability of the Sulfuricurvum sp. Csm1 enzyme (encoded by SEQ ID NO:186, SEQ ID NO:147) to execute a DSB at the rice CAO1 locus was tested. Following bombardment of rice callus with the plasmids used in this experiment, DNA was extracted from hygromycin-resistant rice callus pieces and subjected to T7EI assays. Following PCR amplification of the rice CAO1 genomic locus, one callus piece from this experiment was identified that contained an indel at the expected site by T7EI analysis. PCR products from rice callus piece no. 93-47 were analyzed by Sanger sequencing to identify sequences at the CAO1 locus in this callus. Figure 3A shows the results of these sequence assays for a 42 base pair deletion in callus piece no. 93-47 at the expected site of the CAO1 locus targeted by each guide RNA, demonstrating faithful DSB generation at this site by the Sulfuricurvum sp. Csm1 enzyme.

[0268] In experiment 97 (Table 7), the ability of Microgenomates (Roizmanbacteria) bacterium Csm1 enzyme (encoded by SEQ ID NO:193, SEQ ID NO:134) to effect DSB at the rice CAO1 locus was tested. Following bombardment of rice callus with the plasmid used in this experiment, DNA was extracted from hygromycin-resistant rice callus pieces and subjected to T7EI assays. Following PCR amplification of the rice CAO1 genomic locus, T7EI analysis identified three callus pieces from this experiment that contained indels at the expected site. Callus pieces no. 97-112, 97-130, and 97-141 showed banding patterns in the T7EI experiment analysis consistent with faithful DSB generation at this site by Microgenomates (Roizmanbacteria) bacterium Csm1 enzyme. DNA extracted from callus pieces no. 97-112 and 97-141 was subjected to sequence analysis (Figure 3A), which showed an identical 8-base pair deletion in these calli, indicating faithful generation of a DSB at this site by the Microgenomates (Roizmanbacteria) bacterium Csm1 enzyme.

Claims

1. 1. A method for modifying a nucleotide sequence at a target site in the genome of a eukaryotic cell, comprising: introducing into said eukaryotic cell: (i) a DNA-targeting RNA or a DNA polynucleotide encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises (a) a first segment comprising a nucleotide sequence complementary to a sequence of a target DNA and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide; (ii) a Cpf1 or Csm1 polypeptide, or a polynucleotide encoding a Cpf1 or Csm1 polypeptide, wherein the Cpf1 or Csm1 polypeptide comprises (a) an RNA-binding portion that interacts with a DNA-targeting RNA and (b) an activity portion that exhibits site-specific enzymatic activity; wherein the Cpf1 or Csm1 polypeptide is selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236; The genome of the eukaryotic cell is a nuclear, plastid, or mitochondrial genome; method.

2. 1. A method for modifying a nucleotide sequence at a target site in the genome of a prokaryotic cell, comprising: introducing into the prokaryotic cell: (i) a DNA-targeting RNA or a DNA polynucleotide encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises (a) a first segment comprising a nucleotide sequence complementary to a sequence of a target DNA and (b) a second segment that interacts with a Cpf1 or Csm1 polypeptide; (ii) a Cpf1 or Csm1 polypeptide, or a polynucleotide encoding a Cpf1 or Csm1 polypeptide, wherein the Cpf1 or Csm1 polypeptide comprises (a) an RNA-binding portion that interacts with a DNA-targeting RNA and (b) an activity portion that exhibits site-specific enzymatic activity; wherein the Cpf1 or Csm1 polypeptide is selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236; the genome of the prokaryotic cell may be a chromosome, a plasmid, or other DNA sequence within the cell; the prokaryotic cell is not a natural host for the gene encoding the Cpf1 or Csm1 polypeptide; method.

3. The method of claim 1 , wherein the eukaryotic cell is a plant cell.

4. Cultivating the plant under conditions in which the Cpf1 or Csm1 polypeptide is expressed and cleaves the nucleotide sequence at the target site to produce a modified nucleotide sequence; and Selecting plants containing the modified nucleotide sequence. The method of claim 3 further comprising:

5. 2. The method of claim 1, wherein the DNA-targeting RNA is a guide RNA (gRNA).

6. 3. The method of claim 2, wherein the DNA-targeting RNA is a guide RNA (gRNA).

7. 2. The method of claim 1, wherein the modified nucleotide sequence comprises an insertion of heterologous DNA into the genome of the cell, a deletion of a nucleotide sequence from the genome of the cell, or a mutation of at least one nucleotide in the genome of the cell.

8. 3. The method of claim 2, wherein the modified nucleotide sequence comprises an insertion of heterologous DNA into the genome of the cell, a deletion of a nucleotide sequence from the genome of the cell, or a mutation of at least one nucleotide in the genome of the cell.

9. 2. The method of claim 1, wherein the modified nucleotide sequence comprises the insertion of a polynucleotide encoding a protein that confers antibiotic or herbicide resistance to transformed cells.

10. 10. The method of claim 9, wherein the polynucleotide encoding a protein that confers antibiotic or herbicide resistance comprises SEQ ID NO:76 or encodes a protein comprising SEQ ID NO:

77.

11. 1. A nucleic acid molecule comprising a polynucleotide sequence encoding a Cpf1 or Csm1 polypeptide, the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 21, 22, 24, 25, and 174-206, or a fragment or variant thereof; or the polynucleotide sequence encodes a Cpf1 or Csm1 polypeptide selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 20, 23, 106-173, and 230-236; and The polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide is operably linked to a promoter that is heterologous to the polynucleotide sequence encoding the Cpf1 or Csm1 polypeptide. Nucleic acid molecule.

12. (i) a sequence selected from the group consisting of SEQ ID NOs: 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 21, 22, 24, 25, and 174-206, or a fragment or variant thereof; (ii) a nucleic acid molecule encoding an effector domain; A fusion protein encoded by a nucleic acid molecule comprising:

13. A Cpf1 or Csm1 polypeptide encoded by the nucleic acid molecule of claim 11.

14. The nucleic acid of claim 11, wherein the fragment or variant encodes a Cpf1 or Csm1 polypeptide comprising one or more mutations at one or more positions corresponding to positions 917 or 1006 of FnCpf1 (sequence number 3) or positions 701 or 922 of SmCsm1 (sequence number 160) when aligned for maximum identity.

15. A plant cell, a eukaryotic cell or a prokaryotic cell comprising the nucleic acid molecule of claim 11.

16. A plant cell, a eukaryotic cell or a prokaryotic cell comprising the fusion protein of claim 12.

17. A eukaryotic cell produced by the method of claim 1.

18. The eukaryotic cell of claim 17 , wherein the eukaryotic cell is a plant cell.

19. A plant comprising the nucleic acid molecule of claim 11.

20. A plant comprising the fusion protein or polypeptide of claim 12.

21. A plant produced by the method of claim 3.

22. A seed of the plant according to claim 19.

23. A seed of the plant according to claim 21.

24. The nucleic acid molecule of claim 11 , wherein the polynucleotide sequence encoding a Cpf1 or Csm1 polypeptide is codon-optimized for expression in a plant cell.

25. The method of claim 3 , wherein the polynucleotide sequence encoding a Cpf1 or Csm1 polypeptide is codon-optimized for expression in a plant cell.

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