Compositions and methods for high yield lettuce

Abstract

The present disclosure is directed to lettuce plants comprising non-naturally occurring mutations in the Gibberellin 2-Beta Dioxygenase 1 (GA2ox1) or Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) genes and having enhanced biomass. The GA2ox1 mutations maintain a similar flowering time compared to wild-type plants, while GA2ox4 mutations lead to a faster flowering time compared to wild-type plants. The lettuce plants may also comprise mutations in two or more Polyphenol Oxidase (PPO) genes. The present disclosure is also directed to methods for making and breeding such lettuce plants.

Description

COMPOSITIONS AND METHODS FOR HIGH YIELD LETTUCE

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 752,302, filed January 31, 2025, which is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to high yield lettuce varieties with increased biomass due to non-naturally occurring mutations in lettuce Gibberellin 2-Beta Dioxygenase genes, as well as methods for making and breeding the same.SEQUENCE LISTING STATEMENT

[0003] This application contains a computer readable Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file was created on January 7, 2026, is named 257432_001141. xml and is 163,368 bytes in size.BACKGROUND

[0004] The lettuce industry is a significant sector within the global agricultural market, driven by the high demand for fresh, nutritious, and versatile leafy greens. Lettuce (Lactiica saliva) is a staple in various culinary applications, ranging from salads to garnishes, and is valued for its crisp texture and mild flavor. However, conventional lettuce varieties often present limitations in terms of biomass production, which directly impacts yield and profitability for growers.

[0005] One of the primary challenges faced by the lettuce industry is the need for new varieties of lettuce plants that exhibit greater fresh weight biomass compared to conventional varieties. Increased biomass not only enhances the marketable yield but also improves the efficiency of resource use, including water, nutrients, and space. This is particularly crucial for field-grown lettuce, where maximizing biomass without extending the flowering time is essential to maintain the crop's lifecycle and ensure timely harvests.

[0006] Moreover, there is a growing demand for lettuce varieties that are optimized for hydroponic systems. Hydroponically grown lettuce offers numerous advantages, such as reduced water usage, faster growth cycles, and the ability to cultivate crops in controlled environments. However, conventional lettuce varieties often do not perform optimally under hydroponic conditions. There is a need for hydroponically grown lettuce plants that can produce biomassmore rapidly and flower earlier than traditional varieties, thereby enabling more efficient and frequent harvests.

[0007] The development of new lettuce varieties that address these specific needs — greater fresh weight biomass for field-grown lettuce with similar flowering times to conventional varieties, and faster biomass production and earlier flowering for hydroponically grown lettuce — would represent a significant advancement in the lettuce industry. These innovations would not only enhance yield but also contribute to more sustainable and efficient agricultural practices.

[0008] This disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY

[0009] One aspect of the present disclosure is directed to a lettuce plant comprising a non-naturally occurring mutation in a lettuce Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene, where the lettuce plant has a similar flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

[0010] Another aspect of the present disclosure is directed to a method of making a normal flowering lettuce plant with increased biomass. This method comprises introducing a loss-of-function mutation into a Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene of a lettuce plant cell and regenerating a lettuce plant from the lettuce plant cell where the lettuce plant has a similar flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation. This method may also comprise transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNA targeting the GA2oxl gene and a genome editing nuclease and editing the plant cell’s genome to induce the loss-of-function mutation in the GA2oxl gene. This method may further involve introducing loss-of-function mutations into two or more Polyphenol Oxidase (PPO) genes.

[0011] A further aspect of the present disclosure is directed to a method of breeding a normal flowering lettuce plant with increased biomass. This method involves providing a candidate plant with a non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 1 (GA2oxB) gene, breeding the candidate plant with at least one other lettuce plant, and selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene.

[0012] Another aspect of the present disclosure is directed to a lettuce plant comprising a non-naturally occurring mutation in a lettuce Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene,where the lettuce plant has a faster flowering time, an increased average leaf size, increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

[0013] A further aspect of the present disclosure is directed to a method of making a rapid flowering lettuce plant with increased biomass. This method involves introducing a loss-of-function mutation into a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene of a lettuce plant cell and regenerating a lettuce plant from the lettuce plant cell, where the lettuce plant has a faster flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation. This method may also involve transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNA targeting the GA2ox4 gene and a genome editing nuclease and editing the plant cell’s genome to induce the loss-of-function mutation in the GA2ox4 gene. This method may further involve introducing loss-of-function mutations into two or more Polyphenol Oxidase (PPO) genes.

[0014] Yet another aspect of the present disclosure is directed to a method of breeding a faster flowering lettuce plant with increased biomass. This method involves providing a candidate plant with a non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene, breeding the candidate plant with at least one other lettuce plant, and selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene.

[0015] As disclosed herein, numerous GA2ox genes were identified in lettuce that were found to be expressed differently in different tissues. Using the genetic approaches described in the present disclosure, lettuce plant cells and lettuce plants were produced with loss-of-function mutations in GA2oxl and GA2ox4 genes. It was surprisingly shown that loss-of-function mutations in certain GA2oxl resulted in lettuce plants with increased biomass and similar flowering time as wild type plants, whereas loss-of-function mutations in GA2ox4 resulted in lettuce plants with increased biomass and earlier flowering time compared to wild type plants.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGs. 1 A-B are photographs of aeroponically grown genome edited lettuce seedlings. FIG. 1A is a photograph of GA2ox6 edited lettuce plant events 53-15 and 53-16. FIG. IB is a photograph of 24-day old GA2ox4 edited lettuce plant events 52-06-01 and 52-06-16 compared to wild type lettuce plants.

[0017] FIG. 2 is a photograph of 24-day old GA2ox4 edited aeroponically grown lettuce R1 event plants 52-06-01 (homozygous three base pair deletion (D3; SEQ ID NO:84)) and 52-06-16 (homozygous eight base pair deletion (D8; SEQ ID NO:85)) compared to wild type lettuce plants grown at the same time.

[0018] FIG. 3 is a photograph showing a comparison of bolting phenotypes of 10-week-old lettuce plants grown in soil in the greenhouse. Plants from left to right include: GA2ox6 R1 event plant 53-10-12 (homozygous seven base pair deletion (D7; SEQ ID NO:86)), GA2oxl R1 event plant 54-09-08 (homozygous three base pair deletion (D3; SEQ ID NO:77)), a wild-type lettuce plant, and GA2oxl R1 event plant 54-09-05 (homozygous five base pair deletion (D5-2; SEQ ID NO:83)).

[0019] FIG. 4 is a photograph showing a comparison of hydroponically grown GA2oxl R1 event lettuce plants 54-29-05 (homozygous five base pair deletion (D5-1; SEQ ID NO:78)) showing larger leaf size and biomass in comparison to control lettuce plants with no genome editing.

[0020] FIG. 5 is a photograph showing a comparison of individual lettuce plants grown hydroponically: (left) GA2oxl R1 event plant 54-29-05 showing larger leaf size and biomass, in comparison to (middle) a control lettuce plant that had gone through transformation and regeneration with no genome editing, and (right) a wild-type plant.

[0021] FIG. 6 is a graph showing the fresh weight of two-week-old GA2oxl and GA2ox4 R3 lettuce plants grown aeroponically. From left to right are fresh weights of GA2oxl Event 54.29.5.31 (homozygous five base pair deletion (D5-1; SEQ ID NO:78)) lettuce plants, GA2oxl Event 54.1.5.19 (wild type) lettuce plants, GA2ox4 Event 52.06.18.23 (homozygous eight base pair deletion (D8; SEQ ID NO:85)) lettuce plants, GA2ox6 Event 53.10.5.6 (homozygous seven base pair deletion (D7; SEQ ID NO:87)) lettuce plants, and wild-type plants (GF WT-5; parental variety Green Forest).

[0022] FIG. 7 is a graph showing the fresh weight of three-week-old GA2oxl, GA2ox4 and GA2ox6 R3 lettuce plants grown aeroponically. From left to right are fresh weights of GA2oxl Event 54.29.5.31 lettuce plants, GA2oxl Event 54.1.5.19 (wild type) lettuce plants, GA2ox4 Event 52.06.18.23 lettuce plants, GA2ox6 Event 53.10.5.6 lettuce plants, and wild-type plants (parental variety Green Forest).

[0023] FIG. 8 is a graph showing the fresh weight of four-week-old GA2oxl, GA2ox4 and GA2ox6 R3 lettuce plants grown aeroponically. From left to right are fresh weights of GA2oxl Event 54.29.5.31 lettuce plants, GA2oxl Event 54.1.5.19 (wild type) lettuce plants,GA2ox4 Event 52.06.18.23 lettuce plants, GA2ox6 Event 53.10.5.6 lettuce plants, and wild-type plants (parental variety Green Forest).

[0024] FIG. 9 is a graph showing a comparison of fresh weight of six, eight, nine, eleven and twelve- week-old GA2oxl, GA2ox4 and GA2ox6 R3 lettuce plants grown aeroponically. From left to right are fresh weights of GA2oxl Event 54.29.5.31 lettuce plants, GA2oxl Event 54.1.5.19 (wild type) lettuce plants, GA2ox4 Event 52.06.18.23 lettuce plants, GA2ox6 Event 53.10.5.6 lettuce plants, and wild-type plants (parental variety Green Forest).

[0025] FIG. 10 is a photograph of GA2oxl Event 54.29.5 (homozygous for the D5-1 mutation (SEQ ID NO:78)) lettuce heads cut in half after harvest in field grown plants. Leaf size and head height are both increased for the GA2oxl for edited plants. Additional field data and observations are described in Table 9 (Example 3).

[0026] FIGs. 11 A-H show part of the wild-type nucleotide sequence for GA2oxl (SEQ ID NO:1) aligned with the nucleotide sequence of genome edited mutations of GA2oxl. FIG.11 A shows an alignment of a D4 mutation (SEQ ID NO:76) having a four base pair deletion of nucleotides 291-294 of SEQ ID NO: 1. FIG. 1 IB shows an alignment of a D3 mutation (SEQ ID NO:77) having a three base pair deletion of nucleotides 290-292 of SEQ ID NO:1. FIG. 11C shows an alignment of a D5-1 mutation (SEQ ID NO:78) having a five base pair deletion of nucleotides 290-294 of SEQ ID NO: 1. FIG. 1 ID shows an alignment of a DI 1 mutation (SEQ ID NO:79) having an eleven base pair deletion of nucleotides 290-300 of SEQ ID NO:1. FIG.1 IE shows an alignment of a D12-1 mutation (SEQ ID NO:80) having a twelve base pair deletion of nucleotides 288-299 of SEQ ID NO: 1. FIG. 1 IF shows an alignment of a D12-2 mutation (SEQ ID NO:81) having a twelve base pair deletion of nucleotides 290-301 of SEQ ID NO: 1. FIG. 11G shows an alignment of a D 14 mutation (SEQ ID NO: 82) having a fourteen base pair deletion of nucleotides 291-304 of SEQ ID NO: 1. AND FIG. 11H shows an alignment of a D5-2 mutation (SEQ ID NO:83) having a five base pair deletion of nucleotides 294-298 of SEQ ID NO:1.

[0027] FIGs. 12A-B show sequence alignments of wild-type nucleotide sequence for GA2ox4 (SEQ ID NO: 19) and genome edited mutations of GA2ox4. FIG. 12A shows an alignment of a D3 mutation (SEQ ID NO: 84) having a three base pair deletion of nucleotides 207-209 of SEQ ID NO: 19. And FIG. 12B shows an alignment of a D8 mutation (SEQ ID NO:85) having an eight-base pair deletion of nucleotides 200-207 of SEQ ID NO: 19.

[0028] FIGs. 13A-C show sequence alignments of wild-type nucleotide sequence for GA2ox6 (SEQ ID NO:21) and genome edited mutations of GA2ox6. FIG. 13A shows an alignment of a D7 mutation (SEQ ID NO: 86) having a seven base pair deletion of nucleotides518-524 of SEQ ID NO:21. FIG. 13B shows an alignment of a D6 mutation (SEQ ID NO:87) having a six base pair deletion of nucleotides 516-521 of SEQ ID NO:21. And FIG. 13C shows an alignment of a D10 mutation (SEQ ID NO:88) having a ten base pair deletion of nucleotides 518-527 of SEQ ID NO:21.

[0029] FIGs. 14A-B are illustrations showing the positions of the non-haem dioxygenase domain and the 2OG-Fe(II) oxygenase domain in LsGA2oxl (FIG. 14A; SEQ ID NO:1) and LsGA2ox4 (FIG. 14B; SEQ ID NO: 19).DETAILED DESCRIPTION

[0030] The present disclosure is directed to cells of lettuce plants, lettuce plants, lettuce seeds, lettuce leaves, lettuce heads, and lettuce plant parts having one or more non-naturally occurring Gibberellin 2-Beta Dioxygenase (“GA2ox”) gene mutations. The one or more GA2ox gene mutations may impart desirable phenotypes, such as increased yield, increased lettuce head size, increased leaf size, and increased lettuce leaf biomass. In some embodiments, the Gibberellin 2-Beta Dioxygenase (^GA2ox’ ) gene mutations are in Gibberellin 2-Beta Dioxygenase 1 (“GA2oxl”). Mutations in GA2oxlmay increase yield and biomass while maintaining a similar flowering time in lettuce plants comprising the mutation compared to wildtype lettuce plants without the mutation. In some embodiments, the Gibberellin 2-Beta Dioxygenase mutations are in Gibberellin 2-Beta Dioxygenase 4 C(jA2ox4" . Mutations in GA2ox4 may impart the desirable phenotypes of increasing yield and biomass while imparting a more rapid flowering time in lettuce plants comprising the mutation compared to wild-type lettuce plants without the mutation. Methods of obtaining lettuce plants, cells, and plant parts having non-naturally occurring mutations in GA2oxl and GA2ox4 genes are disclosed herein. Also disclosed are lettuce plants, cells, and plant parts having mutations in GA2oxl or GA2ox4 genes in combination with mutations in two or more Polyphenol Oxidase (PPO) gene(s) and methods for making same.

[0031] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person of ordinary skill in the art.

[0032] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to aperson of ordinary skill in the art upon reading this disclosure. In another example, reference to “a cell” includes both a single cell and a plurality of cells.

[0033] The term “about” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, such as within 10% or within 5% of a given value or range.

[0034] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0035] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives.

[0036] The terms “nucleic acid”, “nucleotide”, or “polynucleotide” sequence are used interchangeably, and refer to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acids include polyribonucleic acid (“RNA”) and polydeoxyribonucleic acid (“DNA”), both of which may be single-stranded or double-stranded. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi -synthetic DNA. DNA may be linear, circular, or supercoiled.

[0037] As used herein, a “gene” refers to an assembly of nucleotides that typically encodes a polypeptide or protein and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific functional RNA, protein, or polypeptide, optionally including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and / or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and / or regulatory sequences derived from different sources. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. “Heterologous” or “exogenous” DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Heterologous genes can comprise nativegenes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation or transfection procedure.

[0038] Transfection” and “transfected” are used inter-changeably herein for any kind of introduction of a material, including a nucleic acid (DNA / RNA), ribonucleoprotein (“RNP”) complex, amino acid, chemical, metabolite, nanoparticle, microparticle, combinations, and the like into at least one cell of interest by any kind of physical (e.g., bombardment) or chemical way of introducing the relevant at least one material.

[0039] A “reference sequence” means a nucleic acid or amino acid used as a comparator for another nucleic acid or amino acid, respectively, when determining sequence identity. A reference sequence can be a wild-type sequence.

[0040] “Sequence identity,” “percent identity,” or “% identical” refers to the exactness of a match between a reference sequence and a sequence being compared to it when optimally aligned. For example, sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Multalin program (Corpet, “Multiple Sequence Alignment with Hierarchical Clustering,” Nucleic Acids Res. 16:10881-90 (1988), which is hereby incorporated by reference in its entirety) or the Megalign® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Sequences may also be aligned using algorithms known in the art including, but not limited to, CLUSTAL V algorithm or the BLASTN or BLAST 2 sequence programs.Gibberellin 2-Beta Dioxygenase Genes

[0041] Gibberellin 2-Beta Dioxygenases are involved in the biosynthesis and deactivation of gibberellins by catalyzing the hydroxylation of gibberellins at the 2-beta position, leading to the formation of inactive gibberellin forms. This process helps regulate the levels of active gibberellins within the plant, thus controlling growth processes. Gibberellins are essential plant hormones that influence numerous aspects of growth, including seed germination, stem elongation, leaf expansion, and flowering.

[0042] One aspect of the present disclosure is directed to a lettuce plant comprising a non-naturally occurring mutation in a lettuce Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene, where the lettuce plant has a similar flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

[0043] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0044] In some embodiments, the present disclosure relates to modifying a plant or a plant cell to reduce or eliminate the expression and / or activity of a Lactuca sativa Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene or GA2oxl protein.

[0045] In some embodiments, the nucleotide coding sequence for a lettuce (Lactuca sativa) GA2oxl gene is LOCI 11914174 (SEQ ID NO:1; Nucleotides 160..1173 of GenBank Accession No. XM_023909934.3, which is hereby incorporated by reference in its entirety) and is set forth in Table 12 infra.

[0046] In some embodiments, the GA2oxl gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO: 1. In some embodiments the GA2oxl gene comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1.

[0047] In some embodiments, the lettuce GA2oxl gene encodes the amino acid sequence of SEQ ID NO:2, or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:2. The amino acid sequence for the GA2oxl protein of LOCI 11914174 (SEQ ID NO:2; GenBank Accession No. XP_023765702.1, which is hereby incorporated by reference in its entirety) is set in Table 12 infra.

[0048] In some embodiments, the GA2oxl protein comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the GA2oxl protein comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the amino acid sequence of GA2oxl of SEQ ID NO:2. In some embodiments the GA2oxl protein comprises an amino acid sequence of SEQ ID NO:2 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0049] Another aspect of the present disclosure is directed to a lettuce plant comprising a non-naturally occurring mutation in a lettuce Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene, where the lettuce plant has a faster flowering time, an increased average leaf size, increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

[0050] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0051] In some embodiments, the present disclosure relates to modifying a plant or a plant cell to reduce or eliminate the expression and / or activity of a Lactuca sativa Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene or GA2ox4 protein.

[0052] In some embodiments, the nucleotide coding sequence for a lettuce (Lactuca sativa) GA2ox4 gene is LOC111890054 (SEQ ID NO:19; Nucleotides 1..1337 of GenBank Accession No. XM_023886216.3, which is hereby incorporated by reference in its entirety) and is set forth in Table 12 infra.

[0053] In some embodiments, the GA2ox4 gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO: 19. In some embodiments the GA2ox4 gene comprises the nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID N0:19.

[0054] In some embodiments, the lettuce GA2ox4 gene encodes the amino acid sequence of SEQ ID NO:20, or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:20. The amino acid sequence for the GA2ox4 protein of LOCI 11890054 (SEQ ID NO:20; GenBank Accession No. XP_023741984.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 infra.

[0055] In some embodiments, the GA2ox4 protein comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, the GA2ox4 protein comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the amino acid sequence of GA2ox4 of SEQ ID NO:20. In some embodiments the GA2ox4 protein comprises an amino acid sequence of SEQ ID NO:20, or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0056] In some embodiments, the nucleotide coding sequence for a lettuce (Lactuca sativa) GA2ox6 gene is LOCI 11882624 (SEQ ID NO:21; Nucleotides 134..1147 of GenBank Accession No. XM_023879001.3, which is hereby incorporated by reference in its entirety) and is set forth in Table 12 infra.

[0057] In some embodiments, the GA2ox6 gene comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO:21. In some embodiments the GA2ox6 gene comprises the nucleotide sequence of SEQ ID NO:21, or anucleotide sequence that has at least 95% sequence identity to the nucleotide sequence of SEQ ID N0:21.

[0058] In some embodiments, the lettuce GA2ox6 gene encodes the amino acid sequence of SEQ ID NO:22, or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:22. The amino acid sequence for the GA2ox6 protein of LOCI 11882624 (SEQ ID NO:22; GenBank Accession No. XP_023734769.1, which is hereby incorporated by reference in its entirety) is set in Table 12 infra.

[0059] In some embodiments, the GA2ox6 protein comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, the GA2ox6 protein comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the amino acid sequence of GA2ox6 of SEQ ID NO:22. In some embodiments the GA2ox6 protein comprises an amino acid sequence of SEQ ID NO:22 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0060] As described herein, one or more non-naturally occurring mutations are introduced into one or more of a Lactuca sativa GA2ox gene. In some embodiments, the GA 2 ox gene is GA2oxl. In some embodiments, the GA2ox gene is GA2ox4. In some embodiments, the GA2ox gene is GA2ox6. In some embodiments, the one or more non-naturally occurring mutations in the GA2ox gene or genes are loss-of-function mutations.

[0061] The term “non-naturally occurring mutation” means a human-induced change in the genetic sequence compared to a wild type sequence. The mutation may be, without limitation, from one or more nucleotide insertions, one or more nucleotide substitutions, one or more nucleotide deletions, or any combination thereof. In some embodiments, the non-naturally occurring mutation is a genome edited mutation.

[0062] In some embodiments, the non-naturally occurring mutation is a loss-of-function mutation. As used herein, the phrase “loss-of-function mutation” refers to a mutation that results in a gene or gene product no longer being able to perform its normal function, or no longer having its normal level of activity, in whole or in part, compared to a wild-type (un-mutated) counterpart. Loss-of-function mutations are also referred to as inactivating mutations that typically result in the gene product having less or no function, / .< ., being partially or wholly inactivated. Loss-of-function mutations include insertions and deletions that interrupt or change the coding region of a gene, such as causing a premature stop codon or altering the splicing of a nucleotide sequence. In some embodiments, loss-of-function mutations are those that cause the gene and / or protein to not be expressed or not be properly expressed, or those that inactivate theprotein, such as from the introduction of a frameshift to the coding region leading to a premature stop codon.

[0063] In some embodiments, the one or more non-naturally occurring mutations reduce or eliminate expression of a GA2ox gene. In some embodiments, the one or more non-naturally occurring mutations reduce or eliminate expression of a GA2oxl gene. In some embodiments, the one or more non-naturally occurring mutations reduce or eliminate expression of a GA2ox4 gene. In some embodiments, the one or more non-naturally occurring mutations reduce or eliminate expression or activity of a GA2ox protein. In some embodiments, the one or more non-naturally occurring mutations reduce or eliminate expression or activity of a GA2oxl protein. In some embodiments, the one or more non-naturally occurring mutations reduce or eliminate expression or activity of a GA2ox4 protein.

[0064] An “indel” is an insertion, a deletion, or a combination of one or more insertion(s) and deletion(s) of nucleic acid sequences as compared to a reference or wild type sequence.

[0065] In some embodiments, the loss-of-function mutation is an insertion mutation. In some embodiments, the loss-of-function mutation is a deletion mutation. In some embodiments, the loss-of-function mutation is a combination of one or more insertion mutations. In some embodiments, the loss-of-function mutation is a combination of one or more deletion mutations. In some embodiments, the loss-of-function mutation is a combination of one or more insertion mutations and one or more deletion mutations. In some embodiments, the non-naturally occurring mutation is a frameshift mutation.

[0066] A loss-of-function mutation that introduces a premature stop codon in the coding sequence or eliminates or changes an amino acid residue that is essential for catalytic function of an enzyme is referred to herein as a “knockout” mutation. In some embodiments, the loss-of-function mutation is a knockout mutation.

[0067] In some embodiments, the polypeptides or proteins of this or any other embodiment described herein comprise one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid insertions, deletions, or other modifications (e.g., substitution of one amino acid for another) compared to a wild-type sequence. In some embodiments, the loss-of-function mutation is a knockout mutation that disrupts the key structural domain of GA2ox, including the non-haem dioxygenase domain or the 2OG-Fe(II) oxygenase domain. Altering the reading frame of the protein with an insertion or deletion (or combination thereof) that leads to a premature stop codon is one means of altering the non-haem dioxygenase domain or the 2OG-Fe(II) oxygenase domain. Other mutations that do not change the reading frame (such as a missense mutation or in-frame deletion) but eliminate or alter essential amino acids, e.g., in the non-haem dioxygenasedomain or the 2OG-Fe(II) oxygenase domain are also considered loss-of-function mutations. The non-haem dioxygenase domain and the 2OG-Fe(II) oxygenase domain of GA2oxl and GA2ox4 are shown in FIGs. 14A-B.

[0068] In some embodiments, the non-naturally occurring mutation is in the GA2oxl gene and is selected from SEQ ID NOs:76-83 or combinations thereof. In some embodiments, the non-naturally occurring mutation is in the GA2ox4 gene and is selected from SEQ ID NOs:84-85 or combinations thereof. In some embodiments, the non-naturally occurring mutation in the GA2oxl gene is a 1-14 bp deletion in SEQ ID NO: 1, or a sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the non-naturally occurring mutation in the GA2ox4 gene is a 1-14 bp deletion in SEQ ID NO: 19, or a sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (or any number or range therein) to the nucleotide sequence of SEQ ID NO: 19.

[0069] While specific exemplary mutations leading to loss-of-function of GA2ox genes are described herein, a person of ordinary skill in the art will appreciate, based on the discussion herein, that other mutations will lead to loss-of-function mutations, all of which are encompassed by the present disclosure.

[0070] In some embodiments, the same loss-of-function mutation in a GA2ox gene occurs in both chromosomal alleles of that GA2ox gene. In other words, the mutation is a homozygous mutation. In some embodiments, the one or more loss-of-function mutations are in both chromosomal alleles of the GA2oxl gene. In some embodiments, the one or more loss-of-function mutations are in both chromosomal alleles of the GA2ox4 gene. In other embodiments, the loss-of-function mutation in a GA2ox gene occurs in only one chromosomal allele of that GA2ox gene. In other words, the mutation is a heterozygous mutation. In some embodiments, the one or more loss-of-function mutations are in at least one chromosomal allele of the GA2oxl gene. In some embodiments, the one or more mutations are in at least one chromosomal allele of the GA2ox4 gene.

[0071] In yet another embodiment, two different loss-of-function mutations in the same GA2ox gene can occur in each chromosomal allele of the same GA2ox gene such that both alleles comprise different loss-of-function mutations in the GA2ox gene. In some embodiments, the one or more loss-of-function mutations are in at least one chromosomal allele of the nucleic acid sequence. In some embodiments, the one or more loss-of-function mutations are in both chromosomal alleles of the nucleic acid sequence.Lettuce with Reduced GA2ox Gene Expression or Protein Levels

[0072] Additional aspects and embodiments of the present disclosure are directed to reducing GA2ox expression in lettuce plants, cells, plant parts, seeds, and lettuce heads. The “expression” of a GA2ox gene refers to the transcription of a GA2ox gene. GA2ox gene expression levels may be measured by any means known in the art such as, without limitation, digital PCR, qRT-PCR (quantitative real time PCR), semi-quantitative PCR, RNA-seq, and Northern blot analysis.

[0073] In some embodiments, the expression of a GA2ox gene is reduced by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, or any number or range therein, in comparison to the expression of a GA2ox gene in a wild-type lettuce cell, plant, plant part, seed, or lettuce head without a non-naturally occurring mutation. In some embodiments, the expression of a mutated GA2ox gene is undetectable.

[0074] In some embodiments, the expression of a GA2oxl gene is reduced by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, or any number or range therein, in comparison to the expression of a GA2oxl gene in a wild-type lettuce cell, plant, plant part, seed, or lettuce head without a non-naturally occurring mutation. In some embodiments, the expression of a mutated GA2oxl gene is undetectable.

[0075] In some embodiments, the expression of a GA2ox4 gene is reduced by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, or any number or range therein, in comparison to the expression of a GA2ox4 gene in a wild-type lettuce cell, plant, plant part, seed, or lettuce head without a non-naturally occurring mutation. In some embodiments, the expression of a mutated GA2ox4 gene is undetectable.

[0076] The expression “amount” or “level” of a protein refers to the abundance of a particular protein, for example GA2oxl or GA2ox4, which may be measured by any means known in the art such as, without limitation, Western blot analysis, ELISA, other forms of immunological detection, or mass spectrometry.

[0077] In some embodiments, the amount of a GA2oxl protein is reduced by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, or any number or range therein, in comparison to the amount of GA2oxl protein in a wild-type lettuce cell, plant, plant part, seed, or lettuce head. In some embodiments, the amount of a mutated GA2oxl protein is undetectable.

[0078] In some embodiments, the amount of a GA2ox4 protein is reduced by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, or any number or range therein, in comparison to the amount of a GA2ox4 protein in a wild-type lettuce cell, plant, plant part, seed, or lettuce head. In some embodiments, the amount of a mutated GA2ox4 protein is undetectable.Lettuce with Reduced GA2ox Activity

[0079] Additional aspects and embodiments of the present disclosure are directed to lettuce cells, plants, plant parts, seeds, and lettuce heads with reduced GA2ox activity. GA2ox protein “activity” or “GA2ox activity” refers to the enzymatic activity of the GA2ox protein(s). GA2ox protein activity may be measured biochemically by methods known in the art including, but not limited to, mass spectrometry, e.g., to identify the presence or absence of specific oxidized products. In some embodiments, GA2ox protein activity is measured functionally, for example, by assessing its effects on phenotypic traits of a lettuce cell, plant, plant part, seed, or lettuce head, such as growth rate, biomass production, and flowering time.

[0080] In some embodiments, GA2ox activity is reduced or eliminated in lettuce plants of the present disclosure such that lettuce plants, seeds, and plant parts harvested from the lettuce plants described herein (z.e., comprising one or more loss-of-function mutations of a nucleic acid sequence encoding a GA2ox gene) demonstrate one or more of: normal flowering time (GA2oxL), more rapid flowering time (GA2ox4), increased average leaf size, increased fresh weight, and / or an increased size lettuce head at harvest when compared to lettuce plants, seeds, and plant parts of a wild-type lettuce without the mutation.

[0081] In some embodiments, the lettuce cell, plant, plant part, seed, or lettuce head of the present disclosure has GA2oxl activity that is reduced by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or any number or range therein, of the activity of total GA2oxl in wild-type lettuce cells, plants, plant parts, seeds, or lettuce heads. In some embodiments, the lettuce cell, plant, plant part, seed, or lettuce head has undetectable total GA2oxl activity.

[0082] In some embodiments, the lettuce cell, plant, plant part, seed, or lettuce head of the present disclosure has GA2ox4 activity that is reduced by 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or any number or range therein, of the activity of total GA2ox4 in wild-type lettuce cells, plants, plant parts, seeds, or lettuce heads. In some embodiments, the lettuce cell, plant, plant part, seed, or lettuce head has undetectable total GA2ox4 activity.

[0083] In some embodiments, the lettuce cell, plant, plant part, and / or seed further comprises non-naturally occurring mutations in two or more lettuce Polyphenol Oxidase (PPO) genes. Exemplary lettuce PPO nucleotide and amino acid sequences are described in U.S. Patent No. 12,203,086, which is hereby incorporated by reference in its entirety. In some embodiments, the two or more PPO genes are selected from PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-O, PPO-R, PPO-S, and combinations thereof. In some embodiments, the non-naturally occurring mutations in the two or more PPO genes are loss-of-function mutations. In some embodiments, a lettuce plant part, seed, or progeny comprise the one or more non-naturally occurring mutations of the GA2oxl gene and the non-naturally occurring mutations in the two or more PPO genes. In some embodiments, the non-naturally occurring mutations in the two or more PPO genes are loss-of-function mutations. In some embodiments, a lettuce plant part, seed, or progeny comprise the one or more non-naturally occurring mutations of the GA2ox4 gene and the non-naturally occurring mutations in the two or more PPO genes.Methods of Inducing Non-Naturally Occurring Mutations in Lettuce

[0084] Modifying a gene in a lettuce cell, plant, plant part, and / or seed, so that the cell, plant, plant part, and / or seed possesses a gene with a loss-of-function mutation, may be done by any method known in the art. That is, any method known in the art for introducing mutations into a cell in a gene sequence may be used to achieve the lettuce plant cell of the present disclosure. Such methods should be effective for introducing mutations into one or more of the GA2ox genes of lettuce, including, without limitation the GA2oxl or GA2ox4 genes.

[0085] In some embodiments, a mutation may be induced by a human by treatment of a plant or plant part with a mutagenic agent. Any suitable mutagenic agent can be used for embodiments of the present disclosure. For example, mutagens creating point mutations, deletions, insertions, rearrangements, transversions, transitions, or any combination thereof may be used. Suitable radiation mutagens include, without limitation, ultraviolet light, x-rays, gamma rays, and fast neutrons. Suitable chemical mutagens include, but are not limited to, ethyl methanesulfonate (“EMS”), methylmethane sulfonate (“MMS”), N-ethyl-N-nitrosourea (“ENU”), triethylmelamine (“TEM”), N-methyl-N-nitrosourea (“MNU”), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N’-nitro-nitrosoguanidine 25 (“MNNG”), nitrosoguanidine, 2-aminopurine, 7, 12 dimethyl-benz(a)anthracene (“DMBA”), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (“DEO”), diepoxybutane(“DEB”), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl) aminopropylamino] acridine dihydrochloride (“ICR- 170”), sodium azide, formaldehyde, or combinations thereof.

[0086] A mutation may be detected using a method such as “TILLING” or “Targeting Induced Local Lesions in Genomes” which is a general reverse genetic method providing an allelic series of induced mutation by random chemical or physical mutagenesis, that can be used to identify mutations in a gene or region of interest. In a common use of the TILLING methodology, plant material, such as seeds, are subjected to chemical mutagenesis, which creates a series of mutations within the genomes of the seeds’ cells. The mutagenized seeds are grown into adult Ml plants and self-pollinated. DNA samples from the resulting M2 plants are pooled and are then screened for mutations in a gene of interest. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the gene of interest. See for example, Colbert et al., “High-Throughput Screening for Induced Point Mutations,” Plant Physiology 126:480-484 (2001) and Krasileva et al., “Uncovering Hidden Variation in Polyploid Wheat,” Proc. Nat. Acad. Sci. 114-E913-E921 (2017), each of which is hereby incorporated by reference in its entirety.

[0087] In some embodiments, a GA2ox or PPO gene mutation is induced by a human in a plant cell of the present disclosure by genome editing. Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or deleted, or any combination thereof, from a genome using artificially engineered nucleases, or “molecular scissors.” The nucleases typically create double-stranded breaks (“DSBs”) at desired locations in the genome and harness the cell’s endogenous mechanisms to repair the induced break by processes of homology dependent repair (“HDR”) or nonhomologous end-joining (“NHEJ”). Any method of genome editing may be used in the embodiments of the present disclosure.

[0088] CRISPR / Cas type RNA-guided endonucleases provide an efficient system for inducing genetic modifications in genomes of many organisms. Non-limiting examples of genome editing nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a (Cpfl), Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, Mad7, SynNucl, or homologs, modified versions, and endonuclease inactive versions thereof. An example of a fusion protein to Cas9 is a cytidine deaminase-Cas9 fusion protein used in cytidine base editing to mutate nucleotides in target genes without generating double-strand breaks as described in Komor et al., “Programmable Editing of a Target Base inGenomic DNA without Double-Stranded DNA Cleavage,” Nature 533:420-424 (2016), which is hereby incorporated by reference in its entirety. The use of CRISPR guide RNA in conjunction with CRISPR / Cas technology to target RNA is also described in Wiedenheft et al., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR / Cas Systems,” Science 339:819-23 (2013); and Gaj et al., “ZFN, TALEN, and CRISPR / Cas-based Methods for Genome Engineering,” Cell 31:397-405 (2013), each of which is hereby incorporated by reference in its entirety.

[0089] There are typically two distinct components to a CRISPR system, a guide RNA (“gRNA”) and a genome editing endonuclease. The gRNA uses a CRISPR RNA (“crRNA”) comprising a DNA targeting segment that can be engineered to contain a complementary stretch of nucleotide sequence (e.g., at least 10 nucleotides) to target a DNA site for binding and subsequent modification by CRISPR genome editing nuclease. The length of a crRNA may range from about 15 nucleotides to about 60 nucleotides. The crRNA can be chemically synthesized and can also be engineered to include a ribonucleotide analog or a modified form thereof, or an analog of a modified form, or non-natural nucleosides. Exemplary portions of gRNA sequences that target lettuce GA2ox genes are provided in Table 3, infra, without limitation. Other gRNAs are also contemplated and can be generated by methods such as those described herein and in Example 2.

[0090] In some embodiments, a gRNA is directed to the lettuce GA2oxl gene. In some embodiments, a gRNA is directed to the lettuce GA2ox4 gene. In some embodiments, a gRNA that targets lettuce GA2oxl comprises one of SEQ ID NOs:55-63. In some embodiments, a gRNA that targets lettuce GA2ox4 comprises one of SEQ ID NOs:64-65.

[0091] In some embodiments, a gRNA is directed to one or more lettuce PPO genes. In some embodiments, gRNAs are targeted to sequences shared in common between multiple PPO genes in order to target and edit more than one PPO gene at a time resulting in mutations in more than one PPO gene. In some embodiments, gRNAs can be directed to a PPO gene sequence that is not shared with other O genes resulting in mutations in only one PPO gene. In some embodiments, the gRNAs that target lettuce PPO genes comprise, without limitation, one or more of SEQ ID NOs:70-75, shown in Table 3, infra.

[0092] Depending on the genome editing nuclease used, the gRNA can also comprise a trans-activating crRNA (“tracrRNA”). Such is the case with Cas9, for example. The tracrRNA is a small RNA sequence that forms a binding handle used by the CRISPR protein. The tracrRNAcan be chemically synthesized and can also be engineered to include a ribonucleotide analog or a modified form thereof, or an analog of a modified form, or non-natural nucleosides.

[0093] The term gRNA also includes single guide RNAs (“sgRNA”), which combine the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA. sgRNAs can be synthesized or expressed as a continuous RNA transcript. Alternatively, two-part gRNAs can be assembled from two separate RNAs by combining a crRNA-containing RNA with a tracrRNA. In the sgRNA or the two-part gRNA, crRNA and tracrRNA are present either in their native form, or a modified form. Either type of gRNA may be about 60 nucleotides to about 120 nucleotides long. These gRNAs can be chemically synthesized and can also be engineered to include a ribonucleotide analog or a modified form thereof, or an analog of a modified form, or non-natural nucleosides.

[0094] When the gRNA and the gene editing endonuclease are introduced into the cell, the genomic target sequence can be modified or permanently disrupted to create a loss-of-function mutation. A complex of a genome editing nuclease with a gRNA is called a ribonucleotide particle or ribonucleoprotein (RNP) complex. The RNP complex is recruited to the target sequence by the base-pairing between the gRNA sequence, which has a region of complementarity to the target sequence in the genomic DNA. In some embodiments, the target sequence is a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 19, or portions thereof.

[0095] For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (“PAM”) sequence immediately following the target sequence. The binding of the RNP complex localizes the genome editing nuclease to the genomic target sequence so that the genome editing nuclease can cut both strands of DNA causing a DSB. Cas9 generates DSBs through the combined activity of two nuclease domains, RuvC and HNH. Cas9 will cut 3-4 nucleotides upstream of the PAM sequence. CRISPR specificity can be controlled by level of homology and binding strength of the specific gRNA for a given gene target, or by modification of the Cas endonuclease itself. For example, a D10A mutant of the RuvC domain, retains only the HNH domain and generates a DNA nick rather than a DSB.

[0096] A software tool can be used to optimize the choice of gRNA within a target sequence, and to minimize total off-target activity across the rest of the genome. The cleavage efficiency at each off-target sequence can be estimated, e.g., using an experimentally-derived weighting scheme. Each possible gRNA is then ranked of its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the leastoff-target cleavage. An exemplary software tool to use for estimating gRNA cleavage efficiency is Geneious software (Geneious, San Diego, CA).

[0097] Other nucleases can also be used for genome editing. ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences, and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. ZFNs include an engineered zinc finger DNA-binding domain fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be used to induce double-stranded breaks (DSBs) in specific DNA sequences.

[0098] TALEN is a sequence-specific endonuclease that includes a transcription activator-like effector (“TALE”) and a FokI endonuclease. The transcription activator-like effector is a DNA binding protein that has a highly conserved central region with tandem repeat units of 34 amino acids. The base preference for each repeat unit is determined by two amino acid residues called the repeat-variable di-residue, which recognizes one specific nucleotide in the target DNA. Arrays of DNA-binding repeat units can be customized for targeting specific DNA sequences. As with ZFNs, dimerization of two TALENs on targeted specific sequences in a genome results in GAI-dependent introduction of double stranded breaks, stimulating homology directed repair (“HDR”) and non-homologous end joining (NHEJ) repair mechanism.

[0099] Meganucleases with re-engineered homing nucleases can also be used to effect genome modification in plants in the methods described herein. Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). This site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-Scel meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance.Meganucleases are considered to be the most specific naturally occurring restriction enzymes. Among meganucleases, the LAGLID ADG family of homing endonucleases has become a valuable tool for the study of genomes and genome engineering over the past fifteen years. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed.

[0100] In some embodiments, the mutations and methods of generating mutations described herein are applicable to homologues of GA2oxl, GA2ox4, and PPO genes described herein, but from other lettuce varieties. In some embodiments, homologues encompassed are nucleic acid sequences at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical with the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 19.Methods of Generating Lettuce Cells and Lettuce Plants with GA2ox Mutations

[0101] Another aspect of the present disclosure is directed to a method of making a normal flowering lettuce plant with increased biomass. This method comprises introducing a loss-of-function mutation into a Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene of a lettuce plant cell and regenerating a lettuce plant from the lettuce plant cell where the lettuce plant has a similar flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation. This method may also comprise transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNA targeting the GA2oxl gene and a genome editing nuclease and editing the plant cell’s genome to induce the loss-of-function mutation in the GA2oxl gene. This method may further involve introducing a loss-of-function mutation into each of two or more Polyphenol Oxidase (PPO) genes.

[0102] Another aspect of the present disclosure is directed to a method of making a normal flowering lettuce plant with increased biomass. This method comprises introducing a loss-of-function mutation into a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene of a lettuce plant cell and regenerating a lettuce plant from the lettuce plant cell where the lettuce plant has a similar flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation. This method may also comprise transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNA targeting the GA2ox4 gene and a genome editing nuclease and editing the plant cell’s genome to induce the loss-of-function mutation in the GA2ox4 gene. This method may further involve introducing a loss-of-function mutation into each of two or more Polyphenol Oxidase (PPO) genes.

[0103] These aspects of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0104] In some embodiments, the methods of genome editing of lettuce described herein are achieved without inserting exogenous DNA into the plant cell. For example, a ribonucleotide particle or ribonucleoprotein (RNP) complex is preassembled and delivered to a target lettuce plant cell. Use of ribonucleoprotein complexes (RNP) for genome editing can eliminate integration of nucleic acid into the plant genome and obviate the need for backcrossing and screening of progeny to obtain plants without exogenous DNA.

[0105] In some embodiments, the RNP complex is prepared in vitro using a molar ratio of genome editing nuclease to gRNA of 1 :7. In some embodiments, the molar ratio of genome editing nuclease to gRNA ranges from 3:1, 2:1, 1:1, 1:2, 1:3, 1:6, 1:7, 1:8, or 1:9, as non-limiting examples.

[0106] In some embodiments, a plurality of RNP complexes is used to enable genome editing of multiple genes for traits of interest. In some embodiments, each RNP complex of the plurality of RNP complexes comprises a genome-editing nuclease and a gRNA sequence, where the plurality of RNP complexes comprise different gRNA sequences targeting at least two different genes. In some embodiments, the plurality of RNP complexes comprise different gRNA sequences, each targeting at least 2 or more different genes. The methods of the present disclosure allow the simultaneous editing of multiple different gene targets without the need to combine them by breeding.

[0107] In some embodiments, introduction of RNP complexes into plants may be performed by introducing the RNP complexes into protoplasts. Introduction of polynucleotide constructs or RNPs into plants may be performed by introducing the constructs or RNPs into protoplasts. Protoplasts may be made by any means known in the art such as, but not limited to, that found in Engler & Grogan, “Isolation, Culture and Regeneration of Lettuce Leaf Mesophyll Protoplasts,” Plant Sci. Lett. 28:223-229 (1983); Nishio, “Simple and Efficient Protoplast Culture Procedure of Lettuce, Lactuca sativa L.,” Jap. J. Breeding 3%(2). \65-\T\ (1988), which are hereby incorporated by reference in their entirety. In some embodiments, equal ratio of each RNP complex is incubated with the protoplasts. In some embodiments, 1 nmol gRNA is used per 10,000; 50,000; 100,000; 150,000; 200,000; 300,000 protoplasts; or any amount or range in between. In some embodiments, 1 nmol gRNA is used per 100,000 protoplasts.

[0108] In some embodiments, protoplast cells are transfected with genome editing components (e.g., RNP complexes). Plant protoplasts are enclosed only by a plasma membrane and will therefore take up macromolecules like RNP complexes. These protoplasts can be capable of regenerating whole plants.

[0109] In some embodiments, said introducing comprises transfecting the plant cell with at least one ribonucleoprotein (RNP) complex comprising a guide RNA and a genome editing nuclease, and editing the plant cell’s genome to induce the one or more loss-of-function mutations. In some embodiments, said introducing comprises transfecting the plant cell with two or more different RNP complexes. In some embodiments, the two or more RNP complexes comprise at least two guide RNA sequences selected from SEQ ID NOs:55-75.

[0110] In some embodiments, the protoplast cell genome is edited to induce loss-of-function mutations in a GA2oxl gene. In some embodiments, the protoplast cell genome is edited to induce loss-of-function mutations in a GA2ox4 gene. In some embodiments, the protoplast cell genome is also edited to induce loss-of-function mutations in one or more of PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S genes, and combinations thereof. In some embodiments, the protoplast cell genome is edited to induce loss-of-function mutations in a GA2oxl gene and loss-of-function mutations in one or more of PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S genes, and combinations thereof. In some embodiments, the protoplast cell genome is edited to induce loss-of-function mutations in a GA2ox4 gene and loss-of-function mutations in one or more of PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S genes, and combinations thereof.[oni] In some embodiments, the PPO genes are selected from a PPO-A gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:27, a. PPO-B gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:29, a PPO-C gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:31, a PPO-D gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:33, PPO-E gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 35, a PPO-G gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:37, a PPO-J gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:39, a PPO-M gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:41, a PPO-N gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:43, a PPO-O gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:45, a PPO-P gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:47, a PPO-Q gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:49, a PPO-R gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:51, a PPO-S gene having at least 80%, 85%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:53, and combinations thereof.

[0112] In some embodiments, the PPO genes are selected from a PPO-A gene encoding a PPO-A protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:28, a PPO-B gene encoding a PPO-B protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:30, a PPO-C gene encoding a PPO-C protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:32, a. PPO-D gene encoding a PPO-D protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:34, a PPO-E gene encoding a PPO-E protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:36, a PPO-G gene encoding a PPO-G protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38, a PPO-J gene encoding a PPO-J protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:40, aPPO-M gene encoding a PPO-M protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:42, a PPO-N gene encoding a PPO-N protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:44, a PPO-O gene encoding a PPO-O protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:46, a PPO-P gene encoding a PPO-P protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:48, a PPO-Q gene encoding a PPO-Q protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:50, a PPO-R gene encoding a PPO-R protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52, a PPO-S gene encoding a PPO-S protein having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54, and combinations thereof.

[0113] Another aspect of the present disclosure relates to a method of editing a GA2oxl or GA2ox4 gene of a lettuce cell and optionally two or more of PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S genes. This method involves introducing into a lettuce cell a polynucleotide construct comprising a first nucleic acid sequence encoding a gene editing nuclease; a first promoter that is functional in plants, where the first promoter is operably linked to the first nucleic acid sequence; a second nucleic acid sequence encoding one or more gRNA(s) targeting i) either the GA2oxl or GA2ox4 gene, and ii) optionally two or more of polyphenol oxidase PPO-A, PPO-B, PPO-C, PPO-D,PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S genes; and a second promoter that is functional in plants, operably linked to the second nucleic acid sequence.

[0114] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0115] Useful promoters for driving expression of the nucleic acid sequence encoding gRNAs targeting PPO genes of interest are any pol III and / or pol II promoters operable in plants including, but not limited to, an Arabidopsis thaliana U6 promoter, a 35S promoter, an Arabidopsis thaliana AtUBQlO promoter, and a CsVMV promoter. The promoter that drives expression of the gRNA(s) may be the same or different than the promoter that drives expression of the gene editing nuclease. In some embodiments, the gene editing nuclease is a Cas9 nuclease, a MAD7 nuclease, a Cpfl nuclease, or a chimeric nuclease. In some embodiments, the first promoter and the second promoter are each independently selected from AtU6 promoter, 35S promoter, and CsVMV promoter. In some embodiments, the method further comprises a third nucleic acid sequence encoding a selectable marker operably linked to a third promoter that is functional in plants.

[0116] Delivery of polynucleotide constructs for modification of a plant genome can be accomplished by plant transformation, including, for example, infection with a microbe, such as Rhizobia o Agrobacterium infection. The Ti (or Ri) plasmid of Agrobacterium enables the highly successful transfer of a foreign nucleotide molecule into plant cells. A variation of Agrobacterium transformation uses vacuum infiltration in which whole plants are used (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety). In some embodiments, transformation involves fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al., “Liposome-Mediated Delivery' of Tobacco Mosaic Virus RNA into Tobacco Protoplasts: A Sensitive Assay for Monitoring Liposome-Protoplast Interactions,” Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which is hereby incorporated by reference in its entirety).

[0117] Wounding of a target plant tissue prior to or during DNA delivery, for example using Agrobacterium or a Rhizobia species, such as Ensifer adhaerens. may also be employed to cause transformation. Various methods of wounding are employed in plant transformation methods, including for example, microprojectile bombardment; treatment with glass beads; cutting, scratching or slicing; sonication; or silicon carbide fibers or whiskers.

[0118] In some embodiments, said introducing comprises: transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNAtargeting the GA2oxl gene and a genome editing nuclease; and editing the plant cell’s genome to induce the loss-of-function mutation in the GA2oxl gene. In some embodiments, said introducing comprises: transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNA targeting the GA2ox4 gene and a genome editing nuclease; and editing the plant cell’s genome to induce the loss-of-function mutation in the GA2ox4 gene. In some embodiments, said introducing comprises: transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising (i) at least one guide RNA targeting at least two PPO genes and a genome editing nuclease, and / or (ii) at least two guide RNAs targeting at least two PPO genes and a genome editing nuclease, and editing the plant cell’s genome to induce the loss-of-function mutations in each of the two or more PPO genes.

[0119] Transformation or transfection of protoplasts may be performed using any method known in the art including, but not limited to, polyethylene glycol treatment (Lelivelt et al., “Plastid Transformation in Lettuce (Lactuca sativa L.) by Polyethylene Glycol Treatment of Protoplasts,” Meth. Mol. Biol. 1132:317-330 (2014); Lelivelt et al., “Stable Plastid Transformation in Lettuce (Lactuca sativa L.),” Plant Mol. Biol. 58:763-774 (2005), which are hereby incorporated by reference in their entirety); using Sheen’s protocol (Sheen, J. (2002) at URL genetics.mgh.harvard.edu / sheenweb / ); Yoo & Sheen, “Arabidopsis Mesophyll Protoplasts: A Versatile Cell System for Transient Gene Expression Analysis,” Nat. Protocol. 2(7): 1565- 1572 (2007), which are hereby incorporated by reference in their entirety); microinjection, gene gun delivery (RNP biolistics or proteolistics), electroporation, gold nanoparticles, starch nanoparticles, silica nanoparticles, starch nanoparticles, and the like, and Agrobacterium-mediated delivery (for a review of these methods, see Demirer & Landry, “Delivering Genes to Plants SBE Supplement,” (2017) SBE Supplement: Plant Synth. Biol. 40-45, which is hereby incorporated by reference in its entirety).

[0120] Once genome editing has been performed by introduction of RNPs or polynucleotide constructs, plants can be regenerated from the edited lettuce plant cell and grown into plants. Methods of cultivating protoplasts into plants may be done by any means known in the art. See, for example, Enomoto and Ohyama, “Regeneration of Plants from Protoplasts of Lettuce and its Wild Species,” In: Bajaj Y.P.S. (eds) Plant Protoplasts and Genetic Engineering!. Biotechnology in Agriculture and Forestry, vol 8. Springer, Berlin, Heidelberg (1989), which is hereby incorporated by reference in its entirety.

[0121] A lettuce cell, plant part, or plant comprising genome edits as described herein may be identified by comparing the sequence of the region of the gene targeted by the RNPcomplex with the sequence from a control plant. A control plant or plant cell may comprise a wild-type plant or cell, / .< ., of the same genotype as the starting material for the genome editing. In some embodiments, DNA is extracted from a lettuce cell, plant part, plant, or fruit, and the sequence around the target genome sites for the gRNA is evaluated. In some embodiments, Inference of CRISPR Edits (ICE) is used for analysis of genome edits (see Hsiau et al., “Inference of CRISPR Edits from Sanger Trace Data, ” bioRxiv 251082 (2019), which is hereby incorporated by reference in its entirety). In some embodiments, the plant cell having genome edits is regenerated without the use of a selectable marker.

[0122] In some embodiments, the method of mutating the GA2ox genes leaves no pest sequences in the genome of the lettuce plant or plant cell (such as Agrobacterium sequences or selectable marker sequences). In some embodiments, the lettuce plant, plant part, or seed is free of exogenous DNA. In some embodiments, the lettuce plant, plant part, or seed is free of plant pest sequences.Lettuce Varieties

[0123] The terms “lettuce cell” or “lettuce plant cell” are used interchangeably, and include cells, protoplasts, cell tissue cultures from which lettuce plants can be regenerated, calli, clumps, and cells that are intact in lettuce or parts of lettuce including, but not limited to seeds, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, hypocotyls, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, micellar tissue, ovaries, and other lettuce tissue or cells. In some embodiments, the lettuce cell is a protoplast.

[0124] In some embodiments, the lettuce cell, plant, plant part, or seed is modified by genome editing. In some embodiments, the lettuce cell is a regenerable lettuce cell. In some embodiments, a lettuce plant comprises the lettuce cell. In some embodiments, a lettuce plant, plant part, seed, or fruit is propagated (sexually or asexually) from a lettuce plant, plant part, or seed of any of the embodiments of the present disclosure.

[0125] There are numerous types of lettuces grown throughout the world that are applicable to the present disclosure and, indeed, any type or variety of lettuce is contemplated by the present disclosure. Suitable types of lettuces include green, white, red and variegated lettuces.

[0126] Suitable varieties of lettuces for use in any of the embodiments described herein is a lettuce such as, but not limited to, a variety of romaine, iceberg, red leaf, leaf lettuce, romaine lettuce, Frisee lettuce, butter lettuce, Batavia lettuce, Bibb lettuce, or a Boston lettuce. Additionallettuce varieties include, but are not limited to Little Gem, Green Leaf, Red Leaf, Oakleaf, Lollo Rosso, Lollo Bionda, Batavia, Summer Crisp, Merveille des Quatre Saisons, Deer Tongue, Buttercrunch, Tom Thumb, Yugoslavian Red, Reine des Glaces, Forellenschluss, Cimmaron, Red Sails, Salad Bowl, Black Seeded Simpson, Grand Rapids, Tango, Parris Island Cos, Jericho, Winter Density, Rouge d'Hiver, Drunken Woman, Outredgeous, Flashy Trout Back, Green Ice, New Red Fire, Red Romaine, Green Forest, Butter King, May Queen, Marvel of Four Seasons, Red Velvet, Ruby Red, Valmaine, Vivian, Coastal Star, Nevada, Redina, Salinas, Paraiso, San Andreas, and Sierra. In certain embodiments, the lettuce is a romaine lettuce, such as, but not limited to, Green Forest, Platinum, Gold Mine, Paris Island, Avalanche, Rubicon, Musena, Costal Star, Ideal Cos, Topenga, Ridgeline, Green Towers, Helvius, Jerico, Fresh Heart, Claremont, Show Stopper, Spretnak, Caesar, Salvius, Marilyn, Defender, Concept, King Henry, Pipeline, Rome 59, Valley Heart, Wildcat, Bali, or Mondo.Methods of Breeding GA2OX Mutant Genes into Multiple Types of Lettuce Cultivars

[0127] A further aspect of the present disclosure is directed to a method of breeding a normal flowering lettuce plant with increased biomass. This method involves providing a candidate plant with a non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene, breeding the candidate plant with at least one other lettuce plant, and selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene.

[0128] Yet another aspect of the present disclosure is directed to a method of breeding a faster flowering lettuce plant with increased biomass. This method involves providing a candidate plant with a non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene, breeding the candidate plant with at least one other lettuce plant, and selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the non-naturally occurring mutation of a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene.

[0129] These aspects of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0130] The various GA2ox mutations and optionally the PPO mutations of the present disclosure can be transferred to other varieties of lettuce through breeding to develop new, unique lettuce cultivars and hybrids. Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or inbred line which is the recurrent parent. The source of the trait to be transferred is called the “donor parent”, or“candidate plant” such as the GA2oxl or GA2ox4 mutant lettuce lines described in the present disclosure.

[0131] After the initial cross of a plant of the present disclosure to another plant, individuals possessing the GA2oxl or GA2ox4 mutations of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting plants can be selfed to produce plants with homozygous GA2oxl or GA2ox4 mutations. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait, such as the GA2oxl or GA2ox4 mutations of the present disclosure, transferred from the donor parent.Backcrossing methods can also be used with the lettuce plants of the present disclosure to improve or introduce one or more characteristic into the lettuce cultivar of the present disclosure.

[0132] In some embodiments, breeding comprises crossing, making hybrids, backcrossing, self-crossing, double haploid breeding, and / or combinations thereof.Molecular Breeding Evaluation Techniques

[0133] In some embodiments, selecting comprises analyzing DNA from the lettuce plant, germplasm, pollen, or seed of the lettuce plant for the presence of the non-naturally occurring mutations of the GA2oxl gene and detecting the non-naturally occurring mutations of the GA2oxl gene. In some embodiments, selecting comprises analyzing DNA from the lettuce plant, germplasm, pollen, or seed of the lettuce plant for the presence of the non-naturally occurring mutations of the GA2ox4 gene and detecting the non-naturally occurring mutations of the GA2ox4 gene. In some embodiments, selecting comprises analyzing DNA from the lettuce plant, germplasm, pollen, or seed of the lettuce plant for the presence of the non-naturally occurring mutations of the two or more PPO genes and detecting the non-naturally occurring mutations of the two or more PPO genes.

[0134] In some embodiments, the combination of GA2oxl mutations with optionally two or more PPO mutations, or the combination of GA2ox4 mutations with optionally two or more PPO mutations, or the breeding of such mutations into new lettuce plants, or cultivars are performed using molecular markers to track the mutations. The term “marker” refers to a nucleotide sequence or a fragment of such sequence, e.g., a single nucleotide deletion, used as a point of reference at an identifiable physical location on a chromosome (e.g., restriction enzyme cutting site, gene) whose inheritance can be tracked. Markers can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, cDNA, etc.). The term can also refer to nucleic acid sequences complementary to or flanking a marker. The term can also refer to nucleic acid sequences used as a molecular markers probe, primer,primer pair, or a molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence and is capable of amplifying sequence fragments using PCR and modified PCR reaction methods.

[0135] A marker may be tracked using a marker assay. The term “marker assay” refers generally to a molecular markers assay, such as PCR, KASP, PACE or SSR, for example, used to identify whether a certain DNA sequence or SNP, for example, is present in a sample of DNA. For example, a marker assay can include a molecular markers assay, e.g., KASP assay, which can be used to test whether a GA2oxl, a GA2ox4, and / or a PPO mutation is present. Markers corresponding to genetic polymorphisms between members of a population can be detected by methods commonly used in the art including, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLPs), detection of amplified variable sequences of the plant genome, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs), and detection of randomly amplified polymorphic DNA (RAPD). In other embodiments, nucleic acids may be detected with other high throughput hybridization technologies including microarrays, gene chips, LNA probes, nanoStrings, and fluorescence polarization detection among others. Other forms of nucleic acid detection can include next generation sequencing methods such as DNA SEQ or RNA SEQ using any known sequencing platform including, but not limited to: Roche 454, Solexa Genome Analyzer, AB SOLiD, Illumina GA / HiSeq, Ion PGM, MiSeq, among others.

[0136] Detection of markers can be achieved at an early stage of plant growth by harvesting a small tissue sample (e.g., branch, or leaf disk). This approach is preferable when working with large populations as it allows breeders to weed out undesirable progeny at an early stage and conserve growth space and resources for progeny which show more promise. In some embodiments the detection of markers is automated, such that the detection and storage of marker data is handled by a machine. Recent advances in robotics have also led to full service analysis tools capable of handling nucleic acid / protein marker extractions, detection, storage and analysis.

[0137] In some embodiments, the method further comprises: selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the one or more non-naturally occurring mutations of a GA2oxl gene and the non-naturally occurring mutations in the two or more PPO genes. In some embodiments, the method further comprises: selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the one or more non-naturally occurringmutations of a GA2ox4 gene and the non-naturally occurring mutations in the two or more PPO genes.Lettuce Plant Phenotypes Due to GA2ox Mutations

[0138] The GA2ox mutations described herein confer multiple beneficial phenotypes on the lettuce plant providing significant agronomic advantages, including improved yield for commercial lettuce production in the field and earlier harvests under hydroponic growth conditions. For mutations in GA2oxL these phenotypes include, without limitation, an increased average leaf size, increased fresh weight, and / or an increased size lettuce head at harvest, while maintaining a similar flowering time as a wild type plant of the same variety grown under the same environmental conditions. For mutations in GA2ox4, these phenotypes include, without limitation, an increased average leaf size, increased fresh weight, and / or an increased size lettuce head at harvest, while having a faster flowering time compared to a wild type plant of the same variety grown under the same environmental conditions. A “wild type” plant is a plant of the same variety not having the mutation in a lettuce GA2ox gene. As used herein, a wild type plant is also a control plant.

[0139] For example, in some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2oxl mutation that exhibits an increased average leaf size relative to leaves of a control plant. In some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2ox4 mutation that exhibits an increased average leaf size relative to leaves of a control plant. Leaf size can be measured at any time during growth of the lettuce plant or after harvest. Typically, the size of three or more leaves is measured for calculating average leaf size. To determine the average leaf size of a lettuce plant, one would first select a representative sample of leaves from multiple plants within the same growth stage and environmental conditions. Each leafs length and width could be measured, e.g., by using a ruler or digital caliper or image analysis software. The leaf area can be calculated by multiplying the length by the width, or via image analysis. After obtaining the leaf area measurements for all sampled leaves, the average leaf size is calculated by summing the individual leaf areas and dividing by the total number of leaves measured. In some embodiments, the lettuce plant with the GA2oxl or GA2ox4 mutation exhibits an average leaf size increase ranging from 4% to 500% over the average leaf size of a control plant. In some embodiments, average leaf size is increased by at least 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%, or any number or range therein. Insome embodiments, the average leaf size of a lettuce plant comprising a GA2oxl mutation is at least 4% larger than the average leaf size of a wild-type lettuce plant without the mutation. In some embodiments, the average leaf size of a lettuce plant comprising a GA2ox4 mutation is at least 4% larger than the average leaf size of a wild-type lettuce plant without the mutation.

[0140] In some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2oxl mutation that exhibits an increased average fresh weight relative to the fresh weight of a control plant. In some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2ox4 mutation that exhibits an increased average fresh weight relative to the fresh weight of a control plant. Fresh weight can be measured at any time during growth of the lettuce plant or after harvest. Whole plants (such as lettuce heads) or plant parts (such as leaves) can be measured for calculating fresh weight. Average fresh weight can be measured, or individual plants or plant parts can be measured in comparison to a wild type control. In some embodiments, fresh weight of a lettuce plant can be determined by selecting a representative sample of plants from multiple plants within the same growth stage and environmental conditions. Each plant's fresh weight could be measured, e.g., by using a precision scale. After obtaining the fresh weight measurements for all sampled plants, the average fresh weight is calculated by summing the individual fresh weights and dividing by the total number of plants or plant parts measured. In some embodiments, the lettuce plant or plant part with the GA2oxl or GA2ox4 mutation exhibits an increased fresh weight ranging from 4% to 500% over the fresh weight of a control plant or plant part. In some embodiments, average fresh weight is increased by at least 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500%, or any number or range therein. In some embodiments, the average fresh weight of a lettuce plant comprising a GA2oxl mutation is at least 4% greater than the average fresh weight of a wild-type lettuce plant without the mutation. In some embodiments, the average fresh weight of a lettuce plant comprising a GA2ox4 mutation is at least 4% greater than the average fresh weight of a wild-type lettuce plant without the mutation.

[0141] Leaf size or fresh weight biomass can be measured at various time periods during the growth of the lettuce plant, including but not limited to 3 days after germination, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, and at harvest. In some embodiments, the average fresh weight of a two-week old plant is at least 4% greater than the average fresh weight of a wild-type lettuce plant without the mutation. In some embodiments, the average fresh weight of a three-week old plant is at least10% greater than the average fresh weight of a wild-type lettuce plant without the mutation. In some embodiments, the average fresh weight of a three-week old plant is at least 15% greater than the average fresh weight of a wild-type lettuce plant without the mutation.

[0142] In some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2oxl mutation that exhibits an increased head size relative to a control plant at harvest. In some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2ox4 mutation that exhibits an increased head size relative to a control plant at harvest. The size of the lettuce head can be measured at the time of harvest, typically around 9 to 11 weeks for outdoor-grown lettuce and 4 to 6 weeks for hydroponically grown lettuce. To determine the increased head size, one would first select a representative sample of plants from multiple plants within the same growth stage and environmental conditions. At harvest, the diameter and height of each lettuce head are measured using a ruler, a digital caliper, or image software. The head size can be calculated by considering both the diameter and height, or by using image analysis software for more precise measurements. The average head size is then calculated by summing the individual head sizes and dividing by the total number of plants measured. In some embodiments, the lettuce plant with the GA2oxl mutation is at least 4% larger than the lettuce head of a wild-type lettuce plant without the mutation at harvest. In some embodiments, the lettuce plant with the GA2ox4 mutation is at least 4% larger than the lettuce head of a wild-type lettuce plant without the mutation at harvest.

[0143] In some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2oxl mutation that exhibits a similar flowering time relative to the flowering time of a control plant. In some embodiments, the present disclosure is directed to a lettuce plant comprising a GA2ox4 mutation that exhibits a faster flowering time relative to the flowering time of a control plant. Flowering time, also referred to as time to bolting, is defined herein as the number of days from planting to the onset of flowering or the initiation of the bolting process, where the plant begins to produce a flowering stem. Flowering time can be measured during the growth of the lettuce plant by recording the number of days from planting to the onset of flowering or bolting. Typically, the flowering time of three or more plants is measured for calculating the average flowering time. To determine the average flowering time of a lettuce plant, one would first select a representative sample of plants from multiple plants within the same growth stage and environmental conditions. The number of days to flowering for each plant is recorded. After obtaining the flowering time measurements for all sampled plants, the average flowering time is calculated by summing the individual days to flowering and dividing by the total number of plants measured.

[0144] In some embodiments, the lettuce plant with the GA2oxl mutation exhibits a flowering time within 0 to 3 days of the average flowering time of a control plant. In some embodiments, the lettuce plant with the GA2oxl mutation exhibits a flowering time within 0, 1, 2, or 3 days (or any number or range therein) of the average flowering time of a control plant. As used herein, a “similar flowering time” or a “regular flowering time” is defined as a flowering time that is either within 0-3 days of a control plant without a GA2ox mutation when grown under the same environmental conditions or where the average number of plants that have flowered is within 20-30% of the average number of plants that have flowered in the control variety when grown under the same environmental conditions. In some embodiments, the lettuce plant with the GA2ox4 mutation exhibits a flowering time 4 or more days earlier (or faster) than the average flowering time of a control plant. In some embodiments, the faster flowering time is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days earlier, or any number or range therein than the average flowering time of a control plant. As used herein, a “faster flowering time” or an “earlier flowering time” is defined as a flowering time that is either 4 or more days sooner than the flowering time of a control plant without a GA2ox mutation when grown under the same environmental conditions or where the average number of plants that have flowered is greater than 30% of the average number of plants that have flowered in the control variety when grown under the same environmental conditions.

[0145] The lettuce plants of the present disclosure may be grown in any environment conducive to lettuce production including, without limitation, field growth, hydroponic growth and aeroponic growth. In field growth, soil preparation, irrigation schedules, nutrient management, and strategies for pest and disease control can be used to enhance growth and yield. Hydroponic growth involves cultivating plants in a nutrient-rich water solution without the use of soil, allowing for precise control over nutrient delivery and environmental conditions. Aeroponic growth involves suspending plants in the air and periodically misting their roots with a nutrient-rich solution. Any method of cultivation suitable for lettuce production is contemplated.

[0146] In embodiments comprising two or more PPO mutations, the PPO mutations described herein confer multiple beneficial phenotypes on the lettuce plant including, without limitation, reduced browning and increased levels of polyphenolics. Additional beneficial phenotypes are disclosed in U.S. Patent No. 12,203,086, which is hereby incorporated by reference in its entirety.

[0147] For example, a lettuce plant with two or more PPO mutations exhibits a reduced-browning phenotype. As used herein, “reduced-browning” means that when lettuce leaves areharvested, cut, sliced, or processed in a manner where cell wall destruction takes place, browning will be detectably less than in a control (wild type) lettuce variety. Any reduction in browning (such as a reduction in browning visible to the naked eye relative to a control) may be advantageous.

[0148] In some embodiments, the rate of browning of lettuce leaves produced from a PPO mutant plant is reduced relative to leaves a control plant. In another embodiment, the total quantity or degree of browning of lettuce leaves produced from a PPO mutant plant is reduced relative to leaves from a control plant.

[0149] Any detectable level of reduced browning that is detectable to the naked eye may constitute a reduction in browning. Beyond this, reduced browning may be detected by a device, such as a chromameter, even if not visible to the human eye. Browning may be determined by known methods including, but not limited to, spectroscopy (e.g., light absorption, laser-induced fluorescence spectroscopy, time-delayed integration spectroscopy, large aperture spectrometer); colorimetry (e.g., tristimulus, “spekol” spectrocolorimeter); and visual inspect! on / scoring.

[0150] The PPO mutant plant may be considered reduced-browning if the PPO mutant sample visual score is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% less than the control.

[0151] The skilled person would appreciate that browning may vary depending on a number of factors including, but not limited to, the manner and ambient conditions in which plant material is bruised. For example, lettuce leaves stored at 4°C may show different browning characteristics from lettuce leaves stored at 24°C, as detected by the eye or by an instrument, such as a chromameter, or like devices.

[0152] In embodiments comprising two or more PPO mutations, the lettuce plants of the present disclosure also exhibit higher levels of polyphenolics in comparison to a wild type variety. In one embodiment, the level of polyphenolics is 5%, 10%, 15%, 20% or more than 20% higher than a wild type variety. The lettuce plants of the present disclosure comprising PPO mutations also retain higher levels of polyphenolics after harvest in comparison to a wild type variety. In one embodiment, the level of polyphenolics is 5%, 10%, 15%, 20%, or greater than 20% higher than a wild type variety at 7, 14, or 21 days after harvest.

[0153] Although certain embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions,substitutions, and the like can be made without departing from the spirit of the present disclosure and these are therefore considered to be within the scope of the present disclosure.EXAMPLESExample 1 - Gibberellin 2-Beta Dioxygenase Genes & Gene Expression in Lettuce

[0154] Gibberellin 2-Beta Dioxygenase (GA2ox) gene sequences from Arabidopsis thaliana (TAIR Locus ID NOs: AT1G78440, AT1G30040, AT2G34555, AT1G47990, AT3G17203, AT1G02400, AT1G50960, AT4G21200, AT5G58660, and AT3G47190, from arabidopsis.org, each of which is hereby incorporated by reference in its entirety) or Nicotiana tabacum (GenBank Accession Nos. AB 125232 and AB 125233, each of which is hereby incorporated by reference in its entirety) were used as bait to query the most recent lettuce genome assembly accessible through the Lettuce Genome Resource website (lgr.genomecenter.ucdavis.edu and Reyes-Chin-Wo et al., “Genome Assembly with in vitro Proximity Ligation Data and Whole-Genome Triplication in Lettuce,” Nature Communications 8:14953 (2017), each of which is hereby incorporated by reference in its entirety). The genome assembly described in this paper is available on CoGe, Organism id36218, Lactuca sativa (lettuce) and from NCBI under Bioproject PRJNA173551, each of which is hereby incorporated by reference in its entirety. This strategy can also be used to query databases for additional varieties of lettuce.

[0155] In Table 1 below, lettuce GA2ox gene sequences identified by sequence identity to Arabidopsis sequences are marked with aA, and lettuce GA2ox gene sequences identified by sequence identity to Nicotiana sequences are marked with a #. Additionally, by using search terms such as Gibberellic Acid 2-Oxidase, Gibberellin 2-Beta Dioxygenase, or Gibberellin 2-Oxidase inside the KEGG enzymes search engine from the Lettuce Genome Resource website, more GA2ox genes were identified (marked with an asterisk *). Using all of the above resources and methods, at least thirteen putative GA2ox genes in lettuce were identified as set forth in Table 1. GA2ox genes with sequence identity to each of the GA2ox genes of Table 1 can be identified in additional varieties of lettuce through sequence comparison searches.Table 1. Lettuce Gibberellin 2-Beta Dioxygenase Genes & Gene Expression in Leaves &

[0156] Gibberellin 2-Beta Dioxygenase genes 1 & 4 can be recognized by the presence of characteristic domains including 2OG-Fe(II) oxygenase and non-haem dioxygenase. See FIGs. 14A-B.

[0157] In Table 1, each Gibberellin 2-Beta Dioxygenase gene has been identified by its Locus number in GenBank of the sequenced lettuce genome variety Salinas (iceberg lettuce). See Reyes-Chin-Wo et al., “Genome Assembly with in vitro Proximity Ligation Data and Whole-Genome Triplication in Lettuce,” Nature Communications 8.1:1-11 (2017), which is hereby incorporated by reference in its entirety. Each coding and amino acid sequence is also referenced by its specific GenBank Accession and version number in Table 12 as set forth in Example 6 below. A summary of the gene expression levels in leaf tissue of some of the genes in romaine lettuce variety Green Forest is also provided in Table 1.

[0158] Additional expression data was compiled for each of the lettuce GA2ox genes based on reads per kilobase per million mapped reads (RPKM) from The Lettuce Genome Database (lettucegdb.com) as shown in Table 2.Table 2. Lettuce Gibberellin 2-Beta Dioxygenase Gene Expression

[0159] LsGA2oxl, LsGA2ox4, and LsGA2ox6 were selected for genome editing.Example 2 - Genome Editing of GA2ox Genes in Lettuce

[0160] Guide RNAs were designed for each of LsGA2oxl, LsGA2ox4, and LsGA2ox6 genes using Geneious software (Dotmatics, Boston, MA). Geneious allows the user to find CRISPR sites in a specific gene sequence with predicted activity score based on Doench et al., “Rational Design of Highly Active sgRNAs for CRISPR-Cas9-Mediated Gene Inactivation,” Nat. Biotechnol. 32(12): 1262-1267 (2014) and Doench et al., “Optimized sgRNA Design to Maximize Activity and Minimize Off-Target Effects of CRISPR-Cas9,” Nat. Biotechnol.34(2): 184-191 (2016), each of which is hereby incorporated by reference in its entirety.Geneious also allows users to score gRNAs against an off-target database of your choice. As GA2ox catalytic domains are present all along the gene sequence gRNAs targeting the 5’ end of the gene was preferred. Based on high activity, high specificity and location, a minimum of three gRNAs per gene target were chosen and tested in protoplasts from Green Forest Romaine lettuce. gRNAs exhibiting the best gene editing efficiency evaluated by next generation sequencing were then chosen to use in transfection experiments. Although specific gRNA sequences are provided in Table 3 below, gRNAs targeting other regions of each gene, or the regulatory regions of the genes (e.g., promoter sequences, TATA box, etc.) could also be designed and used following this procedure. gRNA sequences with appropriate PAM motifs were identified based on the genome editing nuclease to be used (e.g., Cpfl or Cas9, etc.).

[0161] Ribonucleotide protein (RNP) complexes composed of purified CRISPR Cas9 or Cpfl protein combined with a gRNA targeting GA2ox genes was introduced to lettuce protoplasts via polyethylene glycol mediated transfection (Yoo et al., 2007). Briefly, lettuce protoplasts were isolated from mesophyll cells and adjusted to a density of 1 x 106protoplast per ml in MMG media. An aliquot of 100 pl (1 x 105protoplasts) was combined with 1.5 pl RNP mixture followed immediately by 100 pl of freshly prepared PEG 4000. Protoplasts were incubated for 12-20 minutes and transfections were stopped by adding 5.5-6x volume of W5 solution then mixed gently. Protoplasts were gently pelleted, washed 1-3 times, resuspended in alginate solution (2.5-3.5%) and expelled dropwise or plated into calcium-containing media withmannitol. Protoplasts were incubated with liquid culture media and induced to regenerate into calli then plants. It is also important to note that by utilizing DNA-free transfection, no foreign DNA is introduced into the lettuce genome.

[0162] Examples of gRNA sequences designed to target LsGA2oxl, LsGA2ox4 and LsGA2ox6 are provided in Table 3 as SEQ ID NOs:55-69 (shown using their corresponding DNA sequences). The PAM (Protospacer Adjacent Motif), a short DNA sequence that follows the DNA region targeted for cleavage by the CRISPR-Cas9 enzyme, is shown. The specific sequence of the PAM varies depending on the type of CRISPR system being used. For example, Streptococcus pyogenes Cas9 (SpCas9) recognizes the PAM sequence “NGG”, where “N” can be any nucleotide. The PAM sequence is adjacent to the 5’ end of the sequences Cpfl and adjacent to the 3’ end of the sequence for Cas9. Table 3 also includes gRNA sequences for targeting single or multiple LsPPO genes (SEQ ID NOs:70-75) as described in U.S. Patent No.12,203,086, which is hereby incorporated by reference in its entirety.Table 3. Guide RNAs for LsGA2ox & LsPPO Genome Editing&&

[0163] Protoplast cell transfections were performed using ribonucleoprotein complexes (RNPs). Protoplasts were isolated from six week old wild type Romaine lettuce plants (about 1 g of leaf tissue) and transfected following Sheen’s protocol (Sheen, “A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts,” available on line at URLgenetics. mgh. harvard. edu / sheenweb / (2002); Yoo et al., "Arabidopsis Mesophyll Protoplasts: A Versatile Cell System for Transient Gene Expression Analysis,” Nature Protocols 2: 1565-1575 (2007), which are hereby incorporated by reference in their entirety). Transfected protoplasts were incubated at 25 °C in the dark for about 60 hours.

[0164] Sixty hours post-transfection, protoplasts from three independent transfection reactions were pooled together and genomic DNA (gDNA) was extracted with 400 pl urea buffer (6.9 M Urea, 350 mM NaCl, 50 mM Tris-Cl pH 8.0, 20 mM EDTA pH 8.0, 1% Sarkosyl) followed by a phenol: chloroform: isoamyl alcohol and a chloroform: isoamyl alcohol steps. DNA precipitation was done at -80°C for 20 minutes in an equal volume of isopropanol. Finally, DNA was washed once with 70% ethanol and resuspended in 20 pl of distilled (DI) water. gDNA concentration was estimated using a nanodrop 8000 (Thermo Fisher Scientific, Waltham, MA) then diluted at 30 ng / pl for further analysis.

[0165] Next Generation Sequencing (“NGS”) was used to deeply sequence the specific PCR products for each target gene and assess gene editing frequency by comparing the number of pair reads showing a mutation (indels) at the target site to the number of pair reads depicting a wild type pattern. NGS was performed by the Center for Computational and Integrative Biology DNA Core Facility at Massachusetts General Hospital, Boston, USA using standard protocols.

[0166] Plants were regenerated from protoplasts using published methods. See for example, (Curtis, “Lettuce (Lactuca sativa L.),” in Agrobacterium Protocols, Second Edition, Volume 1, eds. Kan Wang, pp449-458 (2006), which is hereby incorporated by reference in itsentirety). These plants have no exogenous DNA introduced due to the use of the RNPs for genome editing.

[0167] Once plants have been regenerated from protoplasts (RO generation), genomic DNA was extracted from leaf tissue using a CTAB method. DNA was subjected to PCR of the target genes then submitted to sequencing to identify the genome editing pattern. Table 4 shows the mutations present in each RO Green Forest lettuce line. The identifying ID for seed produced from these RO plants (R1 generation) is also provided. Deletions are indicated by an uppercase “D” if they are homozygous or if they are biallelic for a different mutation in the opposite chromosome. A lowercase “d” indicates that the deletion is heterozygous. The number following the “D” or “d” indicates the number of nucleotides deleted. Insertions are indicated by an uppercase “I” if they are homozygous or if they are biallelic for a different mutation in the opposite chromosome. A lowercase “i” indicates that the insertion is heterozygous. The number following the “I” or “i” indicates the number of nucleotides inserted. Bi-allelic lines having a different mutation in each chromosomal allele of a specific gene are indicated with a / symbol. For example, GA2oxl line 9 (D3 / D14) has a mutation with a deletion of three nucleotides in one allele of GA2oxl, and another mutation with a deletion of 14 nucleotides in the other allele of GA2oxl. Some lines seem to be chimeric with more than two mutation patterns as GA2ox6 lines 10, 11, 13, 16 and 22.Table 4. Genome Edits and Editing Efficiency in Three GA2ox Genes in Lettuce

[0168] Once R1 seeds had matured and were harvested, R1 plants with various gene edits in GA2oxl, GA2ox4, or GA2ox6 were grown aeroponically. Seeds were sown on rockwool sheets and let to germinate for one week in a growth chamber set to 24°C, 16h light / 8h dark photoperiod (FIG.1A) before being transferred to an aeroponic system (AEssence, AEssenceGrows, Santa Clara, CA) in a fully randomized manner (FIG. IB) and grown for an additional 17 or more days before biomass was evaluated by measuring fresh weight.

[0169] FIG. 2 shows biomass increase of 24-day old R1 plants of GA2ox4 genome edited line 52-06-01 and 52-06-16 in comparison to wild-type lettuce. Biomass differences from various R1 lines are shown in Table 5, below. As lines were still segregating, number of plants exhibiting the same mutation were limited but these preliminary data showed that some mutant lines had a positive effect on biomass / weight.Table 5. Biomass Differences in R1 GA2ox Edited Lines vs Wild-Type Controls

[0170] As manipulation of the gibberellic acid pathway may lead to flowering defects, plants were allowed to grow for a longer period in the greenhouse to visualize the effects of the mutation(s) on flowering time. Flowering time of R1 plants with gene edits in various GA2ox genes was evaluated at 10 weeks of growth in greenhouse. Lettuce plants edited for GA2oxl had fewer plants that had an early bolting phenotype while most plants with edits in GA2ox4 and GA2ox6 flowered early as depicted in Table 6. Most plants included in this experiment were either bi-allelic or homozygous plants. Only eight plants with edits in GA2ox6 wereheterozygous and these heterozygous plants were all the mid bolting-early flowering type. FIG.3 shows examples the flowering phenotypes of 10-week-old GA2oxl, GA2ox4, and GA2ox6 mutant plants in comparison to wild type. As shown in Table 6 below, 49% of plants with mutations in GA2oxl had not yet bolted or were starting to bolt at 10 weeks, whereas only 16% or 21% of plants with mutations in GA2ox4 or GA2ox6, respectively, had not yet bolted or were starting to bolt at 10 weeks. These results indicated that lettuce plants with mutations in GA2oxl had similar flowering times as wild-type plants, whereas lettuce plants with mutations in GA2ox4 or GA2ox6 had faster flowering times in comparison to wild-type plants.Table 6. Phenotypic Evaluation of GA2ox Gene Edits for Flowering Time

[0171] As GA2oxl mutants were showing less effect on flowering time they were chosen as best candidates for biomass increase in both field production and indoor farming (e.g., aeroponic or hydroponic growth). However, the faster flowering time of GA2ox4 and GA2ox6 mutants also make them especially useful for indoor farming where it is beneficial to maximize crop turnover and reduce the time to harvest. This accelerated growth cycle allows for more frequent planting and harvesting, leading to increased productivity and potentially higher yields within the same space and time frame. Additionally, faster flowering can help in optimizing resource use, such as light, water, and nutrients, making the overall indoor farming operation more efficient and cost-effective.

[0172] The biomass increases of different R2 generation GA2oxl mutant lines in comparison to wild type grown over a 21 -day aeroponic (Aessence system) growth period versus a 24-day soil growth period are shown in Table 7. Lines 54-9-3 and 54-29-5 exhibited a more constant biomass increase in both growth environments. Line 54-29-5 was prioritized for further evaluation. Line 54-29-5 includes the D5 mutation, a loss-of-function mutation resulting from a frame-shift mutation, which modifies the open reading frame to result in a prematurely aborted translation of the protein.Table 7. Phenotypic Evaluation of T2 generation Genome edited Lines of GA2oxl

[0173] To investigate R2 line 54-29-5 even further, sixty seeds were sown then transferred to the Aessence aeroponic system for evaluation. FIGs. 4 and 5 show photographic comparisons of 20-day old R2 generation plants of GA2oxl genome edited plant line 54-29-5 in comparison to wild type and non-edited lines. The fresh weight biomass for this experiment is shown in Table 8. R2 line 54-29-5 with homozygous gene edit D5 in GA2oxl showed a 76-81% increase in fresh weight biomass versus control lines, and a 49% biomass increase vs PPO genome edited line GV110.Table 8. Evaluation of R2 line 54-29-5 with homozygous gene edit D5 in GA2oxl

[0174] Once R3 seeds were available for selected lines of each GA2ox mutant of GA2oxl, GA2ox4 and GA2ox6, a larger experiment was performed before beginning larger scale trialing with external partners. These experiments were set up aeroponically for a total of four weeks of growth, and at the greenhouse for a total of 12 weeks of growth.

[0175] FIGs. 6-8 show biomass of GA2ox mutants and controls (non-edited and wild type) after two, three and four weeks of aeroponic growth. In summary, all lines showed a slight increase in biomass compared to the control plants starting at two weeks, and peaking at threeweeks before control plants catch up at four weeks. The increase in early biomass observed in this experiment is consistent with prior experiments using earlier generations of plants. Plants with mutations in GA2oxl and GA2ox4 exhibit higher increases in biomass than plants with mutations in GA2ox6.

[0176] FIG. 9 shows fresh weight (g) biomass evaluations of R3 generation plants between six and twelve weeks of greenhouse growth. When plants were germinated and grown in soil in a greenhouse to mid-maturity and formation of a head of lettuce, biomass increase are significant once plants reached eleven weeks.Example 3 - Field Evaluation of GA2oxl Edited Lettuce

[0177] Extensive field trials have been initiated in 2024 with a field trial cooperator located in Salinas, CA. Four trials in diverse locations of the Salinas Valley have been conducted already with additional trials planned in the desert (Yuma, AZ) late 2024. Trials are usually running for a two-three months period depending on the season. At harvest, multiple parameters were observed including all the ones described in Table 9 below. The objective with the high biomass lettuce grown in the field is to see a statistically significant increase of at least 10% in weight with no significant changes of the other attributes relevant to lettuce such as core length and tip bum.

[0178] R3 generation field data from mid-February to early May Salinas trial are presented in Table 9. As Green Forest Romaine lettuce is typically grown in the Salinas Valley April to mid-May this first trial was executed at the right period and data resulting from this trial should be the best. Data was collected from 6 lettuce heads with Green Forest wild type lettuce weighing between 1.75 to 2 lbs. while our improved biomass GA2oxl mutant weighed 2 lbs. and greater. In a second trial executed in Salinas Valley in June and July the average weight of wild type dropped a bit being 1.5 lbs + / - 0.03 (SE) against 1.67 lbs + / - 0.05 (SE) determined for the edited line, which was still larger than wild type.Table 9. Phenotypic Evaluation of GA2oxl Edited Variety in Field Grown PlantsExample 4 - Genome Edits of GA2oxl Orthologs in Multiple Varieties of Lettuce & Lettuce Plants with a Combination of Ga2oxl and PPO edits

[0179] Several additional varieties of lettuce were edited with gRNAs directed to GA2oxl. Editing was performed by transforming lettuce using Agrobacterium-mediated transfer of T-DNAs comprising a Cas9 genome editing nuclease and gRNAs targeting GA2oxl (SEQ ID NOs:58, 61 and 63) using methods described in U.S. Patent No. 12,203,086, which is hereby incorporated by reference in its entirety.

[0180] As shown in Table 10, Agrobacterium-meddated genome edits in GA2oxl in multiple lettuce varieties were made including Green Forest (Romaine), Coastline Batavia, R101 (standard Romaine), and R108 (blonde Romaine).

[0181] Edits in GA2oxl in Green Forest lettuce variety GV110 were in addition to genome edited mutations in PPO-A, PPO-B, PPO-D, PPO-E, PPO-G, and PPO-S genes.Table 10. GA2oxl Gene Edits in Multiple Varieties of Lettuce Using Agrobacterium

[0182] Protoplasts of additional varieties of lettuce were also edited in the Ga2oxl gene using RNPs. These varieties included R107 (blonde Romaine) and Valley Heart. 16 edited Platinum lines and 11 edited Valley Heart lines were identified as depicted in Table 11 with the following mutations identified: I1 / D3; I1 / D5; I1 / D7; and D4 / D7.Example 5 - Genome Editing of Both GA2ox and PPO Genes in Lettuce

[0183] RNPs can be used to edit GA2ox genes and PPO genes simultaneously in a protoplast using the methods described in Example 2. Alternatively, a lettuce line having PPO mutations can be used to introduce further edits in a GA2ox gene using the RNP method described in Example 2 and described below.

[0184] Lettuce line GV110 comprising genome edited mutations in PPO-A, PPO-B, PPO-D, PPO-E, PPO-G, and PPO-S genes was used to add additional edits in GA2oxl using RNPs with Cas9 and various gRNAs (SEQ ID NOs:53, 61 and 63).

[0185] GA2oxl edits were also introduced into PPO KO Romaine varieties ‘Valley Heart’ and ‘Platinum’ using Cas9 as shown in Table 11 below. PPO mutations are described in U.S. Patent No. 12,203,086, which is hereby incorporated by reference in its entirety Table 11. Editing of Ga2oxl in Multiple Lettuce Varieties Using RNPsExample 6 - Gibberellin 2-Beta Dioxygenase Sequences

[0186] Putative Gibberellin 2-beta dioxygenase genes were identified as described in Example 1. The coding sequence for lettuce (Lactuca saliva Gibberellin 2-beta dioxygenase 1 (GA2oxl) gene LOC111914174 (SEQ ID NO: 1; Nucleotides 160..1173 of GenBank Accession No. XM_023909934.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2oxl protein (SEQ ID NO:2; GenBank Accession No. XP_023765702.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0187] The coding sequence for lettuce (Lactuca sativa) Gibberellin 2-beta dioxygenase 2 (GA2ox2) gene LOCI 11880732 (SEQ ID NO:3; Nucleotides 2..1018 of GenBank Accession No. XM_023877149.2, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox2 protein (SEQ ID NO:4, GenBank Accession No. XP_023732917.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0188] The coding sequence for lettuce (Lactuca sativa) Gibberellin 2-beta dioxygenase 3 (GA2ox3) gene LOCI 11904506 (SEQ ID NO: 17; Nucleotides 52..1065 of GenBank Accession No. XM_023900260.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox3 protein (SEQ ID NO:18; GenBank Accession No. XP_023756028.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0189] The coding sequence for lettuce (Lactuca sativa) Gibberellin 2-beta dioxygenase 4 (GA2ox4) gene LOC111890054 (SEQ ID NO:19; Nucleotides 201..1142 of GenBank Accession No. XM_023886216.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox4 protein (SEQ ID NO:20; GenBank Accession No. XP_023741984.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0190] The coding sequence for lettuce (Lactuca sativa) Gibberellin 2-beta dioxygenase 5 (GA2ox5) gene LOC111903687 (SEQ ID NO:7; Nucleotides 359..1414 of GenBank Accession No. XM_023899436.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox5 protein (SEQ ID NO:8 ; XP_023755204.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0191] The coding sequence for lettuce (Lactuca saliva) Gibberellin 2-beta dioxygenase 6 (GA2ox6) gene LOC111882624 (SEQ ID NO:21; Nucleotides 134..1147 of GenBank Accession No. XM_023879001.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox6 protein (SEQ ID NO:22; GenBank Accession No. XP_023734769.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0192] The coding sequence for lettuce (Lactuca saliva Gibberellin 2-beta dioxygenase 7 (GA2ox7) gene LOCI 11909227 (SEQ ID NO:5; Nucleotides 131..1120 of GenBank Accession No. XM_ 023905019.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox7 protein (SEQ ID NO:6; GenBank Accession No. XP_ 023760787.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0193] The coding sequence for lettuce Lactuca sativa) Gibberellin 2-beta dioxygenase 8 (GA2ox8) gene LOC111897140 (SEQ ID NO:9; Nucleotides 570..1571 of GenBank Accession No. XM_023893105.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox8 protein (SEQ ID NO:10; GenBank Accession No. XP_023748873.1, which is hereby incorporated by reference in its entirety), is set forth in Table 12 below.

[0194] The coding sequence for lettuce (Lactuca sativa) Gibberellin 2-beta dioxygenase 9 (GA2ox9) gene LOC111875983 (SEQ ID NO:11; Nucleotides 592..1575 of GenBank Accession No. XM_023872505.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2ox9 protein (SEQ ID NO:12; GenBank Accession No. XP_023728273.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0195] The coding sequence for lettuce (Lactuca sativa) Gibberellin 2-beta dioxygenase 10 (GA2oxlO) gene LOCI 11905788 (SEQ ID NO: 13; Nucleotides 1..471 of GenBank Accession No. XM_052769064.1, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2oxlO protein (SEQ ID NO:14; GenBank Accession No. XP_052625024.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0196] The coding sequence for lettuce (Lactuca sativa) Gibberellin 2-beta dioxygenase 11 (GA2oxll) gene LOC111905065 (SEQ ID NO:15; Nucleotides 144..1172 of GenBank Accession No. XM_023900748.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2oxl 1protein (SEQ ID NO:16; GenBank Accession No. XP_023756516.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0197] The coding sequence for lettuce Lactuca sativd) Gibberellin 2-beta dioxygenase 12 (GA2oxl2) gene LOC111898951 (SEQ ID NO:23; Nucleotides 1..1434 of GenBank Accession No. XM_023894831.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2oxl2 protein (SEQ ID NO:24; GenBank Accession No. XP_023750599.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.

[0198] The coding sequence for lettuce (Lactuca saliva Gibberellin 2-beta dioxygenase 13 (GA2oxl3) gene LOCI 11919699 (SEQ ID NO:25; Nucleotides 217..1359 of GenBank Accession No. XM_023915277.3, which is hereby incorporated by reference in its entirety), the sequence of which is set forth in Table 12 below. The amino acid sequence for the GA2oxl3 protein (SEQ ID NO:26; GenBank Accession No. XP_023771045.1, which is hereby incorporated by reference in its entirety) is set forth in Table 12 below.Table 12. Nucleotide and Amino Acid Sequences&>

[0199] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

1. WHAT IS CLAIMED IS:

1. A lettuce plant comprising a non-naturally occurring mutation in a lettuce Gibberellin 2-Beta Dioxygenase 1 (GA2oxl) gene, wherein the lettuce plant has a similar flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

2. The lettuce plant of claim 1, wherein the lettuce GA2oxl gene comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 1.

3. The lettuce plant of claim 1 or claim 2, wherein the lettuce GA2oxl gene encodes the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:2.

4. The lettuce plant of any one of the preceding claims, wherein the non-naturally occurring mutation reduces expression or activity of the GA2oxl protein.

5. The lettuce plant of any one of the preceding claims, wherein the non-naturally occurring mutation is a loss-of-function mutation.

6. The lettuce plant of any one of the preceding claims, wherein the non-naturally occurring mutation is in at least one chromosomal allele of the GA2oxl gene.

7. The lettuce plant of any one of the preceding claims, wherein the non-naturally occurring mutation is in both chromosomal alleles of the GA2oxl gene.

8. The lettuce plant of any one of the preceding claims, wherein the non-naturally occurring mutation is selected from SEQ ID NOs:76-83 or combinations thereof.

9. The lettuce plant of any one of the preceding claims, wherein the lettuce plant is free of exogenous DNA.

10. The lettuce plant of any one of the preceding claims, wherein the average leaf size is at least 4% larger than the average leaf size of a wild-type lettuce plant without the mutation.

11. The lettuce plant of any one of the preceding claims, wherein the average fresh weight of a two-week old plant is at least 4% greater than the average fresh weight of a wild-type lettuce plant without the mutation.

12. The lettuce plant of any one of the preceding claims, wherein the average fresh weight of a three-week old plant is at least 10% greater than the average fresh weight of a wild-type lettuce plant without the mutation.

13. The lettuce plant of any one of the preceding claims, wherein the lettuce head at harvest is at least 4% larger than the lettuce head of a wild-type lettuce plant without the mutation at harvest.

14. The lettuce plant of any one of the preceding claims, wherein the average flowering time is within 3 days of that of a wild-type lettuce plant without the mutation.

15. The lettuce plant of any one of the preceding claims, wherein the lettuce is a romaine, iceberg, red leaf, leaf lettuce, romaine lettuce, Frisee lettuce, butter lettuce, Batavia lettuce, Bibb lettuce, or a Boston lettuce variety.

16. The lettuce plant of any one of the preceding claims, wherein the lettuce plant further comprises:non-naturally occurring mutations in two or more lettuce Polyphenol Oxidase (PPO) genes.

17. The lettuce plant of claim 16, wherein the two or more PPO genes are selected from PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S, and combinations thereof.

18. The lettuce plant of claim 16 or claim 17, wherein the non-naturally occurring mutations in the two or more PPO genes are loss-of-function mutations.

19. A lettuce plant part, seed, or progeny from the lettuce plant of any one of the preceding claims, wherein the lettuce plant part, seed, or progeny comprise the one or more non-naturally occurring mutations of the GA2oxl gene and optionally the non-naturally occurring mutations in the two or more PPO genes.

20. A method of making a normal flowering lettuce plant with increased biomass, said method comprising:introducing a loss-of-function mutation into a Gibberellin 2-Beta Dioxygenase 1 (GA2oxP) gene of a lettuce plant cell; andregenerating a lettuce plant from the lettuce plant cell, wherein the lettuce plant has a similar flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

21. The method of claim 20, wherein the lettuce plant cell is a protoplast.

22. The method of claim 20 or claim 21, wherein said introducing comprises: transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNA targeting the GA2oxl gene and a genome editing nuclease; andediting the plant cell’s genome to induce the loss-of-function mutation in the GA2oxl gene.

23. The method of any one of claims 20-22, wherein the guide RNA comprises the corresponding RNA sequence of any one of SEQ ID NOs:55-63.

24. The method of any one of claims 20-23, wherein the at least one mutated allele comprises the insertion of at least one nucleotide, the deletion of at least one nucleotide, or the insertion of at least one nucleotide and deletion of at least one nucleotide, relative to the nucleotide sequence of the GA2oxl gene.

25. The method of any one of claims 20-24, wherein the loss-of-function mutation is in at least one chromosomal allele of the GA2oxl gene.

26. The method of any one of claims 20-25, wherein the loss-of-function mutation is in both chromosomal alleles of the GA2oxl gene.

27. The method of any one of claims 20-26, wherein the method further comprises:introducing a loss-of-function mutations into each of two or more Polyphenol Oxidase (PPO) genes.

28. The method of claim 27, wherein the two or more PPO genes are selected from PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO- PPO-Q, PPO-R, PPO-S, and combinations thereof.

29. The method of claim 27 or claim 28, wherein said introducing comprises: transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising (i) at least one guide RNA targeting at least two PPO genes and a genome editing nuclease, and / or (ii) at least two guide RNAs targeting at least two PPO genes and a genome editing nuclease; andediting the plant cell’s genome to induce the loss-of-function mutations in each of the two or more PPO genes.

30. A method of breeding a normal flowering lettuce plant with increased biomass, said method comprising:providing a candidate lettuce plant with a non-naturally occurring mutation of a Giberellic Acid 2-Oxidase 1 (GA2oxJ) gene;breeding the candidate lettuce plant with at least one other lettuce plant; and selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the non-naturally occurring mutation of the GA2oxl gene.

31. The method of claim 30, wherein said selecting comprises:analyzing DNA from the progeny lettuce seeds, lettuce plants, or lettuce plant parts for the presence of the non-naturally occurring mutations of the GA2oxl gene; and detecting the non-naturally occurring mutations of the GA2oxl gene.

32. The method of claim 30 or claim 31, wherein said breeding comprises crossing, making hybrids, backcrossing, self-crossing, double haploid breeding, and / or combinations thereof.

33. The method of any one of claims 30-32, wherein the GA2oxl gene comprises the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 1.

34. The method of any one of claims 30-33, wherein the GA2oxl gene encodes the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:2.

35. The method of any one of claims 30-34, wherein the progeny lettuce plants have a similar flowering time and increased leaf fresh weight biomass and / or an increased leaf head size at harvest compared to a wild-type lettuce plant without the mutation.

36. The method of any one of claims 30-35, wherein the at least one other plant comprises one or more non-naturally occurring mutations in two or more Polyphenol Oxidase (PPO) genes.

37. The method of any one of claims 30-36, wherein said method further comprises:selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the one or more non-naturally occurring mutations of a GA2oxl gene and the non-naturally occurring mutations in the two or more PPO genes.

38. A lettuce plant comprising a non-naturally occurring mutation in a lettuce Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene, wherein the lettuce plant has a fasterflowering time, an increased average leaf size, increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

39. The lettuce plant of claim 38, wherein the lettuce GA2ox4 gene comprises the nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 19.

40. The lettuce plant of claim 38 or claim 39, wherein the lettuce GA2ox4 gene encodes the amino acid sequence of SEQ ID NO:20, or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:20.

41. The lettuce plant of any one of claims 38-40, wherein the non-naturally occurring mutation is loss-of-function mutations.

42. The lettuce plant of any one of claims 38-41, wherein the non-naturally occurring mutation is in at least one chromosomal allele of the GA2ox4 gene.

43. The lettuce plant of any one of claims 38-42, wherein the non-naturally occurring mutation are in both chromosomal alleles of the GA2ox4 gene.

44. The lettuce plant of any one of claims 38-43, wherein the non-naturally occurring mutation is selected from SEQ ID NOs:84-85 or combinations thereof.

45. The lettuce plant of any one of claims 38-44, wherein the lettuce plant is free of exogenous DNA.

46. The lettuce plant of any one of claims 38-45, wherein the average leaf size is at least 4% larger than the average leaf size of a wild-type lettuce plant without the mutation.

47. The lettuce plant of any one of claims 38-46, wherein the average fresh weight of a two-week old plant is at least 4% greater than the average fresh weight of a wild-type lettuce plant without the mutation.

48. The lettuce plant of any one of claims 38-47, wherein the average fresh weight of a three-week old plant is at least 10% greater than the average fresh weight of a wildtype lettuce plant without the mutation.

49. The lettuce plant of any one of claims 38-48, wherein the lettuce head at harvest is at least 4% larger than the lettuce head of a wild-type lettuce plant without the mutation at harvest.

50. The lettuce plant of any one of claims 38-49, wherein the average time to flowering is earlier by at least 4 days than that of a wild-type lettuce plant without the mutation.

51. The lettuce plant of any one of claims 38-50, wherein the lettuce is a romaine, iceberg, red leaf, leaf lettuce, romaine lettuce, Frisee lettuce, butter lettuce, Batavia lettuce, Bibb lettuce, or a Boston lettuce variety.

52. The lettuce plant of any one of claims 38-51, wherein the lettuce plant further comprises:one or more non-naturally occurring mutations in one or more lettuce Polyphenol Oxidase (PPO) genes.

53. The lettuce plant of claim 52, wherein the two or more PPO genes are selected from PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S, and combinations thereof.

54. The lettuce plant of claim 52 or claim 53, wherein the non-naturally occurring mutations in the two or more PPO genes are loss-of-function mutations.

55. A lettuce plant part, seed, or progeny from the lettuce plant of any one of claims 36-52, wherein the lettuce plant part, seed, or progeny comprise the one or more non-naturally occurring mutations of the GA2ox4 gene and optionally the non-naturally occurring mutations in the two or more PPO genes.

56. A method of making a rapid flowering lettuce plant with increased biomass, said method comprising:introducing a loss-of-function mutation into a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene of a lettuce plant cell; andregenerating a lettuce plant from the lettuce plant cell, wherein the lettuce plant has a faster flowering time, an increased average leaf size, an increased fresh weight, and / or an increased size lettuce head at harvest compared to a wild-type lettuce plant without the mutation.

57. The method of claim 56, wherein the lettuce plant cell is a protoplast.

58. The method of claim 56 or claim 57, wherein said introducing comprises: transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising at least one guide RNA targeting the GA2ox4 gene and a genome editing nuclease; andediting the plant cell’s genome to induce the loss-of-function mutation in the GA2ox4 gene.

59. The method of any one of claims 56-58, wherein the guide RNA comprises the corresponding RNA sequence of any one of SEQ ID NOs:64-65.

60. The method of any one of claims 57-60, wherein the loss-of-function mutation is in at least one chromosomal allele of the GA2ox4 gene.

61. The method of any one of claims 57-61, wherein the loss-of-function mutation is in both chromosomal alleles of the GA2ox4 gene.

62. The method of any one of claims 57-62, wherein the method further comprises:introducing one or more loss-of-function mutations into two or more Polyphenol Oxidase (PPO) genes.

63. The method of claim 63, wherein the two or more PPO genes are selected from PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-J, PPO-M, PPO-N, PPO-O, PPO-P, PPO-Q, PPO-R, PPO-S, and combinations thereof.

64. The method of claim 63 or claim 64, wherein said introducing comprises: transfecting the lettuce plant cell with a genome editing ribonucleoprotein complex comprising (i) at least one guide RNA targeting at least two PPO genes and a genome editing nuclease, and / or (ii) at least two guide RNAs targeting at least two PPO genes and a genome editing nuclease; andediting the plant cell’s genome to induce the loss-of-function mutations in each of the two or more PPO genes.

65. A method of breeding a rapid flowering lettuce plant with increased biomass, said method comprising:providing a candidate lettuce plant with one or more non-naturally occurring mutations of a Gibberellin 2-Beta Dioxygenase 4 (GA2ox4) gene;breeding the candidate lettuce plant with at least one other lettuce plant; and selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the one or more non-naturally occurring mutations of the GA2ox4 gene.

67. The method of claim 66, wherein said selecting comprises: analyzing DNA from the lettuce plant, germplasm, pollen, or seed of the lettuce plant for the presence of the non-naturally occurring mutations of the lettuce GA2ox4 gene; and detecting the non-naturally occurring mutations of the GA2ox4 gene.

68. The method of claim 66 or claim 67, wherein said breeding comprises crossing, making hybrids, backcrossing, self-crossing, double haploid breeding, and / or combinations thereof.

69. The method of any one of claims 66-68, wherein the lettuce GA2ox4 gene comprises the nucleotide sequence of SEQ ID NO: 19, or a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 19.

70. The method of any one of claims 66-69, wherein the lettuce GA2ox4 gene encodes the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:20.

71. The method of any one of claims 66-70, wherein the progeny lettuce plants have a rapid flowering time and increased leaf fresh weight biomass and / or an increased leaf head size at harvest compared to a wild-type lettuce plant without the mutation.

72. The method of any one of claims 66-71, wherein the at least one other plant comprises one or more non-naturally occurring mutations in two or more Polyphenol Oxidase (PPO) genes.

73. The method of any one of claims 66-72, wherein said method further comprises:selecting progeny lettuce seeds, lettuce plants, or lettuce plant parts that comprise the one or more non-naturally occurring mutations of a GA2ox4 gene and the non-naturally occurring mutations in the two or more PPO genes.