Compositions and methods for site-directed genome modification

Abstract

The present disclosure provides novel maize, tomato, and soybean U6, U3, U2, U5, and 7SL snRNA promoters for use in CRISPR / Cas-mediated targeted gene modification in plants. The present disclosure also provides methods of using U6, U3, U2, U5, and 7SL promoters to drive expression of sgRNA polynucleotides that function in CRISPR / Cas systems for targeted gene modification in plants. The present disclosure also provides methods of genome modification that insert blunt-ended DNA fragments into genomic cleavage sites.

Description

[0001] Cross Reference to Related Applications

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 61 / 945,700, filed February 27, 2014.

[0003] Incorporation by Reference of the Sequence Listing

[0004] The sequence listing is included in a file named "MONS350WO_ST25.txt" which is 238 kilobytes in size (calculated in MS-Windows), created on February 27, 2015, and is hereby incorporated by reference in its entirety. The sequence listing is also incorporated by reference in its entirety. TECHNICAL FIELD

[0005] The present disclosure relates to the field of biotechnology. More specifically, the present disclosure provides methods of introducing a recombinant blunt-ended double-stranded DNA fragment into the genome of a plant by introducing a double-stranded break and a novel plant promoter that is beneficial for expression of non-protein-coding small RNAs, e.g., for CRISPR-mediated genome modification, into the genome. BACKGROUND

[0006] Site-specific recombination has potential applications in many broad biotechnology-related fields. Large-fragment nucleases, zinc-finger nucleases (ZFNs), and transcription-activator-like effector nucleases (TALENs), which contain DNA-binding domains and DNA-cleavage domains, are capable of genome modification. While large-fragment nucleases, ZFNs, and TALENs have effectiveness and specificity, these technologies require protein engineering by one or more elements selected for each genomic site where modification is to be made. Recent advances in the application of clustered regularly interspaced short palindromic repeats (CRISPR) demonstrate a genome modification method that is as powerful as similar systems (large-fragment nucleases, ZFNs, and TALENs), but with the advantage of rapid engineering.

[0007] The clustered regularly interspaced short palindromic repeats (CRISPR) system constitutes an adaptive immune system in prokaryotes that targets invading phage nucleases. The system is composed of protein elements (Cas) and guide RNAs (gRNAs) that target the specificity of the nuclease to a specific locus. The system has been successfully engineered to target specific nuclease loci in mammalian, zebrafish, fruit fly, nematode, bacterial, yeast, and plant genomes.

[0008] SUMMARY

[0009] In one aspect, the present application provides a recombinant DNA construct comprising a snRNA promoter (selected from the group consisting of a U6 promoter, a U3 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter); the promoter is operably linked to a sequence encoding a single guide RNA (sgRNA), wherein the sequence of the snRNA promoter comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-201, or SEQ ID NO: 247-283; or a fragment thereof, wherein the fragment is at least 140 bp in length.

[0010] In one embodiment, the sequence of the U6 promoter can comprise any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-166, SEQ ID NO: 200-201, or SEQ ID NO: 283, or a fragment thereof, wherein the fragment is at least 140 bp in length. In another embodiment, the sequence of the U6 promoter can comprise SEQ ID NO: 7. In another embodiment, the sequence of the U6 promoter can comprise a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In yet another embodiment, the sequence of the U3 promoter can comprise any one of SEQ ID NO: 167-171 or SEQ ID NO: 178-182, or a fragment thereof; wherein the fragment is at least 140 bp in length. In yet another embodiment, the sequence of the U2 promoter comprises any one of SEQ ID NO: 183-187, SEQ ID NO: 192-199, or SEQ ID NO: 247-275, or a fragment thereof; wherein the fragment is at least 140 bp in length. In another embodiment, the sequence of the U5 promoter comprises any one of SEQ ID NO: 188-191 or SEQ ID NO: 276-282, or a fragment thereof; wherein the fragment is at least 140 bp in length. In another embodiment, the sequence of the 7SL promoter comprises any one of SEQ ID NO: 172-177, or a fragment thereof; wherein the fragment is at least 140 bp in length. The recombinant DNA construct can further comprise a transcription termination sequence.

[0011] The recombinant DNA construct can also comprise a sequence encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product. In certain embodiments of the recombinant DNA construct, the Cas endonuclease gene product can be additionally operably linked to a nuclear localization sequence (NLS). Further, in certain embodiments of the contemplated recombinant DNA construct, the sequence encoding the Cas endonuclease can be selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 68, and SEQ ID NO: 97, SEQ ID NO: 119, and SEQ ID NO: 136.

[0012] Another aspect of the present application provides a recombinant DNA construct comprising a snRNA promoter selected from the group consisting of a U6 promoter, a U3 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter; the promoter is operably linked to a sequence representing a non-coding RNA, wherein the sequence of the snRNA promoter comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-201, or SEQ ID NO: 247-283, or a fragment thereof, wherein the fragment is at least 140 bp in length. In some embodiments, the non-coding RNA is selected from the group consisting of a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursors encoding the same, a heterochromatin siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double-stranded RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), and a naturally occurring antisense siRNA (nat-siRNA).

[0013] Certain embodiments of the present application also include such recombinant DNA constructs, wherein the sequence of the U3 promoter comprises any one of SEQ ID NOs: 167-171 and SEQ ID NOs: 178-182, or a fragment thereof; wherein the fragment is at least 140 bp in length. In another embodiment of the recombinant DNA construct, the sequence of the U2 promoter comprises any one of SEQ ID NOs: 183-187, SEQ ID NOs: 192-199, or SEQ ID NOs: 247-275, or a fragment thereof; wherein the fragment is at least 140 bp in length. In yet another embodiment of the recombinant DNA construct, the sequence of the U5 promoter comprises any one of SEQ ID NOs: 188-191 or SEQ ID NOs: 276-282, or a fragment thereof; wherein the fragment is at least 140 bp in length. Additionally, the present application provides such embodiments, wherein the sequence of the U6 promoter can comprise any one of SEQ ID NOs: 1-20, SEQ ID NOs: 146-149, SEQ ID NOs: 160-166, SEQ ID NOs: 200-201, or SEQ ID NOs: 283, or a fragment thereof; wherein the fragment is at least 140 bp in length. Another embodiment includes such recombinant DNA constructs, wherein the sequence of the 7SL promoter comprises any one of SEQ ID NOs: 172-177, or a fragment thereof; wherein the fragment is at least 140 bp in length.

[0014] Another aspect of the present application provides cells comprising the above-described recombinant DNA constructs. In certain embodiments, the cell is a plant cell.

[0015] The present application also provides a method of introducing a double-strand break into the genome of a cell comprising introducing into the cell: a) at least one recombinant DNA construct according to claim 1 ; and b) a second recombinant DNA construct comprising a sequence encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product operably linked to a nuclear localization sequence (NLS). In one embodiment of such a method, the sequence of the U6 promoter comprises SEQ ID NO: 7. In another embodiment of the method, the U6 promoter comprises a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In yet another embodiment of the method, the sequence encoding the Cas endonuclease is selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 68, and SEQ ID NO: 97, SEQ ID NO: 119, and SEQ ID NO: 136.

[0016] The present application also provides a method of introducing a double strand break into the genome of a cell comprising introducing into the cell at least one recombinant DNA construct comprising a recombinant DNA construct comprising a snRNA promoter selected from the group consisting of a U6 promoter, a U3 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter; operably linked to a sequence encoding a single guide RNA (sgRNA), wherein the sequence of the snRNA promoter comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-201, or SEQ ID NO: 247-283; or a fragment thereof, wherein the fragment is at least 140 bp in length, and further comprising a sequence encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product.

[0017] In certain embodiments of the method, the sequence of the U6 promoter comprises SEQ ID NO: 7. In other embodiments, the U6 promoter comprises a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments of the method, the sequence encoding a Cas endonuclease is selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 68, and SEQ ID NO: 97, SEQ ID NO: 119, and SEQ ID NO: 136.

[0018] Another aspect of the application provides a method of genome modification comprising: a) introducing a double-strand break into a selection site in the genome of a plant cell, and b) introducing a recombining blunt-end double-stranded DNA fragment into the plant cell, wherein the recombining blunt-end double-stranded DNA fragment is incorporated into the double-strand break by endogenous DNA repair. The method can comprise genome modification such as generating a modified linkage block, joining two or more QTLs, disrupting linkage of two or more QTLs, gene insertion, gene replacement, gene conversion, gene deletion or disruption, transgenic event selection, transgenic trait donor selection, transgenic replacement, or targeted insertion of at least one nucleic acid of interest. In some embodiments of the method, the double-strand break is introduced by a nuclease. In certain embodiments, the nuclease can be selected from the group consisting of: a TALEN nuclease; a CRISPR nuclease; a meganuclease comprising a "LAGLIDADG," "GIY-YIG," "His-Cys Box," or HNH sequence motif; and a zinc finger nuclease. In particular embodiments, the nuclease is a TALEN nuclease, and a TALEN expression construct is introduced into the plant cell, wherein about 0.1 pmol of each TALEN expression construct is introduced into the plant cell.

[0019] Additionally, in the method, the plant cell can be a protoplast, or can have been, or is being, grown in a plant cell culture. In certain embodiments of the method, the plant cell is selected from the group consisting of: a soybean plant cell; a corn plant cell; a rice plant cell; a wheat plant cell; a turfgrass plant cell; a cotton plant cell; and a canola plant cell. In other embodiments of the method, the recombining blunt-end double-stranded DNA fragment does not comprise a region of homology to the selection site in the genome.

[0020] Embodiments of such methods are contemplated wherein about 0.03 to about 0.3 fmol of the recombining blunt-end double-stranded DNA fragment is introduced into the plant cell. In particular embodiments, about 0.15 fmol of the recombining blunt-end double-stranded DNA fragment is introduced into the plant cell. Additionally, the blunt-end double-stranded DNA fragment can comprise a region of micro-homology to a sequence comprising one or both ends of the double-strand break in the genome at the 5' end or the 3' end or both the 5' and 3' ends. Some embodiments include such methods wherein the region of micro-homology is selected from the group consisting of: a sequence of 1 bp, 2 bp, 3 bp, 4, bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, or 10 bp in length. In particular embodiments of the method, the region of micro-homology is 3 bp in length.

[0021] The method can include introducing a double-strand break in step a) above by providing the cell with an endonuclease designed to target a select target site in the genome of the cell. Additionally, the endonuclease can be provided by at least one recombinant DNA construct encoding the endonuclease. In one embodiment, the endonuclease is provided by delivering an mRNA encoding the endonuclease or the endonuclease to the plant cell. In particular embodiments, the endonuclease is selected from the group consisting of: a TALEN endonuclease; a zinc finger endonuclease; a meganuclease; and a CRISPR endonuclease. Additional embodiments can include introducing a double-strand break in step a) by providing the cell with a recombinant DNA construct encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product; and a recombinant DNA construct comprising a U6, U3, U2, U5, or 7SL promoter operably linked to a sequence encoding a single guide RNA (sgRNA) designed to target a select target site in the chromosome of the cell. In particular embodiments, the Cas endonuclease gene product can be additionally operably linked to at least one nuclear localization sequence (NLS).

[0022] In certain embodiments of the method, the sequence of the U6, U3, U2, U5, or 7SL promoter can comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-201, or SEQ ID NO: 247-283, or a fragment thereof; wherein the fragment is at least 140 bp in length and comprises a transcription termination sequence. In particular embodiments, the U6 promoter can comprise a sequence selected from the group consisting of SEQ ID NO: 1-20, SEQ ID NO: 146-149, SEQ ID NO: 160-166, SEQ ID NO: 200-201, and SEQ ID NO: 283, or a fragment thereof; wherein the fragment is at least 140 bp in length, comprises a transcription termination sequence. In alternative embodiments, the U6 promoter can comprise a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In further embodiments, the sequence of the U3 promoter can comprise any one of SEQ ID NO: 167-171 or SEQ ID NO: 178-182, or a fragment thereof; wherein the fragment is at least 140 bp in length. In yet another embodiment, the sequence of the U5 promoter comprises any one of SEQ ID NO: 188-191 or SEQ ID NO: 276-282, or a fragment thereof; wherein the fragment is at least 140 bp in length. Additionally, the sequence of the U2 promoter can comprise any one of SEQ ID NO: 183-187, SEQ ID NO: 192-199, or SEQ ID NO: 247-275, or a fragment thereof; wherein the fragment is at least 140 bp in length. In yet another embodiment, the sequence of the 7SL promoter comprises any one of SEQ ID NO: 172-177, or a fragment thereof, wherein the fragment is at least 140 bp in length.

[0023] Also contemplated are embodiments wherein the recombinant DNA construct encodes a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product; and the recombinant DNA construct comprises a U6, U3, U2, U5, or 7SL promoter operably linked to a sequence encoding a single guide RNA (sgRNA) designed to target a select target site in the chromosome of the cell on the same construct. Other embodiments of the method can include the use of a recombinant DNA construct encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product; and a recombinant DNA construct comprising a U6, U3, U2, U5, or 7SL promoter operably linked to a sequence encoding a single guide RNA (sgRNA) designed to target a select target site in the chromosome of the cell on at least two constructs.

[0024] Another aspect of the application includes a plant cell comprising targeted recombination site-specific integration of a blunt-ended double-stranded DNA fragment. Also provided is a plant, plant part, or plant seed comprising targeted recombination site-specific integration of a blunt-ended double-stranded DNA fragment.

[0025] Yet another aspect of the application includes a method of genome modification comprising: a) introducing a double-stranded break into the genome of a plant cell by introducing into the cell: a) at least one recombinant DNA construct according to claim 1 ; and b) a second recombinant DNA construct comprising a sequence encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product operably linked to a nuclear localization sequence (NLS); and b) introducing a recombinant blunt-ended double-stranded DNA fragment into the plant cell, wherein the recombinant blunt-ended double-stranded DNA fragment is incorporated into the double-stranded break by endogenous DNA repair.

[0026] Another aspect of the application includes a method of genome modification comprising: a) introducing a double-stranded break into the genome of a plant cell described above, and b) introducing a recombinant blunt-ended double-stranded DNA fragment into the plant cell, wherein the recombinant blunt-ended double-stranded DNA fragment is incorporated into the double-stranded break by endogenous DNA repair.

[0027] Yet another aspect of the application includes a recombinant DNA construct comprising at least a first expression cassette comprising a U6, U3, U2, U5, or 7SL promoter operably linked to a sequence encoding a single guide RNA (sgRNA), wherein the sequence of the promoter comprises any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-201, or SEQ ID NO: 247-283, or a fragment thereof; wherein the fragment is at least 140 bp in length. In certain embodiments, the recombinant DNA construct further comprises at least a second expression cassette, wherein the sequence encoding the first sgRNA is different from the sequence encoding the second sgRNA. The recombinant DNA construct can also include such constructs wherein the promoter operably linked to the sequence encoding the first sgRNA is different from the promoter operably linked to the sequence encoding the second sgRNA. In certain embodiments, the construct comprises flanking left and right homology arms (HAs), each of which is about 200-1200 bp in length. In particular embodiments, the homology arms are about 230 to about 1003 bp in length.

[0028] Another aspect of the application provides a method for quantifying nuclease activity by detecting integrated DNA fragments, which is performed by determining the rate of homologous recombination (HR)-mediated targeted integration using digital PCR or quantitative PCR.

[0029] Yet another aspect of the application includes a recombinant DNA construct comprising: a) a first snRNA promoter selected from the group consisting of: a U6 promoter, a U3 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter; the first snRNA promoter operably linked to a sequence encoding a non-coding RNA, and b) a second snRNA promoter selected from the group consisting of: a U6 promoter, a U3 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter; the second snRNA promoter operably linked to a sequence encoding a non-coding RNA, wherein the first snRNA promoter and the second snRNA promoter are different. In certain embodiments, the sequence encoding the first snRNA promoter and the sequence encoding the second snRNA promoter each comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-201, or SEQ ID NO: 247-283, or a fragment thereof; wherein the fragment is at least 140 bp in length. Additionally, in certain embodiments, there is provided a recombinant DNA construct wherein the first and second snRNA promoters are U6 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of: SEQ ID NO: 1-8, SEQ ID NO: 17-20, and SEQ ID NO: 200-201.

[0030] Thus, there is also provided a recombinant DNA construct wherein the first and second snRNA promoters are U6 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of: SEQ ID NO: 12-16, SEQ ID NO: 160-166, and SEQ ID NO: 283. Alternatively, there is provided a recombinant DNA construct wherein the first and second snRNA promoters are U6 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of: SEQ ID NO: 9-11, and SEQ ID NO: 146-149.

[0031] It is also contemplated that where the first and second snRNA promoters are U2 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of recombinant DNA constructs of SEQ ID NOs: 183-187 and SEQ ID NOs: 192-199. Additionally, certain embodiments of the present application include where the first and second snRNA promoters are U2 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of recombinant DNA constructs of SEQ ID NOs: 247-275.

[0032] Yet another embodiment includes where the first and second snRNA promoters are U3 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of recombinant DNA constructs of SEQ ID NOs: 178-182. Yet another embodiment of the present application includes where the first and second snRNA promoters are U3 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of recombinant DNA constructs of SEQ ID NOs: 167-171.

[0033] Alternatively, the recombinant DNA construct can comprise first and second snRNA promoters that are U5 promoters, and wherein the sequences encoding the first and second snRNA promoters are each selected from the group consisting of SEQ ID NOs: 188-191. Alternatively, there is provided where the first and second snRNA promoters are U5 promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of recombinant DNA constructs of SEQ ID NOs: 276-282.

[0034] Certain embodiments of the present application provide where the first and second snRNA promoters are 7SL promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of recombinant DNA constructs of SEQ ID NOs: 175-177. In other embodiments, where the first and second snRNA promoters are 7SL promoters, and the sequences encoding the first and second snRNA promoters are each selected from the group consisting of recombinant DNA constructs of SEQ ID NOs: 172-174.

[0035] Also contemplated are embodiments in which the recombinant DNA construct comprises a first snRNA promoter (which is a U6 promoter), a second snRNA promoter is also present, and is selected from the group consisting of: a U3 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter. Other embodiments include recombinant DNA constructs in which the first snRNA promoter is a U3 promoter, and the second snRNA promoter is selected from the group consisting of: a U6 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter. Alternatively, in the recombinant DNA construct, the first snRNA promoter is a U2 promoter, and the second snRNA promoter can be selected from the group consisting of: a U6 promoter, a U3 promoter, a U5 promoter, and a 7SL promoter; or the first snRNA promoter is a U5 promoter, and the second snRNA promoter is selected from the group consisting of: a U6 promoter, a U2 promoter, a U3 promoter, and a 7SL promoter. Additionally, the recombinant DNA construct can comprise a first snRNA promoter and a second snRNA promoter, the first snRNA promoter is a 7SL promoter, and the second snRNA promoter can be selected from the group consisting of: a U6 promoter, a U2 promoter, a U3 promoter, and a U5 promoter.

[0036] Other contemplated embodiments of the present application include the above-described recombinant DNA construct in which the sequences encoding the first and second snRNA promoters are each selected from the group consisting of: SEQ ID NOs: 1-8, SEQ ID NOs: 17-20, SEQ ID NOs: 200-201, SEQ ID NOs: 183-187, SEQ ID NOs: 192-199, SEQ ID NOs: 178-182, SEQ ID NOs: 188-191, and SEQ ID NOs: 175-177. In certain embodiments of the recombinant DNA construct, the sequences encoding the first and second snRNA promoters are each selected from the group consisting of: SEQ ID NOs: 12-16, SEQ ID NOs: 160-166, SEQ ID NO: 283, SEQ ID NOs: 247-275, SEQ ID NOs: 167-171, SEQ ID NOs: 276-282, and SEQ ID NOs: 172-174.

[0037] The recombinant DNA construct can further comprise a sequence representing one or more additional snRNA promoters selected from the group consisting of: a U6 promoter, a U3 promoter, a U2 promoter, a U5 promoter, and a 7SL promoter; the snRNA promoter is operably linked to a sequence encoding a non-coding RNA, wherein each of the first snRNA promoter, the second snRNA promoter, and the one or more additional snRNA promoters is different. In particular embodiments of the recombinant DNA construct, the sequence representing the one or more additional snRNA promoters is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 146-149, SEQ ID NO: 160-201, or SEQ ID NO: 247-283; or a fragment thereof, wherein the fragment is at least 140 bp in length. Additionally, the recombinant DNA construct can comprise 3, 4, 5, 6, 7, 8, 9, or 10 snRNA promoters.

[0038] In some embodiments of the recombinant DNA construct, the non-coding RNA is an sgRNA targeting a different select target site in the chromosome of the plant cell. The recombinant DNA construct can further comprise a sequence encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product.

[0039] Yet another aspect of the application provides a method of genome modification comprising: a) introducing double-strand breaks into two or more selection sites in the genome of a plant cell by providing the cell with a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease and a recombination DNA construct, wherein the non-coding RNA is an sgRNA targeting different selection target sites in the chromosome of the plant cell, and b) introducing one or more exogenous double-stranded DNA fragments into the plant cell; wherein the exogenous double-stranded DNA fragments are incorporated at the double-strand breaks by endogenous DNA repair. In some embodiments, the one or more exogenous double-stranded DNA fragments are blunt-ended. In certain embodiments of the method, the one or more exogenous double-stranded DNA fragments comprise regions homologous to the selection sites in the genome. In other embodiments, the exogenous double-stranded DNA fragments comprise regions homologous to different selection sites in the genome. BRIEF DESCRIPTION OF DRAWINGS

[0040] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0041] Figure 1 : Nucleotide sequence alignment of four native maize U6 small nuclear RNA (snRNA) genes, including their putative promoters of Chromosome 1, 2, 3, and 8. (A) and (B) The consensus sequence, percentage of conservation, and sequence identity (size of the shown nucleotides is directly proportional to sequence conservation) are provided below the alignment. (B) The solid arrow indicates the transcription start site; upstream of the transcription start site are the "TATA box", the upstream sequence element (USE), and the monocot-specific promoter (MSP) element, each marked with a thick solid box; the seven thymine base chain (poly-T) at the 3' end is the transcription termination signal. The sequences in Figure 1.A and Figure 1.B correspond as follows: ZmU6_Ch1 is represented by SEQ ID NO: 98; ZmU6_Ch2 is represented by SEQ ID NO: 99; ZmU6_Ch3 is represented by SEQ ID NO: 100; ZmU6_Ch8 is represented by SEQ ID NO: 101.

[0042] Figure 2 : shows a modified GUS (beta-glucuronidase) reporter gene with direct repeats of the coding sequence (GUUS) interrupted by the target site (TS) for CRISPR cleavage.

[0043] Figure 3: GUS activity was detected in maize calli after co-bombardment of a GUUS reporter construct with a CRISPR construct designed to introduce a double-stranded break (DSB) into the Zm7 genomic target site.

[0044] Figure 4 : GUS activity was detected in maize calli after co-bombardment of a GUUS reporter construct with a CRISPR construct designed to introduce a DSB into the Zm231 genomic target site. Different genomic targets and single guide RNA (sgRNA) spacer sequences Zml4 were used as negative controls. Fluorescence microscopy images of representative calli co-bombarded with a green fluorescent protein (GFP) expression vector with the GUUS reporter construct, a Cas9 expression vector, and a vector containing various sgRNA cassettes are also shown.

[0045] Figure 5: shows (A) oligonucleotide integration assay; (B) blunt-end oligonucleotides for insertion into maize genomic target sites without microhomology; (C) blunt-end oligonucleotides for insertion into maize genomic target sites with microhomology ends; (D) fragment analysis profile of PCR amplicons spanning the oligonucleotide-chromosome junction in test (top panel) and negative control samples (bottom panel) of the oligonucleotide integration assay (wherein the arrow indicates the expected peak); and (E) DNA sequence of the oligonucleotide-chromosome junction at the Zm_L70c maize genomic target site confirming integration of full-length (integration 1; SEQ ID NO: 103) and truncated oligonucleotide (integration 2; SEQ ID NO: 104), with the expected sequence indicated as SEQ ID NO: 102.

[0046] Figure 6 : shows sgRNA (including a spacer sequence complementary to a native maize genomic target site and an artificial loop (5'-CCAAAAGG-3', SEQ ID NO: 105)) and its predicted secondary structure, designed for Streptococcus thermophilus Cas9-mediated targeting.

[0047] Figure 7: shows (A) removal of a selectable marker gene by multiplexed CRISPR activity followed by targeted integration of a gene of interest (GOI); and (B) CRISPR / Cas multiplex system to assess linkage of multiple QTL candidate genes. Because the likelihood of odds (LOD) is a statistical measure of linkage of genes; a LOD of 3 means that there is a 1000x greater likelihood of a QTL being present in the interval than not.

[0048] Figure 8The graph shows the percentage of target integration (Y-axis) detected at 24 and 48 hours after transformation of maize protoplasts using a CRISPR construct (targeting the natural chromosomal target (Zm7) in maize), and the titration (pmol) (X-axis) of the blunt-end double-stranded DNA fragment added to the transfection mixture. A negative control was run without the Cas9 expression construct.

[0049] Figure 9 A graph showing the change in integration rate (Y-axis) with the amount (in pmol) of the SpCas9 expression construct in the transfection mixture containing maize protoplasts (X-axis).

[0050] Figure 10. Sequence confirmation of the blunt-terminated double-stranded DNA fragment targeted integration into the chromosome of a maize protoplast (transformed using CRISPR / Cas9 and sgRNA expression constructs). For all insertions Figure 10A B, C, the top sequence is the expected sequence of an adapter for the target site and blunt-ended double-stranded DNA fragment (underlined sequence) included in the experiment. 10A. CRISPR / Cas9 construct targeting maize chromosome site Zm7, and having a blunt-ended double-stranded DNA fragment formed by annealed DNA fragments represented by SEQ ID NO: 115 and SEQ ID NO: 116. 10B. CRISPR / Cas9 construct targeting maize chromosome site L70c, and having a blunt-ended double-stranded DNA fragment without microhomologous sequences formed by annealed DNA fragments represented by SEQ ID NO: 45 and SEQ ID NO: 46. 10C. CRISPR / Cas9 construct targeting maize chromosome site L70c, and having a blunt-ended double-stranded DNA fragment containing 3 bp microhomologous sequences formed by annealed DNA fragments represented by SEQ ID NO: 121 and SEQ ID NO: 122 at each end of the DNA fragment.

[0051] Figure 11 A graph showing the change in integration rate (Y-axis) with the amount (in pmol) of the TALEN expression construct targeting the maize chromosome locus L70.4 in the transfection mixture of maize protoplasts (X-axis).

[0052] Figure 12. Schematic diagram of PCR primer positions for NHEJ- and HR-mediated targeted integration and high-throughput screening. Targeted integration of DNA fragments via non-homologous end joining (NHEJ) is shown in Figure 12. Figure 12A As shown, targeted integration of DNA fragments via homologous recombination (HR) is as follows: Figure 12B As shown.

[0053] Figure 13. Schematic of constructs used for homology integration. The blunt ended DNA arrow indicates the 90 bp sequence corresponding to the 90 bp blunt ended double stranded DNA fragment used for NHEJ analysis, LHA refers to the left homology arm, RHA refers to the right homology arm, Zm7 refers to the target site Zm7 targeted by CRISPR / Cas9 + sgRNA. The length of each homology arm is indicated in bp. A. Schematic of HR cassette construct targeting maize chromosomal site Zm7 with LHA and RHA of 240 and 230 bp in length, respectively. B. Schematic of HR cassette construct targeting maize chromosomal site Zm7 with LHA and RHA of 240 and 1003 bp in length, respectively.

[0054] Figure 14. Schematic of constructs used for homology integration. In the figure, the blunt ended DNA arrow indicates the 90 bp sequence corresponding to the 90 bp blunt ended double stranded DNA fragment used for NHEJ analysis, LHA refers to the left homology arm, RHA refers to the right homology arm, L70.4 refers to the target site L70.4 in the maize chromosome targeted by the TALEN pair. The length of each homology arm is indicated in bp. A. Schematic of HR cassette construct targeting maize chromosomal site L70.4 with LHA and RHA of 230 bp in length, respectively. B. Schematic of HR cassette construct targeting maize chromosomal site L70.4 with LHA and RHA of 1027 bp and 230 bp in length, respectively.

[0055] Figure 15. 15A. Graphical representation of data showing the percentage of targeted integration rates using StCas9 CRISPR constructs targeting the native maize chromosomal target sites L70e, L70f and L70g. Control lacks the StCas9 expression cassette construct in the transfection mix. 15B. Sequence alignment of the expected integrated blunt ended double stranded DNA fragment (SEQ ID NO: 144) and one example of an indel of the target site integrated DNA fragment sequence (SEQ ID NO: 145) at the L70f target site.

[0056] Figure 16. 16A. Chromosomal integration rates using constructs with the maize chromosomal 8U6 promoter or one of three separate chimeric U6 promoters driving sgRNA expression in the CRISPR / Cas9 system targeting three different maize chromosomal target sites. Targeted integration was determined by ddPCR analysis using MGB TaqMan probes. 16B. Chromosomal integration rates using constructs with the maize chromosomal 8U6 promoter or one of three separate chimeric U6 promoters driving sgRNA expression in the CRISPR / Cas9 system targeting three different maize chromosomal target sites. Targeted integration was determined by ddPCR analysis using MGB TaqMan probes. Insertion dye was determined by ddPCR analysis.

[0057] Figure 17. 17A. Schematic of PCR screening strategy to detect CRISPR / Cas9 induced mutations at the Tomatine Synthase Inhibitor Target Site 2 (TS2) by NHEJ, resulting in mutation of restriction endonuclease site Smll. 17B. Photograph of PCR amplicons run on an agarose gel showing undigested amplicons and Smll digested amplicons to detect CRISPR / Cas9 induced mutations at the Tomatine Synthase Inhibitor Target Site 2. 17C. Multiple sequence alignment of PCR amplicon sequences of CRISPR / Cas9 induced mutations at the Tomatine Synthase Inhibitor Target Site 2 by NHEJ.

[0058] Figure 18. 18A. Graphical representation of normalized GUS mRNA level data from soybean cotyledon protoplasts analyzed by recombinant expression constructs with U6, U3, and 7SL promoters. 18B. Graphical representation of normalized GUS mRNA level data from corn leaf protoplasts analyzed by recombinant expression constructs with U6, U3, 7SL, U2, or U5 promoters.

[0059] Figure 19 Figure 19. Graphical representation of normalized GUS expression level data from corn leaf protoplasts analyzed by recombinant expression constructs encoding 1) GUS expression construct, 2) dead Cas9-TALE-AD expression construct, and 3) recombinant sgRNA expression constructs with 7SL, U6, U3, U2, or U5 promoters. DETAILED DESCRIPTION

[0061] The present disclosure provides novel promoters for corn (Zea mays) and other plants and methods of use thereof, including targeted genetic modification of plant genomes using transgenic expression of one or more genes, involving the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system present in a number of bacteria. For example, in one embodiment, the present disclosure provides DNA constructs encoding at least one expression cassette comprising a U6 promoter disclosed herein and a sequence encoding a single guide RNA (sgRNA). Methods of initiating CRISPR system modification of a target genome are also provided, as are complementary sequences for use in modifying the genome of a plant using such systems. The present disclosure thus provides tools and methods that allow for insertion, removal, or modification of genes, loci, linkage disorders, and chromosomes within a plant. U3, U2, U5, and 7SL promoters and methods of use thereof, including targeted genetic modification of plant genomes, are also disclosed.

[0062] In another embodiment, the present disclosure provides DNA constructs encoding at least one expression cassette comprising a promoter disclosed herein and a sequence encoding a non-protein coding small RNA (npcRNA). These constructs are used to target nuclear expression of npcRNA molecules.

[0063] The CRISPR system constitutes the adaptive immune system of prokaryotes, which targets DNA and RNA of invading phages with endonucleolytic activity (reviewed in Westra et al., Annu Rev Genet, 46:311-39, 2012). There are three known types of CRISPR systems, type I, type II, and type III. The CRISPR system relies on small RNAs for sequence-specific detection and a targeting foreign nucleic acid to be destroyed. The elements of the bacterial CRISPR system are CRISPR-associated (Cas) genes and one or more CRISPR arrays composed of short palindromic repeats spaced by genomic target sequences (protospacers). The protospacer / repeat elements are transcribed into precursor CRISPR RNA (pre-crRNA) molecules, which are then processed, with the processing initiated by hybridization between a trans-acting CRISPR RNA (tracrRNA) molecule and the pre-crRNA palindromic repeats. The resulting crRNA:tracrRNA molecule consists of one copy of the spacer sequence and one repeat, with a complex of Cas nuclease. The CRISPR / Cas complex then engages a DNA sequence complementary to the crRNA spacer sequence (protospacer), and the RNA-Cas protein complex silences the target DNA through endonucleolytic cleavage of both strands (double-strand break; DSB).

[0064] The natural bacterial Type II CRISPR system requires four molecular elements for targeted cleavage of foreign DNA: a Cas endonuclease (e.g., Cas9), a housekeeping RNase III, a CRISPR RNA (crRNA), and a trans-acting CRISPR RNA (tracrRNA). The latter two elements form a dsRNA complex and bind Cas9, resulting in an RNA-guided DNA endonuclease complex. For targeted genome modification in eukaryotes, this system is simplified to two elements: a Cas9 endonuclease and a chimeric crRNA-tracrRNA, termed a guide RNA (gRNA) or single guide RNA (sgRNA). Experiments initially performed in eukaryotic systems determined that the RNase III element is not necessary to achieve targeted DNA cleavage. The Cas9 minimal two-element system has the unique element of the sgRNA, making this targeted genome modification CRISPR system more cost-effective and more flexible than other targeting platforms (such as meganucleases, zinc finger nucleases, or TALE-nucleases, which require protein engineering for each targeted DNA site modification). Additionally, the ease of design and generation of sgRNAs provides multiple targeted genome modification application advantages for the CRISPR system. For example, the elements of the CRISPR / Cas complex (Cas endonuclease, sgRNA, and optionally foreign DNA for integration into the genome) designed for one or more genomic target sites can be multiplexed in one transformation, or the introduction of the elements of the CRISPR / Cas complex can be spatially and / or temporally separated.

[0065] Expression strategies for sgRNAs

[0066] In certain embodiments, the present disclosure provides novel combinations of promoters and sequences encoding sgRNAs to allow specific introduction of double-stranded DNA cleavage events into endogenous DNA (i.e., the genome). In one embodiment, a U6 promoter of maize is operably linked to an sgRNA-encoding gene for constitutive expression of the sgRNA in a transformed cell. This can be desirable, for example, when the resulting sgRNA transcript remains in the nucleus and is therefore optimally positioned within the cell for guiding nuclear processing. This can also be desirable, for example, when the activity of CRISPR is low or the frequency of finding and cleaving a target site is low. This can also be desirable when the promoter for a particular cell type, such as a germ cell, is not known for a given species of interest. In another embodiment, a U3, U2, U5, or 7SL promoter is operably linked to an sgRNA-encoding gene for sgRNA expression in a transformed cell.

[0067] In another embodiment, a chimeric promoter comprising all or a portion of any of the U6 promoters provided herein can be used to express sgRNAs. Alternatively, a U3, U2, U5, or 7SL chimeric promoter comprising all or a portion of any of these promoters can be utilized. For example, a 3' portion of the U6 promoter of maize chromosome 8 (SEQ ID NO: 7) (including a USE element and TATA box (SEQ ID NO: 17)), operably linked to a 5' portion of the U6 promoter of maize chromosome 1 (SEQ ID NO: 1) (including one MSP element), cloned upstream of a sgRNA, can be used to induce CRISPR-mediated cleavage under different environmental conditions.

[0068] Multiple U6 promoters with different sequences can be used to minimize vector stability issues, which are often associated with sequence repeats. In addition, highly repetitive regions in a chromosome can lead to genetic instability and silencing. Thus, the use of multiple U6 (or other disclosed) promoters in a CRISPR / Cas system for targeted gene modification can facilitate vector stacking of multiple sgRNA cassettes in the same transformation construct, where different sgRNA transcript levels can be optimized for efficient targeting of a single target site. Chimeric U6 promoters can generate novel functional versions with improved or modified expression levels, and four representative chimeric maize U6 promoters (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20) were designed.

[0069] The disclosed U6 promoters can also drive expression of other non-protein coding RNAs (npcRNAs). Non-limiting examples of non-protein coding small RNAs include microRNAs (miRNAs), miRNA precursors, small interfering RNAs (siRNAs), small RNAs (length 22-26 nt) and precursors encoding the same, heterochromatin siRNAs (hc-siRNAs), Piwi-interacting RNAs (piRNAs), hairpin double-stranded RNAs (hairpin dsRNAs), trans-acting siRNAs (ta-siRNAs), and naturally occurring antisense siRNAs (nat-siRNAs).

[0070] Promoters and transcriptional elements of additional small nuclear RNA (snRNA) genes (similar to U6 promoters and transcribed by either RNA polymerase II or RNA polymerase III) can also be identified, such as U3, U2, U5, and 7SL promoters. These alternative promoters can be used in cassette design, especially where these additional elements can facilitate nuclear retention of CRISPR system transcripts. Additional gene transcription elements that can be used in CRISPR cassette design include intronic splice element and transcriptional elements of plant-specific RNA polymerase IV and V promoters.

[0071] Expression strategies for Cas-associated genes

[0072] The present disclosure provides novel promoters for sequence-specific or sequence- guided CRISPR-mediated cleavage by providing for the transcription of, for example, sgRNAs (including a spacer sequence for targeting a protospacer sequence within a genomic target site for cleavage by at least one Cas protein, where the genomic target site is either native or transgenic). In addition, the CRISPR system can be customized to catalyze cleavage at one or more genomic target sites. In certain embodiments, such customized CRISPR systems can have the property of being subject to genetic modification such that the Cas endonuclease protein of the system is recognition, binding, and / or catalytic activity.

[0073] One aspect of the present disclosure is the introduction of an expression vector comprising one or more cassettes encoding a U6 maize promoter or other disclosed promoters such as U3, U2, U5, or 7SL promoter operably linked to an sgRNA (including one copy of a spacer sequence complementary to a protospacer sequence within a genomic target site), and an expression vector encoding a Cas-associated gene into a plant cell to modify the plant cell, such that the plant cell or a plant comprised of such cells will subsequently exhibit a beneficial trait. In one non-limiting example, the trait is a trait such as increased yield, tolerance to biotic or abiotic stress, herbicide tolerance, or other agronomic performance improvement. The ability to generate such plant cells from which plants are derived depends on the introduction of the CRISPR system using the transformation vector cassettes described herein.

[0074] The expression vector encoding a Cas-associated gene can comprise a promoter. In certain embodiments, the promoter is a constitutive promoter, a tissue-specific promoter, a developmentally-regulated promoter, or a cell cycle-regulated promoter. Certain contemplated promoters include promoters that are expressed only in germ cells or reproductive cells, and the like. Such developmentally-regulated promoters have the advantage of limiting the expression of the CRISPR system to only those cells in which DNA is inherited to subsequent generations. Thus, CRISPR-mediated genetic modification (i.e., chromosomal or episomal dsDNA cleavage) is limited to only cells involved in transmitting their genome from one generation to the next. This can be useful if widespread expression of the CRISPR system has genotoxic or has other undesirable effects. Examples of such promoters include promoters of genes encoding DNA ligases, recombinases, replication enzymes, and the like.

[0075] Endonucleases are enzymes that cleave phosphodiester bonds within a polynucleotide strand. Examples of endonucleases that cleave only specific nucleotide sequences are well known in the art and can include, for example, restriction endonucleases. However, the demands of targeted genome engineering as an alternative to classical plant breeding require highly customizable genome editing tools. The CRISPR-associated type II prokaryotic adaptive immune system provides such an alternative. Thus, the DNA constructs provided herein can recognize a specific nucleotide sequence of interest within a target host genome and allow for mutation or integration at that site. In particular embodiments, the DNA construct comprises one or more maize U6 promoters, or chimeras thereof that express high levels of a sequence encoding an sgRNA. DNA constructs that express sgRNAs that target Cas-associated gene products that have endonuclease activity for a specific genomic sequence such that the specific genomic sequence is cleaved and generates a double-strand break that is repaired by a double-strand break repair pathway that can include, for example, non-homologous end joining, homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or combinations thereof, thereby breaking the native locus, can be particularly useful.

[0076] In one embodiment, the CRISPR system comprises at least one Cas-associated gene encoding a CRISPR endonuclease, and one sgRNA comprising one copy of a spacer sequence complementary to a protospacer sequence within an endogenous genomic target site.

[0077] In particular embodiments, the Cas-associated gene can include any type II CRISPR system endonuclease. Such Cas-associated gene products can have properties that make them amenable to genetic modification such that their nuclease activity and recognition and binding to crRNA, tracrRNA, and / or sgRNA can be manipulated.

[0078] The present disclosure also provides the use of CRISPR-mediated double-stranded DNA cleavage to genetically alter the expression and / or activity of a gene or gene product of interest in a tissue- or cell type-specific manner to improve yield or provide another advantageous trait, where the nucleic acid of interest can be endogenous or transgenic in nature. Thus, in one embodiment, the CRISPR system is engineered to mediate a break at a specific site of a gene of interest. Genes of interest include those that change the expression level / protein activity to a desired one. These DNA cleavage events can be in coding sequences or regulatory elements within a gene.

[0079] The present disclosure provides introducing a Type II CRISPR system into a cell. Exemplary Type II Cas-related genes include native and engineered (i.e., modified, including codon-optimized) nucleotide sequences encoding polypeptides having nuclease activity, such as Cas9 of Streptococcus pyogenes, Streptococcus thermophilus, or Bradyrhizobium sp.

[0080] Catalytically active CRISPR-associated genes (e.g., Cas9 endonuclease) can be introduced into or produced by a target cell. As disclosed herein, various methods can be used for implementation.

[0081] Transient expression of CRISPR

[0082] In some embodiments, sgRNAs and / or Cas-related genes are transiently introduced into a cell. In certain embodiments, sufficient amounts of the introduced sgRNAs and / or Cas-related genes are provided to modify the cell, but do not persist beyond a contemplated period of time or after one or more cell divisions. In such embodiments, no additional steps are required to remove or isolate the sgRNAs and / or Cas-related genes from the modified cell. In yet another embodiment of the present disclosure, double-stranded DNA fragments, as well as sgRNAs and / or Cas-related genes, are also transiently introduced into a cell. In such embodiments, sufficient amounts of the introduced double-stranded DNA fragments are provided to modify the cell, but do not persist beyond a contemplated period of time or after one or more cell divisions.

[0083] In another embodiment, mRNA encoding a Cas-related gene is introduced into a cell. In such embodiments, the mRNA is translated to produce sufficient amounts of a Type II CRISPR system endonuclease to modify the cell (in the presence of at least one sgRNA), but does not persist beyond a contemplated period of time or after one or more cell divisions. In such embodiments, no additional steps are required to remove or isolate the Cas-related gene from the modified cell.

[0084] In one embodiment of the disclosure, catalytically active Cas-related gene products are prepared in vitro and then introduced into cells, including prokaryotic or eukaryotic cells. Methods of preparing Cas-related gene products depend on their type and nature, and are known to those of skill in the art. For example, if the Cas-related gene product is a large monomeric DNA nuclease, active forms of the Cas-related gene product can be prepared by bacterial expression in vitro translation, by yeast cells, in insect cells, or by other protein production techniques described in the art. Following expression, the Cas-related gene product is isolated, refolded if necessary, purified, and optionally treated to remove any purification tags, such as His-tags. Once the crude, partially purified, or more fully purified Cas-related gene product is obtained, the protein can be introduced into, for example, plant cells by electroporation, by particle bombardment coated with the Cas-related gene product, by chemical transfection, or by some other means of transmembrane transport. Methods of introducing nucleic acids into bacteria and animal cells are likewise well known in the art. Nanoparticle delivery can also be used to deliver the protein, which can deliver a combination of active proteins and nucleic acids. Once a sufficient amount of Cas-related gene product is introduced, providing an effective amount of in vivo nuclease activity, and appropriate sgRNA, the protospacer sequence within the episome or genomic target site can be cleaved. It will also be recognized that one of skill in the art can create inactivated, but in vivo activated by the natural machinery of Cas-related gene products; such Cas-related gene products are also contemplated by the present disclosure.

[0085] In another embodiment, a construct is created that transiently expresses sgRNAs and / or Cas-associated genes and is introduced into a cell. In yet another embodiment, the vector will produce sufficient amounts of sgRNAs and / or Cas-associated genes to effectively modify one or more desired episomal or genomic target sites by CRISPR-mediated cleavage. For example, the disclosure contemplates that the vector can be prepared by bombardment, electroporation, chemical transfection, or by some other means of transport across the plant cell membrane. Such vectors can have a number of useful properties. For example, in one embodiment, the vector can replicate in a bacterial host, such that the vector can be prepared and purified in sufficient amounts for transient expression. In another embodiment, the vector can encode a drug resistance gene to allow selection of the vector in a host, or the vector can also contain an expression cassette to provide for expression of sgRNAs and / or Cas-associated genes in a plant. In another embodiment, the expression cassette can contain a promoter region, a 5' untranslated region, an optional intron (to assist in expression), multiple cloning sites (to allow for rapid introduction of sequences encoding sgRNAs and / or Cas-associated genes), and a 3' UTR. In particular embodiments, the promoter in the expression cassette can be a U6 promoter of maize. In other embodiments, the promoter can be a chimeric U6 promoter of maize. In some embodiments, it can be advantageous to include unique restriction sites at one or each end of the expression cassette to allow for preparation and isolation of a linear expression cassette, which can then be free of other vector elements. In certain embodiments, the untranslated leader sequence region can be a plant-derived untranslated region. When the expression cassette is transformed or transfected into a monocot plant cell, it is contemplated that a plant-derived intron is used.

[0086] In other embodiments, one or more elements in the vector include a spacer sequence that is complementary to a protospacer sequence contained within the episomal or genomic target site. This facilitates CRISPR-mediated modification within the expression cassette, such that elements such as promoters and transgenes can be removed and / or inserted.

[0087] In another approach, a bacterial or viral vector host can be used to introduce the transient expression vector into a cell. For example, Agrobacterium is such a bacterial vector that can be used to introduce a transient expression vector into a host cell. When using a bacterial, viral, or other vector host system, the transient expression vector is contained within the host vector system. For example, if an Agrobacterium host system is used, the transient expression cassette is flanked by one or more T-DNA borders and is cloned into a binary vector. A number of such vector systems are identified in the art (reviewed by Hellens et al., 2000).

[0088] In embodiments where sgRNAs and / or Cas-associated genes are transiently introduced in sufficient amounts to modify cells, methods of selecting modified cells can be utilized. In one such method, a second nucleic acid molecule comprising a selectable marker is co-introduced with the transient sgRNAs and / or Cas-associated genes. In this embodiment, the co-introduced marker can be part of a molecular strategy to introduce a marker at the target site. For example, the co-introduced marker can be used to disrupt a target gene by insertion between genomic target sites. In another embodiment, the co-introduced nucleic acid can be used to generate a visual marker protein such that transfected cells can be sorted or isolated by some other means. In yet another embodiment, the co-introduced marker can be randomly integrated or directed to integrate at a site independent of the primary genomic target sites by a second sgRNA:Cas protein complex. In yet another embodiment, the co-introduced molecule can be targeted to a specific locus by the double-strand break repair pathway, which can include, for example, non-homologous end joining, homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or combinations thereof, at the genomic target site. In the above embodiments, the co-introduced marker can be used to identify or select cells that can have been exposed to the sgRNAs and / or Cas-associated genes and thus can have been modified by CRISPR.

[0089] Stable expression of CRISPR

[0090] In another embodiment, a CRISPR expression vector is stably transformed into a cell to use the sgRNA and Cas-associated gene products encoded within the vector to cleave DNA sequences at or near genomic target sites in the host genome. In this embodiment, the design of the transformed vector provides flexibility in the timing and conditions of sgRNA and / or Cas-associated gene expression. In addition, the transformed vector can be designed to comprise a selectable or visual marker that provides a means to isolate or effectively select cell lines comprising CRISPR and / or modified by CRISPR.

[0091] Cell transformation systems are described in the art and include a variety of transformation vectors. For example, for plant transformation, two major methods include Agrobacterium-mediated transformation and particle gun bombardment-mediated (i.e., biolistics) transformation. In both cases, the CRISPR is introduced via an expression cassette. The cassette can include one or more of the following elements: a promoter element that can be used to express the sgRNA and / or Cas-associated genes; a 5' untranslated region that enhances expression; an intron element that further enhances expression in certain cell types such as monocot plant cells; a multiple cloning site that provides suitable restriction sites for insertion of sgRNA and / or Cas-associated gene sequences and other desired elements; and a 3' untranslated region that provides efficient termination of expression of the transcript. In particular embodiments, the promoter in the expression cassette will be the U6 promoter of maize. In other embodiments, the promoter will be a chimeric U6 promoter of maize.

[0092] For particle bombardment or transformation with protoplasts, the expression cassette can be an isolated linear fragment or can be part of a large construct that can include bacterial replication elements, bacterial selectable markers, or other elements. The sgRNA and / or Cas-associated gene expression cassette can be physically linked to a marker cassette, or can be mixed with a second nucleic acid molecule that encodes the marker cassette. The marker cassette is composed of the necessary elements to express a visual or selectable marker that allows for efficient selection of transformed cells. In the case of Agrobacterium-mediated transformation, the expression cassette can be adjacent to or flanked by T-DNA borders and contained within a binary vector. In another embodiment, the expression cassette can be outside of the T-DNA. The presence of the expression cassette in the cell can be manipulated by positive or negative selection schemes. In addition, the selectable marker cassette can also be within or adjacent to the same T-DNA borders, or can be elsewhere on a second T-DNA on a binary vector (e.g., 2T-DNA system).

[0093] In another embodiment, cells that are transiently or stably modified by CRISPR are passaged with unmodified cells. The cells can be subdivided into independent clonal source lines, or can be used to regenerate plants of independent sources. Individual plants or clonal populations regenerated from such cells can be used to generate independent source lines. At any of these stages, molecular assays can be utilized to screen the modified cells, plants, or lines. The modified cells, plants, or lines are continued to be propagated, while the unmodified cells, plants, or lines are discarded. In these embodiments, the presence of active CRISPR in the cells is essential to ensure the efficiency of the overall process.

[0094] Transformation methods

[0095] Methods of transforming or transfecting cells are well known in the art. Methods of transforming plants using Agrobacterium or DNA-coated particles are well known in the art and incorporated herein. It is believed that suitable host cell transformation methods for use with the present disclosure in practice include any method by which DNA can be introduced into a cell, such as Agrobacterium-mediated transformation (U.S. Patent Nos. 5,563,055, 5,591,616, 5,693,512, 5,824,877, 5,981,840, and 6,384,301) and acceleration of DNA-coated particles (U.S. Patent Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865), among others. Through the application of techniques such as these, cells of virtually any species can be stably transformed.

[0096] Various methods of selecting transformed cells have been described. For example, a resistance marker such as neomycin phosphotransferase protein confers kanamycin resistance, or 5-enolpyruvylshikimate phosphate synthase confers glyphosate tolerance. In another embodiment, a carotenoid synthase is used to create an orange pigment that is visually identifiable. Each of these three exemplary methods can be effectively used to isolate transformed and / or CRISPR-modified cells or plants or tissues thereof.

[0097] When a nucleic acid sequence encoding a selectable or screenable marker is inserted into the genomic target site, the marker can be used to detect the presence or absence of CRISPR or its activity. This can be useful once a cell has been modified by CRISPR, and it is desirable to recover a genetically modified cell that no longer contains CRISPR, or a plant regenerated from such a modified cell. In other embodiments, the marker can be intentionally designed to integrate into the genomic target site, making it available to track a modified cell independent of CRISPR. The marker can be a gene that provides a visually detectable phenotype, such as in a seed, to allow rapid identification of seeds that carry or lack a CRISPR expression cassette.

[0098] The present disclosure provides methods of regenerating a plant from a cell having a repaired double-strand break at the genomic target site within the protospacer sequence. The regeneration can be used to propagate additional plants.

[0099] The present disclosure additionally provides novel plant transformation vectors and expression cassettes comprising novel U6 promoters and U3, U2, U5, and 7SL promoters and combinations thereof having one or more CRISPR-associated genes and sgRNA expression cassettes. The present disclosure also provides methods of obtaining plant cells, whole plants, and seeds or embryos using CRISPR-mediated cleavage specificity modifications. The present disclosure also relates to novel plant cells comprising CRISPR-associated Cas endonuclease expression constructs and sgRNA expression cassettes.

[0100] Targeting using blunt end oligonucleotides

[0101] In certain embodiments, the CRISPR / Cas9 system can be utilized to target insertion of a blunt-ended double-stranded DNA fragment into a genomic target site of interest. CRISPR-mediated endonuclease activity can introduce a double-stranded break (DSB) into the protospacer sequence of the selected genomic target site and DNA repair, such as microhomology driven non-homologous end joining DNA repair, to insert a blunt-ended double-stranded DNA fragment into the DSB. The blunt-ended double-stranded DNA fragment can be designed using 1-10 bp of microhomology at the 5' and 3' ends of the DNA fragment that correspond to the 5' and 3' flanking sequences at the cleavage site of the protospacer sequence in the genomic target site.

[0102] Molecular breeding using custom CRISPR

[0103] In some embodiments, targeted genetic alteration of the genome is performed using knowledge of the genome. At least one sgRNA can be designed to target at least one region of the genome to break the region from the genome. This aspect of the disclosure can be particularly useful for genetic alteration. The resulting plant can have a modified phenotype or other property depending on the gene or genes that are altered. Previously characterized mutant alleles or introduced transgenes can be targeted for CRISPR-mediated modification to create improved mutant or transgenic lines.

[0104] In another embodiment, the gene targeted for deletion or breakage can be a transgene previously introduced into the target plant or cell. This has the advantage of allowing the introduction of an improved version of the transgene or allowing the breakage of a sequence encoding a selectable marker. In yet another embodiment, the gene targeted for breakage by CRISPR is at least one transgene introduced on the same vector or expression cassette as the other transgene(s) of interest and resides at the same locus as the other transgene(s). One skilled in the art will appreciate that this type of CRISPR-mediated modification can result in the deletion or insertion of additional sequences. Thus, in certain embodiments, a plurality of plants or cells in which deletion has occurred are generated and, after CRISPR-mediated modification, particular plants or cells having minimal alterations to the genome are identified using standard techniques, such that screening such plants or cells can be preferred. Such screening can utilize genotypic and / or phenotypic information. In such embodiments, a particular transgene can be broken while the remaining transgenes remain intact. This avoids the necessity of creating a new transgenic line that contains the desired transgene but not the undesired transgene.

[0105] In another aspect, the disclosure includes a method of inserting a DNA fragment of interest into a specific site of the genome of a plant, wherein the DNA fragment of interest is from the genome of the plant, or is heterologous with respect to the plant. The disclosure allows selection or targeting of a specific region of the genome (i.e., a broad locus) for nucleic acid (i.e., transgene) stacking. Thus, the targeted region of the genome can show linkage of at least one transgene to a haplotype of interest associated with at least one phenotypic trait, and can also result in development of a linkage drag that facilitates transgene stacking and transgene trait integration, and / or allows conventional trait integration while development of the linkage drag.

[0106] Trait integration using custom CRISPR

[0107] Targeted insertion of a DNA fragment of interest at at least one genomic protospacer site by CRISPR-mediated cleavage allows targeted integration of multiple nucleic acids of interest (i.e., trait stacking) to be added to the plant genome at the same site or different sites. The targeted integration site can be selected based on knowledge of underlying breeding values, transgene performance at the location, underlying recombination rates at the location, existing transgenes of the linkage drag, or other factors. Once assembled into a stacked plant, it can be used as a trait donor for crossing with germplasm being advanced in a breeding pipeline, or directly advanced in the breeding pipeline.

[0108] The disclosure includes a method of inserting at least one nucleic acid of interest into at least one site, wherein the nucleic acid of interest is from the genome of a plant, such as a QTL or allele, or is derived from a transgene. Thus, the targeted region of the genome can show linkage of at least one transgene to a haplotype of interest associated with at least one phenotypic trait (as described in U.S. Patent Application Publication No. 2006 / 0282911), development of a linkage drag that facilitates transgene stacking and transgene trait integration, development of a linkage drag that facilitates QTL or haplotype stacking and conventional trait integration, and the like.

[0109] In another embodiment of the disclosure, multiple unique sgRNAs can be used to modify multiple alleles of a specific locus within a linkage block contained on one chromosome by taking advantage of the knowledge of genomic sequence information and the ability to design custom sgRNAs described in the art. A sgRNA specific for or involving a genomic target site upstream of the locus containing the non-target allele is designed or engineered as desired. A second sgRNA specific for or involving a genomic target site downstream of the target locus containing the non-target allele is also designed or engineered. The sgRNAs can be designed such that they are complementary to regions of the genome that have no homology to the non-target locus containing the target allele. The two sgRNAs can be introduced into a cell using one of the methods described above.

[0110] The ability to perform targeted integration relies on the action of the sgRNA:Cas protein complex and the endonuclease activity of the Cas-associated gene product. This advantage provides a method to engineer plants of interest, including plants or cells containing at least one genomic modification.

[0111] Custom sgRNAs can be used in the CRISPR system to generate at least one trait donor to create a custom genomic modification event that is then crossed with at least one second plant of interest that includes a plant in which the CRISPR delivery can bind to the sgRNA of interest for genome editing. In other aspects, one or more plants of interest are directly transformed using the CRISPR system and at least one double-stranded DNA fragment of interest for targeted insertion. It is recognized that this method can be performed in a variety of cells, tissues, and developmental types, including plant gametes. It is also contemplated that one or more elements described herein can be combined with the use of a specific cell, tissue, plant part, and / or developmental stage-specific promoter, such as a meiosis-specific promoter.

[0112] Further, the disclosure contemplates the targeted deletion or disruption of transgenic elements already present within the genome. This allows, for example, the introduction of improved versions of transgenes, or the removal of selectable markers. In yet another embodiment, the gene targeted for disruption by CRISPR-mediated cleavage is at least one transgene introduced on the same vector or expression cassette as another / other transgene(s) of interest and resides at the same locus as the other transgene(s).

[0113] Accordingly, in one aspect, the disclosure provides a method of modifying a locus of interest in a cell, comprising (a) identifying at least one locus of interest within a DNA sequence; (b) creating a modified nucleotide sequence within or adjacent to the locus of interest, according to the disclosure, the nucleotide sequence comprising a protospacer sequence within a genomic target site of a first sgRNA; (c) introducing the sgRNA and a Cas-associated gene into at least one cell, wherein the sgRNA and / or Cas-associated gene is transiently or stably expressed; (d) analyzing the cell DNA for CRISPR-mediated modifications, the DNA constituting the locus of interest or flanking the locus of interest; and (e) identifying the cell or progeny cells thereof as comprising a modification in the locus of interest.

[0114] Another aspect provides a method of modifying a plurality of loci of interest in a cell, comprising: (a) identifying a plurality of loci of interest within a genome; (b) identifying a plurality of genomic protospacer sequence sites within each locus of interest; (c) introducing a plurality of sgRNAs and at least one Cas-associated gene into at least one cell, according to the disclosure, wherein the cell comprises a genomic protospacer sequence site, the sgRNAs and Cas-associated gene are transiently or stably expressed, and one or more modified loci comprising at least one CRISPR-mediated cleavage event are created; (d) analyzing the cell DNA for CRISPR-mediated modifications, the DNA constituting each locus of interest or flanking each locus of interest; and (e) identifying the cell or progeny cells thereof as comprising a modified nucleotide sequence in the locus of interest.

[0115] The disclosure also contemplates sequential modification of a locus of interest according to the disclosure by two or more sgRNAs and Cas-associated genes. The effect of a second CRISPR-mediated genomic modification can maintain, further modify, or remove a gene or other sequence added by the effect of such a first CRISPR-mediated genomic modification.

[0116] Accordingly, the present application includes methods of modifying a locus of interest in a crop such as maize (corn; Zea mays), soybean (Glycine max), cotton (Gossypium hirsutum; Gossypium sp.), peanut (Arachis hypogaea), barley (Hordeum vulgare); oats (Avena sativa); orchardgrass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugarcane (Saccharum sp.); tall fescue (Festuca arundinacea); turfgrass species such as the following species: Agrostis stolonifera, Poa pratensis, Stenotaphrum secundatum; wheat (Triticum aestivum); alfalfa (Medicago sativa); members of the genus Brassica including broccoli, cabbage, carrot, cauliflower, Chinese cabbage; cucumber, bean, eggplant, tobacco, fennel, garden pea, gourd, leek, lettuce, melon, okra, onion, pea, pepper, pumpkin, radish, spinach, squash, sweet corn, tomato, watermelon, ornamental plants, and other fruit, vegetable, tuber, oilseed, and root crops, with oilseed crops including soybean, canola, rapeseed, oil palm, sunflower, olive, maize, cottonseed, peanut, flaxseed, safflower, and coconut.

[0117] Genomic modifications can include modifying a linkage drag, linking two or more QTLs, disrupting linkage of two or more QTLs, gene insertion, gene replacement, gene conversion, gene deletion or disruption, transgenic event selection, transgenic trait donor selection, transgenic replacement, or targeted insertion of at least one nucleic acid of interest.

[0118] Definitions

[0119] The definitions and methods provided define the disclosure and guide one of ordinary skill in the art in the practice of the disclosure. Unless otherwise indicated, the terms are understood according to conventional usage by those of ordinary skill in the relevant art. Definitions for common terms in molecular biology can also be found in Alberts et al., Molecular Biology of The Cell, 5thedition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5thedition, Springer-Verlag: New York, 1991 ; King et al., A Dictionary of Genetics, 6thedition, Oxford University Press: New York, 2247; and Lewin, Genes IX, Oxford University Press: New York, 2007. The DNA base nomenclature shown in 37 CFR § 1.822 is used.

[0120] As used herein, "CRISPR-associated gene" refers to a nucleic acid sequence encoding a polypeptide element of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated system (Cas). Examples include, but are not limited to, Cas3 and Cas9, which encode endonucleases of the CRISPR Type I and Type II systems, respectively.

[0121] As used herein, "single guide RNA (sgRNA)" refers to a crRNA:tracrRNA fusion hybrid single-stranded RNA molecule encoded by a customizable DNA element that typically comprises one copy of a spacer sequence that is complementary to a protospacer sequence of a genomic target site, and a binding domain for a cognate Cas endonuclease of the CRISPR complex.

[0122] As used herein, "genomic target site" refers to a protospacer sequence and a protospacer adjacent motif (PAM) located in a host genome selected to be targeted for mutation and / or double-strand break.

[0123] As used herein, "protospacer sequence" refers to a short DNA sequence (12 to 40 bp) that can be targeted for mutation and / or double-strand break mediated by the enzyme cleavage of a CRISPR system endonuclease, which is directed by complementary base pairing to a spacer sequence in a crRNA or sgRNA.

[0124] As used herein, "protospacer adjacent motif (PAM)" includes a 3 to 8 bp sequence immediately adjacent to a protospacer sequence in a genomic target site.

[0125] As used herein, "microhomology" refers to the presence of identical short sequences of bases (1 to 10 bp) in different polynucleotide molecules.

[0126] As used herein, "codon-optimized" refers to a polynucleotide sequence that has been modified to adopt the codon usage bias of a particular plant. The modified polynucleotide sequence still encodes the same or a substantially similar polypeptide as the original sequence, but uses codon nucleotide triplets that are present in a particular plant with greater frequency.

[0127] As used herein, "non-protein coding RNA (npcRNA)" refers to such non-coding RNA (ncRNA) that is a precursor small non-protein coding RNA, or a fully processed non-protein coding RNA, that is a functional RNA molecule that is not translated into a protein.

[0128] As used herein, the term "chimeric" refers to a product of the fusion of portions of two or more different polynucleotide molecules, or to a gene expression element generated by manipulation of known elements or other polynucleotide molecules. Novel chimeric regulatory elements can be designed or engineered by a variety of methods. In one embodiment of the disclosure, a chimeric promoter can be generated by fusion of the 5' portion of the U6 promoter of maize chromosome 1, which includes at least one monocot-specific promoter (MSP) element, with the 3' portion of the U6 promoter of maize chromosome 8, which includes an upstream sequence element (USE) and a TATA box. The resulting chimeric promoter can have novel expression properties relative to the first or second promoters.

[0129] As used herein, “promoter” refers to a nucleic acid sequence located upstream or 5’ of the translation initiation codon of a gene’s open reading frame (or protein coding region) and involved in recognition and binding of RNA polymerase I, II, or III and other proteins (trans-acting transcription factors) to initiate transcription. A “plant promoter” is a natural or non-natural promoter that functions in a plant cell. A constitutive promoter functions in most or all tissues of a plant throughout its development. A tissue, organ, or cell-specific promoter is expressed only or primarily in a particular tissue, organ, or cell type, respectively. A promoter can exhibit “enhanced” expression, i.e., high level expression in one cell type, tissue, or plant part of a plant compared to other parts of the plant, rather than “specific” expression in a given tissue, plant part, or cell type. A temporally regulated promoter functions only or primarily during certain periods of plant development or at certain times of the day, as is the case, for example, with genes associated with circadian rhythms. An inducible promoter selectively expresses a DNA sequence operably linked thereto in response to the presence of an endogenous or exogenous stimulus, e.g., by a chemical (a chemical inducer) or in response to an environmental, hormonal, chemical, and / or developmental signal. Inducible or regulated promoters include, for example, light, heat, stress, flooding, or drought, plant hormone, wounding, or chemical such as ethanol, jasmonate, salicylic acid, or safener regulated promoters.

[0130] As used herein, “expression cassette” refers to a polynucleotide sequence comprising at least a first polynucleotide sequence capable of initiating transcription of a second polynucleotide sequence operably linked thereto, and optionally a transcription termination sequence operably linked to the second polynucleotide sequence.

[0131] A palindromic sequence is a nucleic acid sequence that reads the same, whether on one strand from 5’ to 3’ or on the complementary strand forming a double helix with it from 3’ to 5’. A palindromic sequence is considered to be equal to its reverse complement. Palindromic sequences can form hairpins.

[0132] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used to describe certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, numerical parameters are reported in a language such as "about" to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, numerical parameters are reported in a language such as "about" to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical values reported are precise values, and in some embodiments, the numerical values reported are approximate values. In some embodiments of the present disclosure, the numerical values are reported to the closest whole number in the units indicated. It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments

[0133] In some embodiments, the use of the terms "a" and "an" and "the" and similar referents in the context of describing the certain embodiments (especially in the context of certain of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. In some embodiments, the use of the term "or" in the context of certain embodiments (e.g., the context of certain of the following claims) is to be interpreted as inclusive or exclusive using the context to determine the intent of the claimant. In some embodiments, the use of the term "or" in the context of certain embodiments (e.g., the context of certain of the following claims) is to be interpreted as "and / or" unless otherwise indicated herein or clearly contradicted by context.

[0134] The terms "comprising," "having," and "including," and other analogous terms are open-ended, both in the general and specific contexts. Any forms or tenses of one or more of these terms, such as "comprise", "comprising", "include", "including", "have", "has", "having", or "includes", are also open-ended. For example, any method that "comprises", "has", "includes" or "comprising", "having", or "including" one or more steps is not limited to possessing only those one or more steps—and can also cover other unlisted steps. Similarly, any composition or device that "comprises", "has", "includes" or "comprising", "having", or "including" one or more features is not limited to possessing only those one or more features—and can cover other unlisted features.

[0135] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0136] The grouping of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. Each group member can be individually or in any combination with other members of the group or any combination of other elements present in the disclosure. One or more members of a group can be included or deleted from a group for reasons of convenience or patentability.

[0137] Having described the disclosure in detail, it will be apparent that modifications, variations, and equivalents of the disclosure herein disclosed can be practiced by persons of ordinary skill in the art without departing from the scope of the disclosure as defined by the appended claims. Furthermore, it should be appreciated that all examples are provided as non-limiting examples. DETAILED DESCRIPTION

[0138] The following examples are included to demonstrate embodiments of the disclosure. Those of skill in the art will understand that modifications, variations and equivalents of the specific embodiments described herein can be practiced by persons of ordinary skill in the art without departing from the concept, spirit and scope of the disclosure. More specifically, it is to be understood that certain agents which are chemically and / or physiologically related to the agents described herein can be substituted for the agents described herein while still obtaining the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0139] Example 1

[0140] Identification of promoters for expression of sgRNAs

[0141] To allow for genome engineering in maize, soybean, and tomato using CRISPR-based gene targeting systems, novel U6 promoters native to these three genomes were identified. After BLAST searching the maize, soybean, and tomato genomes for highly conserved U6 genes, 200-600 bp sequences upstream of these putative U6 genes were selected to test promoter function (Table 1). Four U6 promoters were identified in the maize B73 genome, each on chromosome 1 (SEQ ID NO: 1), chromosome 2 (SEQ ID NO: 3), chromosome 3 (SEQ ID NO: 5), and chromosome 8 (SEQ ID NO: 7), respectively. A multiple sequence alignment of these four maize U6 promoters and the corresponding U6 genes is compiled in FIGs. 1A and B. For each of these maize U6 promoters, conserved U6 promoter motifs (e.g., TATA box, upstream sequence element (USE), and monocot-specific promoter (MSP) elements) are provided (Connelly, Mol. Cell Biol. 14: 5910-5919, 1994) (FIG. IB). The guanine nucleobase after the poly-T stretch is conserved among the four genes and can have a significant role in transcription. The consensus sequence, percent conservation, and sequence logo (size of the nucleotides shown is directly proportional to sequence conservation) are provided below the alignment (FIG. 1). According to the multiple sequence alignment, the conserved motifs of these U6 promoters are within 140 bp of the transcription start site. According to the proximity of these conserved U6 promoter motifs, the 200 bp proximal upstream sequence of the transcription start site of each maize chromosome U6 promoter (chromosome 1 (SEQ ID NO: 2), chromosome 2 (SEQ ID NO: 4), chromosome 3 (SEQ ID NO: 6), and chromosome 8 (SEQ ID NO: 8)) was selected to test effective promoter activity in sgRNA expression cassettes.

[0142] In addition to the four maize U6 promoters, chimeric U6 promoters were designed. Four chimeric maize U6 promoters were designed using different combinations of the maize U6 promoters from chromosomes 1, 2, and 8, each chimeric promoter being 397 bp in length. The breakpoints of the chimeras were determined so that the conserved elements of different chromosomal origin (e.g., USE, MSP, and TATA box) were mixed in the new chimeric U6 promoters, but the relative spacing from the native maize U6 promoters was maintained. For example, the 5' end of the U6 promoter including the MSP and USE was derived from one chromosome, while the 3' end including the TATA box and one or more MSP elements was derived from a second chromosome. While the maize U6 promoter from chromosome 2 is not as strong a promoter as in its native form, it includes more than one MSP element. Thus, chimeras that include primarily chromosome 1 and / or 8 sequences can also include one or more of the chromosome 2 MSP elements. In particular, the 5' portion of chimeric 1 (SEQ ID NO: 17) is derived from the U6 promoter from maize chromosome 1 (SEQ ID NO: 1) (including one MSP element), and the 3' portion of this chimeric is derived from the U6 promoter from maize chromosome 8 (SEQ ID NO: 7) (including the USE element and the TATA box). Similarly, the 5' portion of chimeric 2 (SEQ ID NO: 18) is derived from the U6 promoter from maize chromosome 1 (SEQ ID NO: 1) (including one MSP element), and the 3' portion of this chimeric is derived from the U6 promoter from maize chromosome 8 (SEQ ID NO: 7) (including a second MSP element, the USE element, and the TATA box). The 5' portion of chimeric 3 (SEQ ID NO: 19) is derived from the U6 promoter from maize chromosome 8 (SEQ ID NO: 7) (including one MSP element), and the 3' portion of this chimeric is derived from the U6 promoter from maize chromosome 1 (SEQ ID NO: 1) (including a second MSP element, the USE element, and the TATA box). In addition, for chimeric 3, there is a 3 bp deletion starting at 100 bp from SEQ ID NO: 7, and the 5' end of the chimeric begins with 5'-AAG-3'. Chimeric 4 (SEQ ID NO: 20) is derived from the U6 promoter from maize chromosome 8 (SEQ ID NO: 7) (including the MSP element, the USE element, and the TATA box). However, this chimeric also includes two additional MSP elements derived from the U6 promoters from maize chromosomes 1 and 2 (for a total of 3 MSP elements).

[0143] Table 1. U6 promoters from maize, tomato, and soybean, their chromosomal origin, and length.

[0144]

[0145]

[0146] Example 2

[0147] Identification of Cas9 genes that allow for genome engineering in plants

[0148] CRISPR-mediated, site-directed targeting of a reporter construct was performed in immature maize embryos using a S. pyogenes Cas9 sequence (SEQ ID NO: 28 is the polypeptide sequence of Cas9 with NLS and SEQ ID NO: 96 is the polypeptide sequence of Cas9 without NLS). For expression, the nucleotide sequence of the codon-optimized Cas9 was designed into an expression vector capable of expression in plants. This Cas9 expression vector contains a 35S promoter driving expression of the Cas9 open reading frame, a NLS sequence incorporated into the 3' end of the Cas9 coding region, and a Nos transcription termination sequence (SEQ ID NO: 29).

[0149] Monocot codon-optimized versions of the Cas9 protein (SEQ ID NO: 26) and nucleotide sequence encoding it (SEQ ID NO: 27) were identified from plant-associated bacteria, Sinorhizobium meliloti, and can be used to improve robustness of CRISPR / Cas-mediated genome modification in plants. Cas9 protein (SEQ ID NO: 69) and monocot codon-optimized version of it (SEQ ID NO: 68) were identified from S. thermophilus and can be used to improve robustness of CRISPR / Cas-mediated genome modification in plants. Additional Cas9 genes can also be identified from plant-associated bacteria (e.g., symbiotic or pathogenic bacteria).

[0150] Example 3

[0151] Single guide RNA cassette design

[0152] A set of single guide RNA (sgRNA) expression cassettes targeting the protospacer sequence in a maize genomic target site called Zm7 (5'-GCCGGCCAGCATTTGAAACATGG-3', SEQ ID NO:22) were designed. Different expression cassettes included one of the 397 bp U6 promoters of maize: chromosome 1 (SEQ ID NO:30), chromosome 2 (SEQ ID NO:32), chromosome 3 (SEQ ID NO:34), or chromosome 8 (SEQ ID NO:36); or one of the 200 bp U6 promoters of maize: chromosome 1 (SEQ ID NO:31), chromosome 2 (SEQ ID NO:33), chromosome 3 (SEQ ID NO:35), or chromosome 8 (SEQ ID NO:37). Each expression cassette also contained i) a U6 poly-T terminator, which is conserved in each of the four maize U6 genes; ii) an sgRNA that includes the spacer sequence 5'-GCCGGCCAGCATTTGAAACA-3' (SEQ ID NO:23) corresponding to one copy of the protospacer sequence of the Zm7 genomic target site (SEQ ID NO:22); and iii) a conserved 3' domain of the sgRNA that provides a Cas endonuclease binding domain and ends with a U6 poly-T segment (SEQ ID NO:21).

[0153] Similarly, a set of sgRNA cassettes was designed using one of four maize U6397 bp promoters (SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36; see Table 2), and a spacer sequence of an sgRNA complementary to the protospacer sequence of a maize genomic target site designated Zm231 (SEQ ID NO: 24). Table 3 lists the corresponding SEQ ID NOs for the DNA and RNA sequences of the sgRNAs comprising the Zm7, Zm231, and Zml4 target sites. A negative control sgRNA cassette was designed using the maize U6397 bp promoter of maize chromosome 8 (SEQ ID NO: 36), and a spacer sequence of an sgRNA complementary to the protospacer sequence of a maize genomic target site designated Zml4 (SEQ ID NO: 24). This negative control sgRNA cassette was designed using a spacer sequence of an sgRNA that is not complementary to the protospacer sequence of the Zm231 maize genomic target site. Including an sgRNA comprising a spacer sequence complementary to the Zml4 maize genomic target site does not result in CRISPR / Cas-mediated cleavage of the protospacer sequence of the Zm231 maize target protospacer site. These Zm231 and Zml4 sgRNA cassettes are represented by SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42 (Table 2). Each of these sgRNA cassettes also comprises a U6 poly-T stretch at the 3' end of the sgRNA sequence.

[0154] Table 2. Cassette with indicated maize U6 promoter and sgRNA comprising a spacer sequence complementary to the protospacer sequence of the indicated maize genomic target site.

[0155]

[0156] Table 3. DNA and RNA sequences of S. pyogenes sgRNAs comprising a spacer sequence complementary to the protospacer sequence of the maize genomic target sites Zm7, Zm231, and Zml4.

[0157]

[0158] Example 4

[0159] CRISPR activity in maize - modification of GUS reporter gene analysis

[0160] To determine the activity of CRISPR / Cas-mediated gene targeting efficiency in maize, a transient expression system of a reporter gene in immature maize embryos was used. In addition to the sgRNA cassettes described above, this design incorporated an expression cassette containing the Cas9 endonuclease from S. pyogenes (SEQ ID NO: 28) that included a nuclear localization signal (NLS) sequence and was codon optimized for expression in maize.

[0161] The reporter gene constructs for these experiments were cassettes containing a modified beta-glucuronidase (GUS) coding sequence in which a maize genomic target site for targeted CRISPR cleavage (protospacer and PAM) (e.g., Zm7 (SEQ ID NO: 22), Zm231 (SEQ ID NO: 44), or Zm14 (SEQ ID NO: 43)) was engineered into the reporter gene around the direct repeat sequence internal to the GUS coding sequence. Figure 2 ) If the CRISPR system cleaves the protospacer, the endogenous plant single-strand annealing (SSA) pathway of homologous recombination DNA repair will reconstitute a functional GUS gene when the expression vectors for the CRISPR elements are co-delivered. These modified GUS reporter gene constructs were named GU-Zm7-US, GU-Zm231-US, or GU-Zm14-US, respectively, referring to the maize genomic target site of insertion of the Zm7, Zm231, and Zm14 GUS genes. One of the modified GUS reporter gene cassettes was co-delivered with the other CRISPR elements using expression vectors for one of the sgRNA cassettes and an expression cassette encoding the Cas9 endonuclease (SEQ ID NO: 28). The expression cassettes were mixed using standard protocols and co-coated onto 0.6 M gold particles. These prepared gold particles were then used to bombard 3-day pre-cultured immature maize embryos. The embryos were maintained in culture for 3-5 days after bombardment and then subjected to histochemical staining using X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide) and standard laboratory protocols.

[0162] GUS activity was detected as blue foci Figure 3 and 4 ) using histochemical staining and X-Gluc if CRISPR-mediated Cas9 endonuclease activity occurred at the protospacer site in the modified reporter gene construct.

[0163] The individual expression cassettes were designed to contain one of four maize U6 promoters (from chromosomes 1, 2, 3, and 8) driving expression of an sgRNA containing a spacer sequence complementary to the protospacer sequence of the maize Zm7 genomic target site Figure 3). To prepare samples for expression analysis, 0.6 pM gold particles were coated with 0.6 pmol of one of the Zm7-sgRNA constructs and 0.3 pmol of each of the other constructs (Cas9 expression cassette and Zm7 modification reporter construct (GU-Zm7-US)). Once the coated gold particles were prepared, 1 / 4 of the mixture was used to bombard 3-day-old immature maize embryos using standard protocols. Over 50 immature maize calli were bombarded for each set of evaluated constructs, and staining was performed 5 days post bombardment. After staining, photographs were taken of representative calli (overview of multiple calli and close-up view of individual calli) Figure 3 ) The modification reporter construct GU-Zm7-US was designed to contain the Zm7 genomic target site (SEQ ID NO: 22), and the sgRNA was designed to contain one copy of the Zm7 spacer sequence (SEQ ID NO: 23). The Zm7-sgRNA spacer sequence was incorporated into an expression cassette with one of four 397 bp maize U6 promoters (from chromosome 1 (SEQ ID NO: 30), chromosome 2 (SEQ ID NO: 32), chromosome 3 (SEQ ID NO: 34), or chromosome 8 (SEQ ID NO: 36)). Negative controls for transformation included the modification reporter construct GU-Zm7-US with the Zm7 genomic target site and either: (1) lacking the Cas9 endonuclease expression cassette and the Zm7-sgRNA expression cassette; or (2) lacking only the Zm7-sgRNA expression cassette Figure 3 ) For both of these controls, no blue portions were detected, indicating that the modification reporter construct did not undergo CRISPR-mediated cleavage. The results of the evaluation of the four different 397 bp maize U6 promoters driving expression of the Zm7-sgRNA cassette showed that while all four 397 bp maize U6 promoters functioned (i.e., blue portions were detected in the calli), the different promoters did not function equally (as evidenced by the size and number of blue portions in the calli). The U6 promoter from maize chromosome 8 showed the greatest functionality, followed by the U6 promoter from chromosome 1. The U6 promoters from chromosomes 2 and 3 showed similar functionality to each other (Chr 8 > Chr 1 > Chr 2 = Chr 3).

[0164] The specificity of the CRISPR / Cas9 system in this maize expression system was evaluated by testing mismatches between the protospacer sequence within the genomic target site in the GUUS reporter construct and the spacer sequence included in different sgRNA constructs Figure 4). As shown in the above experiments, 0.6 μΜ gold particles were coated with one or more constructs; 0.3 pmol of the individual modified GUUS reporter construct (GUUS target), 0.16 pmol of the Cas9 endonuclease expression cassette, 0.3 pmol of the individual sgRNA cassette, and 0.03 pmol of the transformation control construct expressing green fluorescent protein (GFP) Figure 4 ) Once the gold particles were prepared, 1 / 4 of the mixture was used to bombard 3-day old immature maize embryos using standard protocols. Over 50 immature maize calli were bombarded for each set of constructs evaluated. The tissue was maintained in culture for 3 days post bombardment. GFP expression was determined by fluorescent microscopy at day 1 and (again) at day 3 to verify that the bombardment and transformation were consistent. After fluorescent microscopy at day 3, the calli were stained for X-Gluc and fluorescent and light micrographs were taken of representative calli Figure 4 ) Fluorescent staining of all calli indicated that the transformation was good.

[0165] Negative controls used in the transformation included the modified reporter construct GU-Zm231-US (1) lacking the Cas9 endonuclease expression cassette and any sgRNA expression cassettes; or (2) having the Zm231-sgRNA expression cassette with the chromosome 8 maize U6 promoter, but lacking the Cas9 endonuclease expression cassette Figure 4 ) Neither of these controls showed the blue portion detected by X-Gluc staining, indicating that the modified reporter construct was not CRISPR-mediated cleaved Figure 4 ).

[0166] The specificity of the CRISPR / Cas9 system can also be evaluated using a control that includes a mismatch between the protospacer site in the modified GUUS reporter construct and the sgRNA spacer sequence. In particular, the mismatch is between the modified reporter construct GU-Zm231-US having the Zm231 genomic target site and (1) the sgRNA expression cassette having the Zm14 spacer sequence and the chromosome 8 maize U6 promoter; or (2) the sgRNA expression cassette having the Zm231 spacer sequence and the chromosome 8 maize U6 promoter Figure 4 ).

[0167] Finally, each of the 397 bp maize U6 promoters (chromosomes 1, 2, 3, and 8) was used to generate sgRNA expression cassettes with the Zm231 genomic target site. Each of these promoters was co-transformed with the modified reporter gene construct GU-Zm231-US made with the Zm231 genomic target site. The results showed that little GUS activity was detected when the sgRNA spacer sequence was mismatched to the genomic target site of the reporter gene construct. In contrast, multiple large blue foci were detected when the sgRNA spacer sequence was matched to the genomic target site of the reporter gene construct Figure 4 ). The U6 promoter of maize chromosome 8 can have the highest efficacy (based on the assumption that efficacy is related to more, larger, and darker blue foci), followed by the U6 promoter of maize chromosome 1. The U6 promoters of maize chromosomes 2 and 3 showed similar efficacy to each other (Chr 8 > Chr 1 > Chr 2 = Chr 3).

[0168] The U6 promoter of maize chromosome 8-driven sgRNAs consistently showed high activity. These findings suggest that different maize U6 promoters have different activities and highlight that the effectiveness of U6 promoters originates from maize chromosome 8 in the CRISPR / Cas system that targets genomic modifications.

[0169] Example 5

[0170] Blunt-end oligonucleotide integration

[0171] The targeted efficacy of blunt-end double-stranded DNA fragments inserted into one of three genomic target sites (referred to as Zm_L70a (SEQ ID NO: 47), Zm_L70c (SEQ ID NO: 59), and Zm_L70d (SEQ ID NO: 61) within the maize genome) was assessed for the CRISPR / Cas9 system. Each of the three genomic target sites is unique to the maize genome. If the CRISPR element is capable of having endonuclease activity and introduces a double-strand break (DSB) into the protospacer sequence of the selected genomic target site, then the endogenous maize non-homologous end joining DNA repair system will insert a blunt-end double-stranded DNA fragment into the DSB.

[0172] Complementary oligonucleotides were pre-annealed to form blunt-end double-stranded DNA fragments that were co-transformed into maize protoplasts along with the CRISPR construct Figure 5A ). The oligonucleotide pairs were designed to either (1) not contain a microhomologous region (see Figure 5B ), or (2) contain 3 bp at each end (5' and 3') that are microhomologous to the corresponding 5' and 3' flanking sequences at the cleavage site of the protospacer sequence in the genomic target site Figure 5C). The microhomologous sequences can facilitate integration of the blunt-ended double-stranded DNA fragments via the microhomology-driven non-homologous end-joining mechanism at the genomic target site. The two oligonucleotide pairs without microhomologous sequences are SEQ ID NO: 45 and SEQ ID NO: 46. Each of the three oligonucleotide pairs comprises a microhomology to their respective genomic target site, and are annealed in pairs with the following oligonucleotides: (1) SEQ ID NO: 62 and SEQ ID NO: 63 (microhomologous to Zm_L70a); (2) SEQ ID NO: 64 and SEQ ID NO: 65 (microhomologous to Zm_L70c); and (3) SEQ ID NO: 66 and SEQ ID NO: 67 (microhomologous to Zm_L70d), to form the blunt-ended double-stranded DNA fragments.

[0173] For integration analysis of these blunt-ended double-stranded DNA fragments, the CRISPR construct used included the Cas9 endonuclease expression cassette described above, and one of the three sgRNA expression cassettes. The three sgRNA expression cassettes were each driven by the 397 bp version of the U6 promoter from maize chromosome 8 (SEQ ID NO: 7), and contained the spacer sequence corresponding to the genomic target site Zm_L70a (SEQ ID NO: 48), Zm_L70c (SEQ ID NO: 58), and Zm_L70d (SEQ ID NO: 60). For these analyses, different combinations of CRISPR elements and oligonucleotides were mixed as follows: 0.6 pmol of the Cas9 expression cassette, 1.6 pmol of one of the sgRNA expression cassettes, and 35 pmol of the pre-annealed oligonucleotide pairs, and transformed into an aliquot of maize leaf protoplast suspension containing approximately 320,000 cells using the standard PEG-mediated protocol. Two days later, the maize protoplasts were collected and analyzed for insertion of the blunt-ended double-stranded DNA fragments at the specific L70 genomic target site, which in each case was targeted by the unique sgRNA selected (Table 4). The negative control was the absence of the Cas9 expression cassette during the maize protoplast transformation.

[0174] To detect blunt-ended double-stranded DNA fragment insertions into maize chromosomes, DNA was extracted and high-throughput thermal amplification (PCR) was performed using multiple primer pairs (Table 5). Since blunt-ended double-stranded DNA fragments can insert into chromosomal DNA cleavage CRISPR in either direction, primers were designed with one strand of blunt-ended double-stranded DNA fragment and two lateral genomic regions, with each primer pair spanning the adapter at the insertion site. PCR amplicons were isolated using a fragment analysis platform (ABI 3730 DNA Analyzer) at Life Technologies (Grand Island, NY). This platform is more sensitive than aggregation-based electrophoresis and offers single-bp resolution to confirm whether the amplicons originated from the template of interest and whether they were specific to the experimental treatment conditions.

[0175] Table 4. DNA and RNA sequences of Streptococcus pyogenes sgRNA containing spacer sequences complementary to the anterior spacer sequences of maize genome target sites L70a, L70c, and L70d.

[0176]

[0177] A representative segment analysis spectrum is as follows Figure 5D As shown in Experiment T3, Table 5. DNA extracted from maize protoplasts was amplified using primers at the Zm_L70c genomic target site (SEQ ID NO: 49, primer specific to the inserted blunt-ended double-stranded DNA fragment, and SEQ ID NO: 55, primer specific to the lateral genomic DNA). The amplification showed a main peak of the expected size and several other peaks of similar size (arrows). The protoplasts were transformed with Cas9, sgRNA (containing a spacer sequence complementary to the anterior spacer sequence at the Zm_L70c maize genomic target site (SEQ ID NO: 83)), and a non-microhomological blunt-ended double-stranded DNA fragment. Figure 5D (Top illustration). In contrast, no amplification products were observed in DNA extracted from the negative control transformation ( Figure 5D (See bottom inset). This PCR profile is consistent with double-strand breaks resulting from non-homologous end-joining error repair, leading to the introduction of short insertions / deletions at the repair site.

[0178] To confirm the integration of the blunt-ended double-stranded DNA fragments at the genomic target site, PCR amplicons were cloned and sequenced (Table 5). The negative control lacking Cas9 protein did not generate a PCR product. Seven of ten experiments showed the expected pattern: the test sample gave a positive PCR product of the expected size, while the control sample had no PCR product. The seven experiments that showed a positive PCR product included experiments that showed integration of both the blunt-ended double-stranded DNA fragment with and without the microhomology. Experiments Tl and T7 were unable to detect targeted integration in either the test or control samples. PCR products were cloned and sequenced from six experiments, confirming the expected DNA fragment-chromosome junctions for blunt-ended double-stranded DNA fragment integration. Sequencing results showed that the site of blunt-ended double-stranded DNA fragment integration had both full-length and truncated DNA fragments (indels) present (see, e.g. Figure 5E Experiment Tl). The sequences were consistent with fragment analysis Figure 5D ), and showed that CRISPR / Cas9 can target naturally occurring sequence-specific chromosomal loci for cleavage in maize protoplasts. These results also show successful integration of blunt-ended double-stranded DNA fragments with and without the microhomology region.

[0179]

[0180] where * = sample was contaminated.

[0181] Example 6

[0182] Targeted genome modification using CRISPR / Cas9 complex genes from Streptococcus thermophilus

[0183] It would be desirable to use CRISPR complex genes derived from S. thermophilus rather than S. pyogenes for CRISPR-mediated genome modification of some plants, such as crops. The inventors developed an expression cassette encoding a codon-optimized nucleotide sequence with two nuclear localization signals (NLS) that are nuclear localization signals of the Cas9 protein of S. thermophilus (SEQ ID NO: 69). The StCas9 was designed to encode N-terminal and C-terminal nuclear localization signals (NLS) at amino acid positions 2-11 and 1133-1142 (SEQ ID NO: 135). In addition, the DNA expression cassette (SEQ ID NO: 136) includes an intron at nucleotide positions 507-695. A series of unique S. thermophilus single guide RNAs (sgRNAs) were designed. The S. thermophilus sgRNAs were designed to link the native S. thermophilus crRNA and tracrRNA with a stem loop (5'-CCAAAAGG-3'; SEQ ID NO: 105) and to include a spacer sequence complementary to a protospacer of a maize genomic target site selected from Zm_L70e (SEQ ID NO: 72), Zm_L70f (SEQ ID: 73), Zm_L70g (SEQ ID NO: 74), or Zm_L70h (SEQ ID NO: 75). The seven nucleotides at the 3' end of each of these genomic target sites represent the S. thermophilus-specific protospacer adjacent motif (PAM, 5'-NNAGAAW-3'; SEQ ID NO: 106). Figure 6 The predicted secondary structure of this S. thermophilus sgRNA (SEQ ID NO: 70) is shown, which has one copy of the spacer sequence (SEQ ID NO: 71) complementary to the protospacer of the maize Zm_L70h genomic target site and a stem loop linker (5'-CCAAAAGG-3'; SEQ ID NO: 105). Table 6 lists the corresponding SEQ ID NOs for the DNA and RNA sequences encoding the S. thermophilus sgRNAs that include a spacer sequence complementary to the protospacer of the maize genomic target sites Zm_L70e, Zm_L70f, Zm_L70g, and Zm_L70h.

[0184] Table 6. DNA and RNA sequences of S. thermophilus sgRNAs that include a spacer sequence complementary to the protospacer of the maize genomic target sites Zm_L70e, Zm_L70f, Zm_L70g, and Zm_L70h.

[0185]

[0186] Streptococcus pyogenes Cas9-mediated genome modification assays were performed essentially as described in Example 5. In particular, 320,000 corn protoplasts were transfected with 0.8 pmol of a Streptococcus pyogenes Cas9 (SEQ ID NO: 136) expression construct, and 1.6 pmol of an sgRNA expression construct driven by a 397 bp version of the U6 promoter from maize chromosome 8 (SEQ ID NO: 7) (including a spacer sequence corresponding to the genomic target site) one of: an sgRNA construct for site L70e (SEQ ID NO: 107), an sgRNA construct for site L70f (SEQ ID NO: 108), and an sgRNA construct for site L70g (SEQ ID NO: 109), and 50 pmol of a pre-annealed blunt-ended double-stranded DNA fragment encoded by SEQ ID NO: 115 and SEQ ID NO: 116. To test transformation efficiency, 2.5 ug of a construct encoding green fluorescent protein (GFP) was included. At the time of harvest, an aliquot of the transfected protoplasts was harvested and the transformation efficiency was calculated as the ratio of GFP positive cells to total cells on a PerkinElmer Operetta® imaging system (PerkinElmer, Waltham, MA). The absence of the StCas9 expression cassette during corn protoplast transformation served as a negative control. Forty-eight hours after transfection, protoplasts were harvested and the blunt-ended double-stranded DNA fragment insertion at genomic target site L70e or L70f or L70g was analyzed by quantitative high-throughput PCR using a BioRad QX200® Droplet Digital PCR (ddPCR) system (BioRad, Hercules, CA) and probes. The transformation efficiency was calculated as the ratio of GFP positive cells to total cells on a PerkinElmer Operetta® imaging system (PerkinElmer, Waltham, MA). The absence of the StCas9 expression cassette during corn protoplast transformation served as a negative control. Forty-eight hours after transfection, protoplasts were harvested and the blunt-ended double-stranded DNA fragment insertion at genomic target site L70e or L70f or L70g was analyzed by quantitative high-throughput PCR using a BioRad QX200® Droplet Digital PCR (ddPCR) system (BioRad, Hercules, CA) and probes. TM Droplet Digital TM PCR (ddPCR TM ) system (BioRad, Hercules, CA) and probes. The blunt-ended double-stranded DNA fragment insertion at genomic target site L70e or L70f or L70g was analyzed by quantitative high-throughput PCR. To determine the percent targeted integration, a set of TaqMan primers and probes were used with the ddPCR system to detect template copy number of the junction of the blunt-ended double-stranded DNA fragment at the chromosomal target site. The junction-specific primers and probes for corn chromosomal sites L70e, L70f, L70g, and L70h are shown in Table 7. To normalize the amount of DNA in the aliquot of transfected protoplasts, the ddPCR system was used with a second set of TaqMan primers and probes (primers encoded by SEQ ID NO: 132 and SEQ ID NO: 134; probe encoded by SEQ ID NO: 133) to determine template copy number in the corn genome at a unique site outside of the target site. The percent targeted integration was calculated as the target site-specific template copy number divided by the corn genome-specific template copy number divided by the transformation efficiency (calculated by using the PE Operetta® imaging system (PerkinElmer, Waltham, MA) to count the ratio of GFP positive cells to total cells). Imaging system (PerkinElmer, Waltham, MA) to determine GFP positive cells versus total cell counts). Data points shown in the figure were determined by the average of four biological replicates. Results are shown in Figure 15, which shows that the percent integration for each of sites L70e, L70f, and L70g was higher than the corresponding control.

[0187] PCR amplicons corresponding to the targeting junctions from the protoplast experiments were sequenced to determine integration of the blunt-ended double-stranded DNA fragments for the selected target sites. Figure 15B An alignment showing the expected integration of a blunt-ended double-stranded DNA fragment at the L70f target site (SEQ ID NO: 144) and one example of a target site integration with some sequence loss of the DNA fragment (SEQ ID NO: 145) is shown. While these sequencing results show an indel, the results confirm integration of the DNA fragment to the L70f target site.

[0188] Table 7. SEQ ID NOs for primers and probes to PCR amplify the junction at the maize chromosomal target site with the inserted DNA fragment.

[0189]

[0190] Example 7

[0191] Targeting multiple unique genomic sites by multiplexing sgRNAs

[0192] A key advantage of the CRISPR system compared to other genome engineering platforms is that multiple sgRNAs directed to a single and unique genomic target site can be delivered as individual elements to enable targeting. Alternatively, multiple sgRNAs directed to a single and unique genomic target site can be multiplexed (i.e., stacked) in a single expression vector to enable targeting. One example of an application where multiple targeted endonucleases can be desired includes the removal of marker genes from transgenic events Figure 7A ). The CRISPR system can be used to remove a selectable marker from a transgenic insert, leaving the gene of interest.

[0193] Another example of an application where such a CRISPR / Cas system can be used is where there is a need for multiple targeted endonucleases, such as in the identification of causal genes behind quantitative traits lacking meiotic recombination hindering in QTL regions separating candidate genes from each other. This can be prevented by transformation with multiple CRISPR constructs that target the gene of interest simultaneously. These constructs can either knock out the candidate genes by frameshift mutations or remove them by deletions. Such transformation can also result in random combinations of intact and mutated loci that allow the identification of causal genes Figure 7B ).

[0194] Example 8

[0195] Integration rates vary with blunt-end DNA fragment concentration and time

[0196] The optimal concentration of blunt-end double stranded DNA fragments included in the analysis mixtures was determined to achieve the highest percentage of targeted integration rates using the corn protoplast system essentially as described in Example 5. For these analyses, the expression construct encoding S. pyogenes Cas9 was modified to include the intron at positions 469-657 in the coding region (SEQ ID NO: 119). Additionally, the protein sequence (SEQ ID NO: 118) contains two NLS sequences (SEQ ID NO: 120), one at the amino terminal end (amino acids 2 to 11 of SEQ ID NO: 118) and one at the carboxy terminal end (amino acids 1379 to 1388 of SEQ ID NO: 118).

[0197] For this analysis, 320,000 corn protoplasts were transfected with 0.8 pmol of the S. pyogenes Cas9 (SEQ ID NO: 119) expression construct, and 1.6 pmol of the sgRNA expression construct driven by the 397 bp version of the U6 promoter of maize chromosome 8 (SEQ ID NO: 7) (including the spacer sequence corresponding to the genomic target site): Zm7 (SEQ ID NO: 23), and 1, 5, 10, 25, 50, and 100 pmol of pre-annealed blunt-end double stranded DNA fragments (SEQ ID NO: 115 and SEQ ID NO: 116). For transformation efficiency, 2.5 ug of a construct encoding green fluorescent protein (GFP) was included and the number of GFP positive protoplasts per 320,000 corn protoplasts was determined. The absence of the Cas9 expression cassette during corn protoplast transformation was included as a negative control. Protoplasts were collected at 24 and 48 hours post-transfection and analyzed using the BioRad QX200 TM Droplet Digital TM PCR (ddPCR TM ) system (BioRad, Hercules, CA) and The probe was used to quantify the insertion of blunt-ended double-stranded DNA fragments at the Zm7 genomic target site by quantitative high-throughput PCR analysis. To determine the percentage of targeted integration, a set of Taqman primers (represented by SEQ ID NO: 137 and SEQ ID NO: 143) and a probe (represented by SEQ ID NO: 138) were used with the ddPCR system to detect the template copy number of the junction of the inserted blunt-ended double-stranded DNA fragments at the chromosomal Zm7 target site. To normalize the amount of DNA in the transfected protoplast aliquots, the ddPCR system was used with a second set of Taqman primers and probe (primers encoded by SEQ ID NO: 132 and SEQ ID NO: 134; probe encoded by SEQ ID NO: 133) to determine the template copy number of the corn genome and a unique site outside of the target site. The calculation of the percentage of targeted integration was the target site-specific template copy number divided by the corn genome-specific template copy number divided by the transformation frequency (determined by GFP-positive cell to total cell counting using the PE Operetta imaging system (PerkinElmer, Waltham, MA)). The data points shown in the figure were determined by the average of four biological replicates. The results are shown in Figure 8 Figure 6, which shows that the peak percentage of targeted integration was obtained using 50 pmol of blunt-ended double-stranded DNA fragments and incubating for 48 hours.

[0198] Example 9

[0199] Integration rate as a function of Cas9 endonuclease concentration

[0200] The optimal concentration of the expression construct encoding S. pyogenes Cas9 included in the protoplast transfection mix was established to achieve the highest percentage of targeted integration with the blunt ended double stranded DNA fragment using the maize protoplast system essentially as described in Example 8. For these analyses, the expression construct encoding the modified S. pyogenes Cas9 was as described in Example 8. For this analysis, 0.1 pmol or 0.4 pmol or 0.8 pmol or 1.6 pmol of the S. pyogenes Cas9 (SEQ ID NO: 119) expression construct, and 1.6 pmol of the U6 promoter of maize chromosome 8 397 bp version (SEQ ID NO: 7) (including the spacer sequence corresponding to the genomic target site) driven sgRNA expression construct: Zm7 (SEQ ID NO: 23), 50 pmol of the pre-annealed blunt ended double stranded DNA fragment (SEQ ID NO: 115 and SEQ ID NO: 116), and a construct encoding GFP were used to transfect 320,000 maize protoplasts. The maize protoplasts were collected 48 hours post transfection and the percentage of targeted integration was assessed using the ddPCR system and Taqman probes as described in Example 8. The results of the Cas9 expression construct titration analysis are shown in Figure 9 where the percentage of targeted integration is shown to increase linearly across the full range of expression construct concentrations tested in pmol.

[0201] Example 10

[0202] Sequence confirmation of blunt ended double stranded DNA fragment insertion

[0203] The PCR amplicons corresponding to the targeted junctions of the protoplast experiments detailed in Example 5 and Example 8 were sequenced to confirm the integration of the blunt ended double stranded DNA fragment into the selected target site Zm7 or L70c.

[0204] For the maize chromosome site Zm7 targeted by the CRISPR / Cas9 construct, and the blunt ended double stranded DNA fragment formed by the annealed oligonucleotides of SEQ ID NO: 115 and SEQ ID NO: 116 (see Example 8), the PCR amplicons were purified by agarose gel and sequenced. The expected sequence is shown in SEQ ID NO: 123 and is shown in Figure 10A . The sequencing results show at least one event of the base pairs of the blunt ended double stranded DNA fragment inserted into the target site (SEQ ID NO: 124). The results also show events with short deletions flanking the DNA insert of the chromosome or the junction as shown in SEQ ID NO: 125 (see Figure 10A ).

[0205] For the CRISPR / Cas9 construct targeted maize chromosomal site L70c, and the blunt-ended double-stranded DNA fragment formed by the annealing oligonucleotides encoded by SEQ ID NO:45 and SEQ ID NO:46 (see Example 5) that did not have microhomology sequences, the PCR amplicon was purified by agarose gel and sequenced. The expected sequence is set forth as SEQ ID NO: 126 and is shown in Figure 10B . The sequencing results show at least one event detected at the target site with base pair perfect insertion of the blunt-ended double-stranded DNA fragment (SEQ ID NO: 127). The results also show an example of an event with short deletions flanking the chromosomal or linker DNA insert (SEQ ID NO: 128) (see Figure 10B ).

[0206] For the CRISPR / Cas9 construct targeted maize chromosomal site L70c, and the blunt-ended double-stranded DNA fragment formed by the annealing oligonucleotides encoded by SEQ ID NO:45 and SEQ ID NO:46 (see Example 5) that did not have microhomology sequences, the PCR amplicon was purified by agarose gel and sequenced. The expected sequence is set forth as SEQ ID NO: 126 and is shown in Figure 10C . The sequencing results show at least one event detected at the target site with base pair perfect insertion of the blunt-ended double-stranded DNA fragment (SEQ ID NO: 127). The results also show an example of an event with short deletions flanking the chromosomal or linker DNA insert (SEQ ID NO: 128) (see Figure 10C ).

[0207] These results indicate that the blunt-ended double-stranded DNA fragment was incorporated at the double-strand break (DSB) created at the target site by the CRISPR / Cas9 system. The DNA fragment was incorporated by non-homologous end joining (NHEJ), an error-prone DNA repair mechanism in nature that heals most somatic double-strand breaks. Consistent with the endogenous NHEJ repair mechanism, the results show that the blunt-ended double-stranded DNA fragment was incorporated with short deletions at the DSB created by the CRISPR / Cas9 elements, as shown by the comparison of SEQ ID NO: 123 and SEQ ID NO: 125 ( Figure 10A ), the comparison of SEQ ID NO: 126 and SEQ ID NO: 128 ( Figure 10B ), and the comparison of SEQ ID NO: 129 and SEQ ID NO: 131 ( Figure 10C) shown. The blunt-ended double-stranded DNA fragment is incorporated into the DSB created by the CRISPR / Cas9 elements in a base pair perfect manner as shown by the comparison of SEQ ID NO: 123 and SEQ ID NO: 124 Figure 10A ), and the comparison of SEQ ID NO: 126 and SEQ ID NO: 127 Figure 10B ), and the comparison of SEQ ID NO: 129 and SEQ ID NO: 130 Figure 10C ) (although the last pair has a 2 bp deletion within the inserted DNA fragment).

[0208] Example 11

[0209] Integration rate as a function of TALEN endonuclease concentration

[0210] The optimal concentration of the desired expression constructs encoding a pair of TALEN endonucleases in the transfection mix was established to achieve the highest percentage of targeted integration of the blunt-ended double-stranded DNA fragment using the maize protoplast system essentially as described in Example 8.

[0211] For these analyses, a pair of expression constructs with TALEN encoding cassettes were tested. The targeted site in the maize chromosome for the TALEN pair was L70.4. For the TALEN analysis, maize protoplast transformation was performed using 0, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.4 pmol of each of the constructs containing the TALEN encoding cassettes. Also included was 50 pmol of the pre-annealed blunt-ended double-stranded DNA fragment (SEQ ID NO: 115 and SEQ ID NO: 116) and 2.5 ug of a construct encoding GFP. The maize protoplasts were collected 48 hours post transfection and the percentage of targeted integration was assessed by high throughput PCR analysis essentially as described in the previous examples. The results of the TALEN expression construct titration analysis are shown in Figure 11 , where a smooth curve of the percentage of targeted integration is shown for approximately 0.1 pmol of each TALEN expression construct included in the transfection reaction.

[0212] Example 12

[0213] Targeted integration by homologous recombination - CRISPR / Cas9

[0214] Genomic modifications by targeted integration of desired DNA sequences will occur at the site of a double-strand break (DSB) in the chromosome. Integration of the DNA sequence is mediated by either the non-homologous end joining (NHEJ) mechanism or homologous recombination using the DNA repair mechanisms of the host cell. DSBs at specific sites in the host cell genome can be achieved using endonucleases such as engineered meganucleases, engineered TALENs, or the CRISPR / Cas9 system.

[0215] A schematic of a high-throughput (HTP) assay for NHEJ and HR-mediated targeted integration is shown in FIG. 12. Targeted integration of a DNA fragment by non-homologous end joining (NHEJ) is shown in Figure 12A Targeted integration of a DNA fragment by homologous recombination (HR) is shown in Figure 12B For HR, a recombination DNA construct comprising a cassette of a DNA fragment with flanking left and right homology arms (left HA and right HA, respectively) is introduced into the host cell. Following NHEJ or HR targeted integration, primers (indicated by the pairs of short arrows in Figure 12A and 12B ) are designed for HTP PCR analysis to detect targeted events in which one primer is inside the inserted DNA fragment and the second primer is in the flanking chromosomal region.

[0216] Homologous recombination (HR)-mediated targeted integration rates were determined using the maize protoplast system described in the above Examples. Target site Zm7 was targeted by a CRISPR / Cas9 nuclease and an sgRNA targeting the maize Zm7 site, as described in Example 8. In addition to the construct encoding the CRISPR / Cas9 and sgRNA cassettes, a construct comprising a homologous recombination cassette was included at a concentration of 4 ug or at a concentration of 6 ug. As described above, a construct encoding GFP was also transfected, and the percentage of GFP-positive cells was used to calculate the targeted integration rate. The control did not include a construct encoding the SpCas9 endonuclease.

[0217] The recombination DNA construct comprising a homologous recombination cassette was designed to have a 90 bp sequence corresponding to a 90 bp blunt-ended double-stranded DNA fragment for NHEJ analysis (encoded by sequences SEQ ID NO: 115 and SEQ ID NO: 116) flanked by left and right homology arms (HAs). The left HA was designed according to the flanking sequence 5’- side of the double-strand break (DSB) site for targeted integration. The right HA was designed according to the flanking sequence 3’-side of the double-strand break (DSB) site for targeted integration. For the Zm7 site, the left HA was 240 bp in length and included two separate right HA sequences, one 230 bp in length and the other 1003 bp in length (see Figure 13A and 13B ).

[0218] Protoplasts were transfected and harvested 48 hours later for integration analysis by high throughput PCR, one primer designed to a region of the DNA fragment sequence (encoded by SEQ ID NO: 115 and SEQ ID NO: 116), the other primer in a region of the chromosome flanking the left homology arm. The expected PCR amplicon size for successful introduction of HR with the Zm7 targeting construct Figure 13A and 13B was 411 bp. In quantitative PCR (qPCR), amplicons larger than about 160 bp cannot be quantitatively determined and thus are not recommended for use. The current experiments clearly show that significantly longer PCR amplicons can also be used in the ddPCR system, which opens up many new opportunities for quantitative biology.

[0219] HR-mediated recombination rates for the maize chromosome site Zm7 are shown in Table 8 and Figure 15. There was no statistically significant difference in the percent of integration between test samples and controls when the construct with the homology arm cassette was at a concentration of 4 ug or 6 ug, and the left HA and right HA were 240 bp and 230 bp, respectively. There was no statistically significant difference in the percent of integration between test samples and controls when the construct with the homology arm cassette was at a concentration of 4 ug, and the left HA was 240 bp and the right HA was 1003 bp (denoted as SL in Table 8). In contrast, there was a statistically significant (p < 0.05) difference in the percent of integration between test samples and controls when the construct with the homology arm cassette was at a concentration of 6 ug, and the left HA was 240 bp and the right HA was 1003 bp (denoted as SL in Table 8). This result shows that targeted integration can be achieved by the mechanism of HR at a DSB site that is targeted by a CRISPR / Cas9 system in the maize genome.

[0220] Table 8. HR-mediated integration rates for maize protoplasts with CRISPR / Cas9 system- mediated DSBs at chromosome site Zm7.

[0221]

[0222]

[0223] ** According to Student's t-test, the test is statistically higher (p < 0.05) than the corresponding control.

[0224] Example 13

[0225] Targeted integration by homologous recombination - TALEN

[0226] Homologous recombination (HR) mediated targeted integration rates were determined using the maize protoplast system described in the examples above. The target site L70.4 was targeted by a pair of TALEN-encoding recombination DNA constructs involving the TALENs targeting the maize L70.4 site as described in example 11. In addition to the constructs encoding the TALEN cassettes, constructs comprising homologous recombination cassettes were included at a concentration of 4 ug or at a concentration of 6 ug. As described above, constructs encoding GFP were also transfected and the percentage of GFP positive cells was used to calculate the targeted integration rate. Controls did not include TALEN-encoding constructs.

[0227] The recombination DNA construct comprising the homologous recombination cassette was designed with a 90 bp sequence corresponding to the 90 bp blunt ended double stranded DNA fragment for NHEJ analysis (encoded by sequences SEQ ID NO: 115 and SEQ ID NO: 116) flanked by left and right homology arms (HA). The left HA was designed according to the sequence flanking the 5 '-side of the double stranded break (DSB) site for targeted integration. The right HA was designed according to the sequence flanking the 3 '-side of the double stranded break (DSB) site for targeted integration. For the L70.4 site, the right HA was 230 bp in length and included two separate left HA sequences, one 230 bp in length and the other 1027 bp in length (see Figure 14A and 14B ).

[0228] Protoplasts were transfected and harvested after 48 hours and integration was analyzed by quantitative, high throughput PCR using the ddPCR system and Taqman probes, one primer designed to a region of the DNA fragment sequence (encoded by sequences SEQ ID NO: 115 and SEQ ID NO: 116) and the other primer in a chromosomal region flanking the left homology arm. The expected PCR amplicon size for successful introduction of HR using the L70.4 targeting construct of Figure 14A was 383 bp. The expected PCR amplicon size for successful introduction of HR using the L70.4 targeting construct of Figure 14B was 1208 bp.

[0229] HR-mediated recombination rates for maize chromosomal locus L70.4 with two separate template DNA constructs are shown in Table 9. When both left and right HA were 230 bp (indicated as SS in Table 9), the concentration of the construct with the homology arm cassette was 4 ug, and there was a statistically significant (p < 0.05) difference in the percentage of integration rates between the test sample and the control. When both left and right HA were 230 bp (indicated as SS in Table 9), the concentration of the construct with the homology arm cassette was 6 ug, and there was no statistically significant difference in the percentage of integration rates between the test sample and the control. When the left HA was 1027 bp and the right HA was 230 bp (indicated as LS in Table 9), the concentration of the construct with the homology arm cassette was 4 ug or 6 ug, and there was no statistically significant difference in the percentage of integration rates between the test sample and the control. This result shows that targeted integration can be achieved by the mechanism of HR at the DSB site that is targeted by the TALEN involved in the specific site in the maize genome.

[0230] Table 9. HR-mediated integration rates for maize protoplasts with TALEN-mediated DSB at chromosomal locus L70.4.

[0231]

[0232] ** According to Student's t-test, the test is statistically higher (p < 0.05) than the corresponding control.

[0233] Example 14

[0234] Targeting using chimeric U6 promoters in the maize genome

[0235] Chimeric U6 promoters were identified as being effective in driving expression of sgRNA constructs and resulting in targeted integration of double-stranded blunt-end DNA fragments at preselected sites in maize chromosomes. These experiments were performed using quantitative chromosome cleavage assays in maize protoplast assays as described in Example 5 and Example 6. The U6 promoters incorporated into the sgRNA constructs were: a) a 397 bp maize chromosome 8 U6 promoter encoded by SEQ ID NO: 7, b) a 397 bp chimeric chl :ch8 U6 promoter encoded by SEQ ID NO: 18, b) a 397 bp chimeric ch8:chl U6 promoter encoded by SEQ ID NO: 19, and c) a 397 bp chimeric ch8:ch2:chl :ch8 U6 promoter encoded by SEQ ID NO: 20. The maize chromosome target sites were L70a, L70c, and L70d as described in Example 5. The CRISPR / Cas9 system utilized an expression cassette with a S. pyogenes Cas9 modified to contain two NLS sequences and an intron and encoded by SEQ ID NO: 119. The double-stranded blunt-end DNA fragment was encoded by SEQ ID NO: 115 and SEQ ID NO: 116.

[0236] In one analysis, quantitative analysis was performed using TaqMan probes 48 hours after transfection of maize protoplasts with CRISPR / Cas9 system components. Results (see Figure 16A ) show targeted integration rates for target site L70a, where sgRNA constructs containing the ch8 U6 promoter or sgRNA constructs containing the chimeric chl :ch8 U6 promoter produced about equal percentages of targeted integration rates. Targeted integration rates for target site L70c, where sgRNA constructs containing the chimeric ch8:chl U6 promoter produced about twice the targeted integration rates as sgRNA constructs containing the ch8 U6 promoter. Targeted integration rates for target site L70d, where sgRNA constructs containing the ch8 U6 promoter had higher targeted integration rates than sgRNA constructs containing the chimeric ch8:ch2:chl :ch8 U6 promoter.

[0237] In another analysis, quantitative analysis was performed using SYBR® Green (BioRad, Hercules, CA) intercalating dye 48 hours after transfection of maize protoplasts with CRISPR / Cas9 system components. Results (see (BioRad, Hercules, CA) intercalating dye. Results (see Figure 16B) that the rate of targeted integration for sgRNA constructs containing the ch8 U6 promoter was nearly equivalent to the rate of targeted integration for sgRNA constructs containing the chimeric chl :ch8 U6 promoter at target site L70a, and the rate of targeted integration for sgRNA constructs containing the chimeric ch8:chl U6 promoter at target site L70c, and the rate of targeted integration for sgRNA constructs containing the chimeric ch8:ch2:chl :ch8 U6 promoter at target site L70d. These data indicate that the rate of targeted integration detected by EvaGreen intercalating dye detection is approximately ten-fold higher than the rate of targeted integration detected using MGB TaqMan probes. This bias is primarily due to differences in the chemistry of the assays analyzed. TaqMan assays use only two primers and an internal probe, with one primer and probe located on the sequence of the inserted DNA fragment. Unfortunately, the double-stranded blunt-ended DNA fragments used for transfection are often degraded by endogenous exonucleases in protoplasts, which results in integration of the DNA fragment at truncated sites where the TaqMan probe binds. These truncated integration events are not detected by TaqMan assays. In another aspect, the TaqMan primer binding site located within the sequence of the inserted DNA fragment is located more internally on the inserted DNA fragment, and remains intact even for the shortest inserted DNA fragments. Since the assay using intercalating Evagreen dye does not require an internal probe, only the TaqMan primers, this assay is not affected by oligonucleotide degradation, and thus detects more integrations than the TaqMan assay. In addition, both methods of determining the percentage of targeted integrations show a similar pattern at the three chromosomal target sites and the three different chimeric U6 promoters driving expression of the sgRNAs.

[0238] These results show that the rate of targeted integration at the maize chromosomal site L70c is significantly slightly higher when the sgRNA construct contains the Ch8::Chl chimeric promoter than when the sgRNA construct contains the ch8 U6 promoter ( Figure 16A and 16B ). These results also show that the rate of targeted integration at the maize chromosomal site L70a is approximately equivalent when the sgRNA construct contains the Chl::Ch8 chimeric promoter than when the sgRNA construct contains the ch8 U6 promoter ( Figure 16A and 16B ). Finally, these results show that the rate of targeted integration at the maize chromosomal site L70d is lower when the sgRNA construct contains the ch8:ch2:chl :ch8 chimeric promoter than when the sgRNA construct contains the ch8 U6 promoter ( Figure 16A and 16B). In summary, at least two of the three chimeric promoters are equivalent to or exceed the best non-chimeric promoter in maize. These promoters will be used in multiplex targeting experiments where diversity of expression elements is essential.

[0239] Example 15

[0240] Targeted mutation in tomato invertase inhibitor

[0241] The CRISPR / Cas9 system was used to knock out the plastidic invertase inhibitor gene (INVINHl) in tomato by introducing a targeted frameshift point mutation following an NHEJ- imperfectly repaired targeted double-strand break. Knocking out this gene by RNAi showed increased fructose content and increased seed weight in an earlier study (Jin et al. Plant Cell 21 :2072-2089, 2009). Reducing or eliminating invertase inhibitor activity by targeted mutagenesis or RNA interference is also used to improve yield and / or quality traits in other crop species (Braun et al. J Exp Bot 65: 1713-1735, 2014).

[0242] For these experiments, tomato protoplasts were transfected with an expression construct containing a cassette encoding SpCas9 with one NLS located at the C-terminus (SEQ ID NO: 28) and an expression construct encoding a sgRNA cassette, where expression is driven by one of four separate tomato U6 promoters: promoter 1 encoded by SEQ ID NO: 146 (which is a fragment of SEQ ID NO: 10), promoter 2 encoded by SEQ ID NO: 147 (which is a fragment of SEQ ID NO: 11), promoter 3 encoded by SEQ ID NO: 148 (which is a fragment of SEQ ID NO: 9), or promoter 4 encoded by SEQ ID NO: 149. The sgRNA targets either the invertase inhibitor site without an Smll site (site 1) or a site in the invertase inhibitor gene with an Smll restriction endonuclease site (labeled site 2). The site 2 sgRNA is encoded by SEQ ID NO: 150. The CRISPR / Cas9 cleavage site within target site 2 includes the Smll restriction endonuclease site. Upon CRISPR / Cas9 induction of a double-strand break at target site 2, NHEJ repair will result in an indel at this site, thus effectively removing the Smll restriction endonuclease site. This mutation of the Smll site is exploited during screening of targeted events by amplifying a 380 bp amplicon (SEQ ID NO: 159) and subjecting the PCR amplicon to Smll digestion. If the Smll site is not mutated, the amplicon can be digested into two fragments of 181 bp and 199 bp. If the Smll site is mutated, the PCR amplicon cannot be digested. This PCR protocol is as follows:Figure 17A Figure 6 shows the results of a gel run of PCR amplicons from tomato protoplasts transfected with CRISPR / Cas9 systems targeting the lycomarase inhibitor.

[0243] Tomato protoplasts were transfected with CRISPR / Cas9 systems targeting the lycomarase inhibitor, and genomic DNA was collected and extracted after 48 hours. The negative control for the CRISPR / Cas9 system was the absence of the expression construct encoding the Cas9 endonuclease. The negative control for the target site was the use of the sgRNA targeting site 1, and no mutation of the Smll site for this sgRNA was expected. PCR amplification was performed using primers SEQ ID NO: 157 and SEQ ID NO: 158, and the resulting PCR amplicons were either not digested or digested with Smll. The reactions were run on an agarose gel, and the results are shown in Figure 17B Figure 6. The negative control for the sgRNA targeting site 1 and the absence of the Cas9 endonuclease only produced PCR amplicons that were intact for the Smll site. When the sgRNA targeted site 2, the Smll site was mutated in the case of the sgRNA cassette containing the tomato U6 promoter 1 or the tomato U6 promoter 2 or the tomato U6 promoter 3, as evidenced by the full-length PCR amplicons (see Figure 17B , arrows show the amplicons without the Smll site). The sgRNA construct targeting site 2 and having the U6 promoter 4 did not appear to show targeting.

[0244] To confirm that the PCR amplicons without the Smll site were in fact due to CRISPR / Cas9-induced NHEJ mutations, the gel-purified and pooled amplicons that appeared to be mutated were then sequenced. Figure 17C A multiple sequence alignment of these PCR amplicons without the Smll site shows that these PCR amplicons from the target site 2 of the lycomarase inhibitor contain an indel, consistent with CRISPR / Cas9-induced mutations. In particular, in the multiple sequence alignment, SEQ ID NO: 151 represents the region of the PCR amplicon that is not mutated (SEQ ID NO: 159). SEQ ID NO: 152 and 153 show an indel with a 1 bp insertion at the cleavage site. SEQ ID NO: 154 shows an indel with a 3 bp deletion at the cleavage site. SEQ ID NO: 155 shows an indel with a 4 bp deletion at the cleavage site. SEQ ID NO: 156 shows an indel with a 6 bp deletion at the cleavage site. In sum, these results show that the CRISPR / Cas9 system using the tomato U6 promoter 1 (SEQ ID NO: 146) or the tomato U6 promoter 2 (SEQ ID NO: 147) or the tomato U6 promoter 3 (SEQ ID NO: 148) to drive the sgRNA includes mutations of the lycomarase inhibitor gene target site 2.

[0245] Example 16

[0246] Promoters driving sgRNA expression

[0247] To identify and select additional promoters useful for driving sgRNA expression of expression cassettes introduced into dicot and monocot plants, RNA polymerase II (Pol II) and RNA polymerase III (Pol III) promoters were identified using BLAST comparison of sequences encoding U6, U3, U5, U2, and 7SL small nuclear RNA (snRNA) against soybean and maize genomes (SEQ ID NOs: 160-201 and SEQ ID NOs: 247-283) (see Table 10). Based on this bioinformatics alignment, 200 or more nucleotides upstream of the 5’ end immediately adjacent to the corresponding snRNA coding region were used as putative promoters driving sgRNA expression of expression cassettes introduced into plant cells for testing.

[0248] Table 10. SEQ ID NOs and origin (tomato or soybean or maize) of putative promoter sequences upstream of snRNA genes.

[0249]

[0250]

[0251]

[0252]

[0253] Example 17

[0254] Normalized RNA transcript level analysis

[0255] To assess the efficacy of the promoters listed in Table 10 to drive sgRNA expression, a series of constructs were prepared comprising a cassette encoding one of the putative promoters (SEQ ID NO: 154 and SEQ ID NO: 160-201) operably linked to a 221 bp fragment of the beta-glucuronidase (GUS) open reading frame, as well as the Pol III promoter (7SL, U6, and U3) poly(T)7 terminator or the sequence 5'-ACAATTCAAAACAAGTTTTAT-3' (SEQ ID NO: 237) for the pol II U2 and U5 promoters (Table 10). Recombinant constructs comprising the promoter-GUS fragment fusion (0.5 pmol) were transfected into soybean cotyledon protoplasts (SEQ ID NO: 202-217) or maize leaf protoplasts (SEQ ID NO: 218-236) along with 300 ng of plasmid as a transformation control encoding Renilla luciferase (RLUC) expressed using the CaMV promoter. Transfected protoplasts were harvested 18 hours post-transfection and RNA levels were determined by TaqMan analysis using probes and primers complementary to the GUS fragment. Internal controls for normalization of the TaqMan analysis included (1) an 18S primer pair / probe set as a control for RNA concentration and (2) RLUC fluorescence as a transformation control.

[0256] In soybean cotyledon protoplasts, all of the tested promoters produced normalized GUS mRNA levels significantly higher than the control (no GUS construct) (one-way ANOVA Student's t-test, p-value < 0.05) Figure 18A ). The lowest normalized GUS mRNA level was using the construct (SEQ ID NO: 210) comprising the U3a promoter (SEQ ID NO: 167). The highest normalized GUS mRNA level was using the construct (SEQ ID NO: 210) comprising the 7SL CR10 promoter (SEQ ID NO: 174). The normalized GUS mRNA levels for all of the tested promoters with this analysis ranged from 11-31 fold higher than the expression level of the no DNA negative control. None of the performance of any class of promoters (U6, U3, or 7SL) exceeded the other, but the U3 promoters generally fell within the lower range of expression observed in the experiment. Liang et al. (J. Genetics and Genomics 41 :63-68, 2014) successfully used a U3 promoter to drive sgRNAs in maize. Thus, while these data indicate that U3 promoters are lower than U6 or 7SL, they are still candidates that can be used to drive sgRNA expression in soybean. These data suggest that any of the U6, U3, or 7SL promoters identified here would be a good candidate for a recombinant expression construct to drive sgRNA expression in plant cells.

[0257] In maize leaf protoplasts, normalized GUS mRNA levels produced by all tested promoters were significantly higher than the control (one-way ANOVA Student's t-test, p-value < 0.05), with expression level values ranging from 26-fold to 141-fold higher than the negative control Figure 18B ). The U6Chr08 promoter construct (SEQ ID NO: 235) produced the highest normalized GUS mRNA expression level, and the U2snRNA_I promoter construct (SEQ ID NO: 227) produced the lowest normalized GUS mRNA expression level, with a difference of about 5.5-fold between them. The U2snRNA_P promoter construct (SEQ ID NO: 226) also prominently had a high normalized GUS mRNA expression level. All the remaining promoters were within the same relative range, with less than 2-fold difference between them Figure 18B ). These data suggest that any of the U6, U3, 7S1, U2, or U5 promoters identified here would be a good candidate for enabling a recombinant expression construct to drive sgRNA expression in plant cells.

[0258] Example 18

[0259] GUS expression analysis of sgRNA expression

[0260] To determine how differences in sgRNA expression levels affect Cas9 activity, a transcriptional activation assay was used that relies on a minimal promoter upstream of a GUS open reading frame in a reporter gene construct (transfected into maize leaf protoplasts). For this assay, the S. pyogenes Cas9 nuclease was mutated at amino acid positions D9A and H599A of the native protein sequence, effectively creating a Cas9 with no endonuclease cleavage activity (also referred to as 'dead Cas9'). In addition, this dead Cas9 was modified to encode a NLS domain at amino acid positions 2-11 of SEQ ID NO: 239 (SEQ ID NO: 120) and an activation domain of a TALE protein at amino acid positions 1135-1471 of SEQ ID NO: 239. The polynucleotide sequence of the dead Cas9 represented by SEQ ID NO: 238 includes an intron at positions 507-695. The reporter gene construct was constructed as follows: where the uidA (GUS) reporter gene is driven by the minimal CaMV promoter with three adjacent sgRNA binding sites (SEQ ID NO: 240) at nucleotide positions 80-98, 117-135, and 154-172 of the sequence of SEQ ID NO: 246. A set of sgRNA (based on the sgRNAs of Cong et al. 2013 Science 339: 819) expression constructs was also constructed, consisting of one promoter per class of snRNA gene, i.e., U6, 7SL, U2, U5, and U3 (Table 11), and which can target the dead Cas9-TALE-AD to one or more of the sgRNA binding sites of the GUS reporter gene construct. The U6 and 7SL promoters normally prime transcription of G, and the U2, U5, and U3 promoters normally prime transcription of A. To ensure proper transcription initiation of the sgRNAs, a G was inserted between the promoter and the spacer sequence for constructs with the U6 or 7SL promoter. For constructs with the U2, U5, or U3 promoter, an A was inserted between the promoter and the spacer sequence. When the dead Cas9-TALE-AD and sgRNA complex binds to the GUS reporter gene construct, the TALE activation domain functions as a transcriptional activator of the minimal CaMV promoter, increasing expression of the GUS transcript, and ultimately increasing the level of GUS protein expression.

[0261] Table 11. Corresponding SEQ ID NOs for sgRNA expression constructs.

[0262]

[0263]

[0264] For this analysis, corn leaf protoplasts were transfected with 0.8 pmol of dead Cas9-TALE-AD expression cassette, 0.5 pmol of GUS expression cassette, 1.6 pmol of one of the sgRNA expression cassettes, 650 ng of luciferase expression cassette, and 300 ng of Renilla luciferase (RLUC) expression cassette. Transfected protoplasts were collected after 18 hours, GUS activity was determined using the 4-methylumbelliferyl-beta-D-glucuronide (MUG, Sigma, St. Louis, MO) fluorometric assay, and luciferase and RLUC activities were determined as controls, normalized to transfection controls. GUS activity is a readout of the frequency of dead Cas9-TALE-AD binding to the reporter plasmid. Each class of snRNA promoter driving the sgRNA gave higher normalized GUS activity compared to controls ( Figure 19 ). The U3 CR08b ( Figure 19 ) promoter in U3_8B) gave the highest normalized GUS activity, about 10X that of controls. Two promoters, 7SL CR07 and U6 Chr08, gave about the same normalized GUS activity, about 4X that of controls. Two promoters, U2 snRNA I ( Figure 19 ) in Us_I) and U5 snRNA E ( Figure 19 ) in U5_e), each gave normalized GUS activity of or slightly more than 2X that of controls. These results indicate that the 7SL, U6, U3, U2, and U5 snRNA promoters are good to excellent candidates for sgRNA expression constructs for the CRISPR / Cas9 system for genome modification.

[0265] The observed differences in normalized GUS expression using the dead Cas9-TALE-AD assay do not reflect the normalized GUS mRNA levels shown in the corn leaf protoplast assay, as detailed in Example 17.

Claims

1. A recombinant DNA construct comprising a U6 promoter that produces a higher level of expression relative to a control lacking the U6 promoter; the promoter is operably linked to a sequence encoding a single guide RNA (sgRNA), wherein the sequence of the U6 promoter is set forth in SEQ ID NO: 7 or SEQ ID NO:

8.

2. The recombinant DNA construct of claim 1, wherein the sequence of the U6 promoter is set forth in SEQ ID NO:

7.

3. The recombinant DNA construct of claim 1, further comprising a transcriptional termination sequence.

4. The recombinant DNA construct of claim 1, further comprising a sequence encoding a promoter that is operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product.

5. The recombinant DNA construct of claim 4, wherein the Cas endonuclease gene product is further operably linked to a nuclear localization sequence (NLS).

6. The recombinant DNA construct of claim 4, wherein the sequence encoding the Cas endonuclease is selected from the group consisting of: SEQ ID NO: 27, SEQ ID NO: 68, and SEQ ID NO: 97, SEQ ID NO: 119, and SEQ ID NO:

136.

7. A method of introducing a double-strand break into the genome of a cell, the method comprising introducing into the cell: a) at least one recombinant DNA construct of claim 1; and b) a second recombinant DNA construct comprising a sequence encoding a promoter that is operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product that is operably linked to a nuclear localization sequence (NLS).

8. The method of claim 7, wherein the sequence of the U6 promoter is set forth in SEQ ID NO:

7.

9. The method of claim 7, wherein the sequence encoding the Cas endonuclease is selected from the group consisting of: SEQ ID NO: 27, SEQ ID NO: 68, and SEQ ID NO: 97, SEQ ID NO: 119, and SEQ ID NO:

136.

10. A method of introducing a double-strand break into the genome of a cell, the method comprising introducing into the cell at least one recombinant DNA construct of claim 4.

11. The method of claim 10, wherein the sequence of the U6 promoter is set forth in SEQ ID NO:

7.

12. The method of claim 10, wherein the sequence encoding the Cas endonuclease is selected from the group consisting of: SEQ ID NO: 27, SEQ ID NO: 68, and SEQ ID NO: 97, SEQ ID NO: 119, and SEQ ID NO:

136.

13. A method of genome modification, the method comprising: a) introducing a double-strand break into a selection site in the genome of the plant cell, and b) introducing a recombinant blunt-ended double-stranded DNA fragment into the plant cell, wherein the recombinant blunt-ended double-stranded DNA fragment is incorporated into the double-strand break by endogenous DNA repair, and wherein in step a) the cell is provided with a recombinant DNA construct encoding a promoter operably linked to a sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Cas endonuclease gene product; and a recombinant DNA construct comprising a U6 promoter according to any one of claims 1-3 operably linked to a sequence encoding a single guide RNA (sgRNA) designed to target a selection target site in the chromosome of the cell to introduce a double-strand break.

14. The method of claim 13, wherein the genome modification comprises modifying a linkage drag, joining two or more QTLs, disrupting linkage of two or more QTLs, gene insertion, gene replacement, gene conversion, gene deletion or disruption, transgenic event selection, or transgenic trait donor selection.

15. The method of claim 13, wherein the genome modification comprises transgenic replacement or targeted insertion of at least one nucleic acid of interest.

16. The method of claim 13, wherein the double-strand break is introduced by an endonuclease.

17. The method of claim 13, wherein the plant cell is a protoplast or is grown in a plant cell culture.

18. The method of claim 13, wherein the plant cell is selected from the group consisting of: a soybean plant cell; a maize plant cell; a rice plant cell; a wheat plant cell; a turfgrass plant cell; a cotton plant cell; and a canola plant cell.

19. The method of claim 13, wherein the recombinant blunt-ended double-stranded DNA fragment does not comprise a region homologous to the selection site in the genome.

20. The method of claim 13, wherein 0.03 to 0.3 fmol of the recombinant blunt-ended double-stranded DNA fragment is introduced into the plant cell.

21. The method of claim 20, wherein 0.15 fmol of the recombinant blunt-ended double-stranded DNA fragment is introduced into the plant cell.

22. The method of claim 13, wherein the blunt-ended double-stranded DNA fragment comprises a region of microhomology to a sequence comprising one or both ends of the double-strand break in the genome at the 5' end or the 3' end or both the 5' and 3' ends.

23. The method of claim 22, wherein the micro-homology region is selected from the group consisting of: a sequence 1 bp, 2 bp, 3 bp, 4, bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, or 10 bp in length.

24. The method of claim 23, wherein the micro-homology region is 3 bp in length.

25. The method of claim 13, wherein the double-strand break is introduced in step a) by providing the cell with an endonuclease designed to target a selected target site in the genome of the cell.

26. The method of claim 13, wherein the Cas endonuclease gene product is further operably linked to at least one nuclear localization sequence (NLS).

27. The method of claim 13, wherein the sequence of the U6 promoter is set forth in SEQ ID NO: 7 or SEQ ID NO:

8.

28. The method of claim 27, wherein the sequence of the U6 promoter is set forth in SEQ ID NO:

7.

29. The method of claim 13, wherein the recombinant DNA construct encodes a promoter operably linked to a sequence encoding a clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas endonuclease gene product; and the recombinant DNA construct comprises a U6 promoter operably linked to a sequence encoding a single guide RNA (sgRNA) designed to target a selected target site in the chromosome of the cell on the same construct.

30. The method of claim 13, wherein the recombinant DNA construct encodes a promoter operably linked to a sequence encoding a clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas endonuclease gene product; and the recombinant DNA construct comprises a U6 promoter operably linked to a sequence encoding a single guide RNA (sgRNA) designed to target a selected target site in the chromosome of the cell on at least two constructs.

31. A method of genome modification, the method comprising: a) introducing a double-strand break into the genome of the plant cell by the method of claim 8; and b) introducing a recombinant blunt-ended double-stranded DNA fragment into the plant cell, wherein the recombinant blunt-ended double-stranded DNA fragment is incorporated into the double-strand break by endogenous DNA repair.

32. A method of genome modification, the method comprising: a) introducing a double-strand break into the genome of the plant cell by the method of claim 10; and b) introducing a recombinant blunt-ended double-stranded DNA fragment into the plant cell, wherein the recombinant blunt-ended double-stranded DNA fragment is incorporated into the double-strand break by endogenous DNA repair.

33. A recombinant DNA construct comprising at least a first expression cassette comprising a U6 promoter operably linked to a sequence encoding a first single guide RNA (first sgRNA), wherein the sequence of the promoter is set forth in SEQ ID NO: 7 or SEQ ID NO:

8.

34. The recombinant DNA construct of claim 33, further comprising at least a second expression cassette, wherein the sequence encoding the first sgRNA is different from the sequence encoding a second sgRNA.

35. The recombinant DNA construct of claim 34, wherein the promoter operably linked to the sequence encoding the first sgRNA is different from the promoter operably linked to the sequence encoding the second sgRNA.

36. The construct of claim 33, comprising flanking left and right homology arms (HAs), each of the homology arms being 200-1200 bp in length.

37. The construct of claim 36, wherein the homology arms are 230 to 1003 bp in length.

Images