WO2018237197A2 - Crispr-mediated selective gene disruption in plants - Google Patents

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) offer an effective way of creating targeted mutagenesis in plants. To alleviate concerns related to genetically modified plants, the present disclosure provides a novel and efficient genome editing system for editing the genomes of complex plants. Specifically, methods for reducing lignin content in switchgrass are disclosed.

Description

CRISPR-MEDIATED SELECTIVE GENE DISRUPTION IN PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] The present application claims priority to U.S. Provisional Application No. 62/523,025, filed on June 21, 2017 which is herein incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[2] This invention was made with Government support under DOE BioEnergy Science Center Contratct #DEAC0500R22725 awarded by the Department of Energy. The Government has certain rights in this invention.

BACKGROUND

[3] In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) have been developed that use targeted nucleases to generate DNA breaks resulting in defined genetic modifications similar to, but much more exact than the modifications seen with conventional antisense, RNAi, and artificial microRNA techniques. The critical difference between the downregulation and CRISPR/Cas9 technologies is that the site- specific nucleases produce defined gene knockouts by altering the genomic DNA sequence, while antisense, RNAi, and artificial microRNA techniques generate knock-downs by repressing transcription of the targeted gene. The site-specific nuclease generates double-strand breaks (DSBs), which are typically repaired by either error-prone non-homologous end-joining (NHEJ) or homolgous recombination (HR). NHEJ causes insertions and deletions (indels) of nucleotides (nt), which result in frameshift mutations causing disturbed translation of a coding gene.

[4] In general, the CRISPR/Cas9 system employs a plasmid DNA to transfect the target plant cells. The plasmid DNA can be introduced into plant cells by Agrobacterium-mediated transformation, polyethylene glycol (PEG) or electroporation treatment of protoplasts, particle bombardment or other methods.

[5] The complex and costly regulatory process for conventional GMOs has made it extremely difficult to commercialize new transgenic cultivars. The situation is even more complicated in outcrossing perennial bioenergy species like switchgrass. However, plants obtained by genome editing technologies are considered low risk because these materials contain small genetic differences that can also occur naturally or through long-standing breeding methods. The United States Department of Agriculture has determined that it will not regulate a waxy corn and a non-browning mushroom produced by CRISPR/Cas9. Therefore, the development of genome editing technologies offers new prospects in improving and

commercializing switchgrass cultivars. The present disclosure provides methods for genome editing and selection in plants with complex genomes that specifically target a single gene without affecting similar genes in the plant. The present disclosure also provides methods for decreasing lignin content. These approaches utilize CRISPR/Cas9, avoiding the regulatory hurdles faced by conventional transgenic crops.

SUMMARY

[6] The present disclosure relates to methods for selectively disrupting a target gene without disrupting other genes in plants, plants produced by such methods and seeds produced by such plants. By selectively disrupting a target gene, disruption of other genes is avoided when other genes have some sequence homology to the target gene.

[7] In some embodiments, a method is provided for selectively disrupting a target gene without disrupting other genes in a plant which includes steps of selecting a target gene, analyzing at least a portion of the nucleotide sequence of the target gene by comparison to the genome of the plant to identify a target sequence that is specific to the target gene, designing a guide RNA having a nucleotide sequence complementary to the target sequence and a scaffold sequence for Cas9 binding, and transforming at least one cell of the plant type with one or more nucleic acid molecules encoding Cas9 and the guide RNA.

[8] In some embodiments, a method is provided for selectively disrupting a target gene without disrupting other genes in a plant and includes the steps of selecting a target family of genes in a plant, selecting a target tissue in the plant, collecting a sample of the target tissue, measuring mRNA expression of each gene of the target family of genes in the target tissue, selecting one target gene from the target family of genes which is preferentially expressed at a higher level than the other genes in the target tissue, analyzing at least a portion of the nucleotide sequence of the target gene by comparison to the genome of the plant to identify a target sequence that is specific to the target gene, designing a guide RNA having a nucleotide sequence complementary to the target sequence and a scaffold sequence for Cas9 binding, and transforming at least one cell of the plant type with one or more nucleic acid molecules encoding Cas9 and the guide RNA.

[9] In some embooimdents, a method is provided for reducing lignin-content in a switchgrass plant and includes the steps of desiging a guide RNA for Pv4CLl, transforming at least one switchgrass cell with one or more nucleic acid molecules encoding CRISPR associated protein 9 (Cas9) and said guide RNA, and generating a plant from said at least one switchgrass cell.

[10] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[11] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentalities disclosed herein.

[12] FIGURE 1 depicts a protein sequence alignment of Pv4CL3a (Pavir.Da00296) and Pv4CL3b (Pavir.Db00533). PYSSGTTGMPKGV AMP-binding motif, GEICIRGR motif, VPP and PVL motifs are shown in red boxes.

[13] FIGURE 2 depicts a phylogenic tree of Pv4CLs and their homologous genes in model plants. The scale indicates amino acid substitutions per position (scale bar = 0.05). Switchgrass: Pv4CLl (Pavir.Fa01395), Pv4CL2 (Pavir.J20148), Pv4CL3 (Pavir.Da00296); Brachypodium: Bd4CLl (Bradi3g05750.1); Foxtail millet: Si4CL2 (Si006172m), Si4CLl (Si016817m); Rice: Os4CL2 (Q42982), Os4CLl (BAD5189); and Maize: Zm4CLl (AY566301). Switchgrass Pv4CLs are shown in red.

[14] FIGURE 3 depicts the relative expression levels of Pv4CLl, Pv4CL2 and Pv4CL3 in leaf, node and internode tissues as measured by qRT-PCR.

[15] FIGURE 4A depicts the nucleotide sequence for Pv4CLl subgenomes "a" and "b" from switchgrass genotype NFCXl with the target site underlined and the PAM sequence boxed. [ 16] FIGURE 4B depicts a sequence alignment of Pv4CLl, Pv4CL2, Pv4CL3a and Pv4CL3b including the target site and PAM sequence in Pv4CLl in a red box and blue box, respectively.

[17] FIGURE 5 depicts a schematic diagram of the PCR/sequencing screening method for edited Pv4CLs.

[18] FIGURE 6 depicts the results of second round PCR sequencing for transformed switchgrass plants.

[19] FIGURE 7 A depicts the sequences for the transformed plants with detailed sequence information. Target site and PAM sequence are shown in boxes from left to right.

[20] FIGURE 7B depicts the sequences for the transformed plants with detailed sequence information.

[21] FIGURE 8 depicts the total lignin content by the acetyl bromide method for control and transgenic plants. Values are mean +/- standard deviation for three replicates. * - P < 0.05; ** - P < 0.01.

[22] FIGURE 9 depicts the aromatic regions of the MR spectra for a control (WT) and pv4cll-26 plant.

[23] FIGURE 10A depicts glucose release over time for a control plant (WT) and mutant plants (pv4cll-25 (#25), pv4cl 7-26 (#26) and pv4cll-29 (#29)). Values are mean +/- standard deviation for triplicate samples.

[24] FIGURE 10B depicts xylose release over time for a control plant (WT) and mutant plants (pv4cll-25 (#25), pv4cll-26 (#26) and pv4cll-29 (#29)). Values are mean +/- standard deviation for triplicate samples.

[25] FIGURE 11 depicts a histologically stained cryosection depicting the cortex (C), cell wall (CW), phloem (Ph), xylem (X), vascular sheath (VS), sylem parenchyma cells (XP), xylem tracheid (XT), xylem vessel (XV). Scale bar = 50 μιη.

[26] FIGURE 12A depicts histological staining of an internode cryosection from a control plant. Scale bar = 50 μιη.

[27] FIGURE 12B depicts histological staining of an internode cryosection from a control plant. Scale bar = 50 μιη.

[28] FIGURE 12C depicts histological staining of an internode cryosection from the pv4cll- 25 plant. Scale bar = 50 μιη. [29] FIGURE 12D depicts histological staining of an internode cryosection from the pv4cll- 25 plant. Scale bar = 50 μιη.

[30] FIGURE 12E depicts histological staining of an internode cryosection from the pv4cll-26 plant. Scale bar = 50 μιη.

[31] FIGURE 12F depicts histological staining of an internode cryosection from the pv4cll-26 plant. Scale bar = 50 μιη.

[32] FIGURE 13 A depicts tillers at the reproductive stage. The yellow boxes indicate the magnified portions shown in FIGURES 13D and 13E, respectively. Scale bar = 0.5 cm.

[33] FIGURE 13B depicts hand-sectioned stem without staining (control). Scale bar = 0.5 cm.

[34] FIGURE 13C depicts hand-sectioned stem without staining (pv4cll-26). Scale bar = 0.5 cm.

[35] FIGURE 13D depicts a magnified view of the control plant. Scale bar = 0.5 cm.

[36] FIGURE 13E depicts a magnified view of the pv4cll-26 plant. Scale bar = 0.5 cm.

[37] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.

DESCRIPTION

[38] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural, the word "a" or "an" means "at least one", and the use of "or" means "and/or", unless specifically stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise.

[39] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. [40] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[41] The present disclosure provides description to the accompanying drawings, in which some, but not all embodiments of the subject matter of the disclosure are shown. Indeed, the subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, rather, these embodiments are provided so that this disclosure satisfies all the legal requirements.

[42] All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[43] In some embodiments, a method is provided for selectively disrupting a target gene without disrupting other genes in a plant which includes steps of selecting a target gene, analyzing at least a portion of the nucleotide sequence of the target gene by comparison to the genome of the plant to identify a target sequence that is specific to the target gene, designing a guide RNA having a nucleotide sequence complementary to the target sequence and a scaffold sequence for Cas9 binding, and transforming at least one cell of the plant type with one or more nucleic acid molecules encoding Cas9 and the guide RNA.

[44] In some embodiments, a method is provided for selectively disrupting a target gene without disrupting other genes in a plant and includes the steps of selecting a target family of genes in a plant, selecting a target tissue in the plant, collecting a sample of the target tissue, measuring mRNA expression of each gene of the target family of genes in the target tissue, selecting one target gene from the target family of genes which is preferentially expressed at a higher level than the other genes in the target tissue, analyzing at least a portion of the nucleotide sequence of the target gene by comparison to the genome of the plant to identify a target sequence that is specific to the target gene, designing a guide RNA having a nucleotide sequence complementary to the target sequence and a scaffold sequence for Cas9 binding, and

transforming at least one cell of the plant type with one or more nucleic acid molecules encoding Cas9 and the guide RNA. [45] In the foregoing embodiments, the plant can be any type of plant. Preferably, the plant is a plant with a complex genome. By way of example but not limitation, the plant can have a polyploidy genome such as a tetraploid genome. In some embodiments, the plant is switchgrass. The at least one cell that is transformed can be any tissue capable of being transformed. By way of example but not limitation, the at least one cell can be a plant, a callus or a protoplast.

Methods for transformation can include conventional methods for delivering nucleic acid molecules such as, by way of example but not limitation, by Agrobacterium-mediated transformation, polyethylene glycol (PEG) or electroporation treatment of protoplasts, particle bombardment or other methods.

[46] The methods for testing mRNA expression can include any conventional methods and are known to one of skill in the art. By way of example but not limitation, the method can be qRT- PCR. In some embodiments, instead of measuring mRNA expression, the expression of a protein encodes by each gene may be measured by known tests in the art and the target gene selected on the basis of preferential expression of such protein from the target gene.

[47] The guide RNA of the present disclosure is any guide RNA is that is sufficient to edit a genomic region of a cell. Such guide RNA may include a Cas9 scaffold portion which can bind Cas9 to effectuate the nuclease activity whereby such guide RNA is sufficient to edit a genomic region in a cell.

[48] The nucleic acid molecules encoding Cas9 and the guide RNA may be the same molecule or separate molecles. The nucleic acid molecules may be exposed to the cells at a concentration and ratio sufficient to edit a genomic region of the cells.

[49] In some embooimdents, a method is provided for reducing lignin-content in a switchgrass plant and includes the steps of desiging a guide RNA for Pv4CLl, transforming at least one switchgrass cell with one or more nucleic acid molecules encoding CRISPR associated protein 9 (Cas9) and said guide RNA, and generating a plant from said at least one switchgrass cell.

[50] In some aspects, the present disclose includes plants derived from the methods disclosed herein and having a disruption in the target gene. Such plants can be generated or propogated by known methods in the art. In some aspects, where the plant has a complex genome, all alleles for the target gene are knocked out. The present disclosure also contemplates seeds produced from such plants. EXAMPLES

[51] The following example demonstrates a specific embodiment employing the methods of the present disclosure to produce mutant plants without the integration of foreign DNA into the genome.

Example 1

[52] For purposes of this Example, the Pv4CL family of genes in switchgrass {Panicum virgatum) was selected as a target for gene knockout using CRISPR/Cas9. The coding sequences of Pv4CLl (EU491511.1) and PvCL2 (JF414903) were known and available from NCBI. Genetic modification of lignin biosynthesis in switchgrass is desirable because switchgrass can be used for biofuel production. Production of a low-lignin switchgrass would be beneficial. A key target is the 4CL ligase, a key enzyme related to the early steps of the monolignol biosynthesis pathway. 4CL catalyzes the conversion from /?ara-coumaric acid to /?ara-coumaroyl-CoA which is a substrate for different branches of the pathway of

phenylproanoid metabolism. Switchgrass is a known outcrossing tetraploid species having a complex genome.

[53] Identification and Phytogeny of Pv4CL genes. Prior to designging a specific guide RNA to target either Pv4CLl alone or both Pv4CLl and Pv4CL2, it was necessary to identify the genomic sequence of Pv4CL to find suitable target sites for the guide RNA. The sequence of Zm4CL (AY566301) from maize, which was known, was blasted against the switchgrass genome using the switchgrass genome dtabase, switchgrass v.1.1 (available at

http://phytozome.jgi.doe.gOv/pz/portal.html#). The blast search using Zm4CL yielded four coding sequences from Panicum virgatum vl.l: Pavir.Fa01395, Pavir.J20148, Pavir.Da00296, and Pavir.Db00533.

[54] These four sequences were compared with the known coding sequences of Pv4CLl and Pv4CL2. Pavir.Fa01395 was found to be identical to Pv4CLl and Pavir.J20148 was found to be identical to Pv4CL2. Pv4CLl was found to be on chromosome 6a while Pv4CL2 was found to be on chromosome la.

[55] However, Pavir.Da00296 and Pavir.Db00533 did not match any known coding sequence for a Pv4CL gene. Both of these sequences were found to have all of the characteristics of 4CL enzymes— the AMP -binding domain, GEICIRGR motif, the VPP, and the PVL domains. The two sequences were found on chromosomes 4a and 4b, respectively. Further, these two sequences were found to have 97% protein identity. Without being bound to theory, in light of the properties of and homology between these two sequences as well as their respective locations, it is expected that these are separate alleles found in disomic inherited tetraploid plants, rather than different genes. The two sequences were denoted Pv4CL3a and Pv4CL3b. A protein sequence alignment of Pv4CL3a and Pv4CL3b is shown in FIGURE 1.

[56] A phylogenic tree for Pv4CLl, Pv4CL2 and Pv4CL3 was generated which included the 4CL sequences from other model plants as shown in FIGURE 2. Phylogenetic analysis was done using Neighbor Joining of MEGA7 (http://www.megasoftware.net). The phylogenic tree was linearized assuming equal evolutionary rates in all lineages. The evolutionary distances were compared using Poisson correction methods and are in the units of the number of amino acid substitutions per site. Switchgrass, brachypodium and foxtail millet protein sequences were obtained from Phytozome v.11, while rice and maize protein sequences were obtained from NCBI. It was observed the Pv4CLl was located in close proximity to Pv4CL3 while it was more distant from Pv4CL2. Identities between the protein sequences of the various 4CL proteins in the phylogenic tree were also determined as shown in Table 1 below. Specifically, Pv4CLl was found to have 60% protein identity with Pv4CL2 while Pv4CL3 was found to have an 83% and 62% identity to Pv4CLl and Pv4CL2, respectively.

Table 1

Protein Sequence Identities Among Pv4CLl and Homologous Proteins

[57] Expression Analysis in Leaf, Node and Internode Tissues. Stems consist of internodes and nodes, which are highly lignified tissues composed of parenchyma and sclerenchyma cells distributed in the interfascicular region and the vascular sheath. In order to determine the major gene for targeting among Pv4CLl, Pv4CL2 and Pv4CL3, the expression of each was determined by qRT-PCR in internode, node and leaf tissues from switchgrass.

[58] Samples of leaves, nodes, and internodes of switchgrass plants of genotype FCX1 were collected at the Rl reproductive stage. Total RNA was extracted from each sample using 1 ml TR1 Reagent^® (Sigma, Missouri, USA) and 0.1 ml of l-bromo-3-chloropropane (MRC, Ohio, USA) according to the manufacturer's instructions. Sample mixtures were placed for 5 minutes at room temperature after shaking well. The sample mixtures were centrifuged for 15 minutes at 4 °C. 400 μΐ of supernatant from each sample was transferred to a new 1.7 ml tube and 300 μΐ of isopropanol was added. The 1.7 ml tube was then centrigued for 8 minutes at 4 °C. The resulting pellet was washed with 75% ethanol.

[59] 2 μg of RNA were reverse transcribed through reverse transcriptase using the Superscript III kit (Invitrogen) and 18 mer oligo dT after treatment with TURBO™ DNase I (Ambion, Austin, TX). Primers were designed to amplify the 3' UTR sequences of Pv4CLl, Pv4CL2 and Pv4CL3 and are listed in Table 2 below. Pvllbi was used as a reference. The Ct values of qRT- PCR were generated by an ABI PRISM 7900 HT sequence detection system (Applied

Biosystems). Changes in gene expression were calculated via the ΔΔ<_¾ method. The relative expression levels are shown in FIGURE 3 which provides the mean +/- standard deviation for three replicates.

Table 2

Primers Used for Pv4CLl, Pv4CL2 and Pv4CL3

[60] As shown in FIGURE 3, Pv4CLl transcripts were more abundant in the internode and the node rather than in the leaf. Pv4CL2 transcripts were barely detectable in the three different tissues and Pv4CL3 was preferentially expressed in the leaf only. In view of these results Pv4CLl was targeted for knockout because it was preferentially detected in highly lignified internodes rather than in leaf tissue while Pv4CL2 was barely detected in leaves, nodes and internodes and Pv4CL3 was highly expressed in leaves which were less lignified than nodes and internodes.

[61] Target Site Identification and Guide RNA Design. Switchgrass phenotype API 3 has been sequenced and the sequence information was released in Phytozome. The sequences of Pv4CLs from AP13 were used to identify corresponding genes in the tissue culture responsive genotype FCX1. The API 3 genomic Pv4CLl sequence was amplified using the

PV4CL1F12/PV4CL1R12 primer pair to single out the target region for genome editing in

FCX1. Flanking the target region of Pv4CLl, four single nucleotide polymorphisms (S Ps) were discovered which distinguish subgenome A from subgenome B. Guide RNA was designed using CHOPCHOP v2 (chop-chop.cbu.uib.no), E-CRISP Design (http://www.e-crisp.org), and the gRNA sequences were double-checked by a local blasting function provided by Bioedit (https://www.bioedit.com). The switchgrass transcripts library was downloaded from

Phtyozome (https://phtyozome.jgi.doe.gov) and NCBI (https://www.ncbi.nlm.nih.gov), and saved in Bioedit. Cadidates were screened and the best spacer was identified using the local blast function of Bioedit. A 20-bp target site was designed before the CGG PAM in order to edit Pv4CLl in both subgenomes as shown in FIGURE 4A. The target site was also compared to the Pv4CL2 and Pv4CL3 genomic sequences from the switchgrass genotype NFCXl as shown in FIGURE 4B. The target site matched 17 nt/20nt as compared to Pv4CL2 and 19 nt/20 nt as compared to Pv4CL3 as shown in FIGURE 4B.

[62] Spacers were cloned into pRGEB32, which contains the rice OsUbi2 promoter in front of Cas9 and the OsU3 promoter in front of the gRNA. The pRGEB32 carrying the 4CL spacer was transferred into the Agrobacterium tumefaciens strain AGL1. Primers used for the gRNA are shown in Table 3 below. Table 3

Primers Used for gRNA

[63] Genetic Transformation of Switchgrass Plants. Transgenic switchgrass plants of genotype NFCXl were obtained by Agrobacterium-mediated transformation using the method of Xi et al. (2009) and the previously obtained Agrobacterium containing the pRGEB32 vector with the 4CL spacer. All transgenic switchgrass plants were regenerated from independent callus lines. Switchgrass plants used in these experiments were grown in the greenhouse at 26 °C with 16 hour light (390 μΕ m^"2 s^"1). 39 independent transgenic plants were obtained.

[64] Identification of Homozygous Mutants. In order to identify the edited Pv4CLl sequence in transgenic switchgrass plants, a serial PCR/sequencing method was designed as shown in FIGURE 5. As shown in FIGURE 5, genomic DNA was extracted from transgenic plantlets and used for PCR amplification with Pv4CLl-, Pv4CL2- and Pv4CL3 -specific primer pairs. The PCR products were then sequenced directly and were compared to the target region of the switchgrass genotype NFCXl . Four Pv4CLl mutant plants were identified and denoted: 25, 26, 28 and 29. No edited mutants were observed for Pv4CL2 or Pv4CL3. The four plants were used for a second round of PCR amplification with Pv4CLl -speicifc primers. The PCR products were cloned into a TA cloning vector. Twenty colonies for each individual edited plant were sequenced.

[65] As shown in FIGURE 6, the second round of sequencing showed that the four plants each had their own mutation patterns: a nucleotide deletion is indicated by a minus sign "-" while an insertion is indicated by a plus sign "+". Four mutation patterns (-29/+18; -27; -32; -213) from the plant pv4cll-25, two mutation patterns (-27; -32/+16) from pv4cll-26, two mutation patterns (-27; -1) from pv4cll-28, and three mutation patterns (-44; -22; -14/+7) from pv4cll-29 were identified as shown in FIGURES 6-7B. Amplicon deep sequencing of the lines confirmed Pv4CLl mutations in the alleles of subgenomes A and B, while no mutations were found in Pv4CL2, Pv4CL3a and Pv4CL3b. The mutant plants were transferred to soil and grown in the greenhouse.

[66] Characterization of Mutant Plants. The internodes of transgenic plants (pv4cll-25, pv4cl- 26 and pv4cll-29) and a control plant at the Rl stage were harvested by removing the leaf blades and sheath. The internodes were chopped into 2-3 cm chips and then ground. The ground samples were washed three times with chloroform/methanol (2: 1, v/v), methanol, and water as described by Chen et al. (2007 and 2013). The remaining cell wall residue (CWR) was lyophilized. The extractive-free CWR was used to quantify lignin content and lignin-monomer compositions. The acetyl bromide (AcBr) method was used to quantify lignin content. 20 mg CWR was treated with 5 ml of 25% (v/v) acetyl bromide in glacial acetic acid for 4 hours at 50 °C. The samples were then cooled and centrifuged at 3500 rpm for 5 minutes. 4 ml of the top layer was then transferred to a 50 ml volumetric flask containing 10 ml of 2 M NaOH and 12 ml of acetic acid. 1 ml of 0.5 M hydroxylamine was added to each flask and the samples were diluted to 50 mL with acetic acid. Absorption spectra (250-350 nm) were determined for each sample and were used to determine the absorption maxima at 280 nm. A molar extinction coefficient of 17.2 was used for all samples. Results for the quantification of total lignin are shown in FIGURE 8. As shown in FIGURE 8, plants pv4cll-25, pv4cll-26, and pv4cll-29 showed 20, 30 and 8% reductions in AcBr lignin content compared to the control.

[67] Thioacidolysis was carried out to analyze lignin monomers. For each of two controls and the transgenic plants, a 20 mg sample of CWR was treated with 3 ml of 0.2 M boron trifluoride etherate in a 8.75: 1 dioxane/ethanethiol mixture for 4 hours at 100 °C. After cooling, deionized water and saturated sodium bicarbonate were added, and the organic solvent was extracted twice with methane chloride and dried with anhydrous sodium sulfate. The solvent was dried under N₂ gas and derivatized by adding 75 μΐ of pyridine and 75 μΐ MSTFA at 37 °C for 30 minutes. The derivative samples were analyzed by FC/MS to measure the monolignol composition. Lignin- derived monomers (S, G and H) were identified and quantified using a Hewlett-Packard 5890 series II gas chromatograph with a 5971 series mass selective detector (column, HP-1, 60 m x 0.25 mm x 0.25 μπι film thickness). Mass spectra were recorded in electron impact mode (70 eV) with 60-650 m/z scanning range. The resulting measurements of lignin monomers and total lignin are shown in Table 4 below. Table 4

[68] As shown in Table 4, compared to the controls, both pv4cll-25 and pv4cll-26 showed 10 and 25% reductions in S lignin, respectively, and 45 and 51% reductions of G lignin,

respectively, while no change of H lignin was observed for either. Similarly, pv4cll-29 showed an 8%) reduction in S and G lignins and no change in H lignin. The S/G ratios were 1.2, 1.4 and 0.9 for pv4cll-25, pv4cll-26, and pv4cll-29, respectively, while the S/G ratio for the controls was 0.9.

[69] Samples were also analyzed by 2D HSQC NMR to determine hydroxycinnamate and interlinkage content. Each samples was solvent-extracted with DI water for 24 hours and an ethanol/toluene mixture (1 :2, v/v) for 12 hours. The extractive-free samples were air-dried in a hood. About 500 mg of the extractive-free samples were ball-milled, and then hydrolyzed using enzyme mixtures which included C-Tec2 and H-Tec2 in 20 mM sodium acetate buffer solution (pH = 5.0) at 50 °C for 48 hours (2 x 24 hours). The recovered lignin-enriched residues were treated with protease for 24 hours to remove residual enzymes. Cellulytic enzyme ligning (CEL) was extracted from the enzymatic residues by 96% toluene for 48 hours. The extracts were concentrated via roto-evaporation and freeze-dried. The obtained lignin sample (-15 mg) was dissolved in 0.1 ml of DMSO-de for NMR analysis using a Shigemi NMR tube. NMR spectra were acquired at 298 K using a Bruker Avance III 400 MHz console equipped with a 5-mm BBO probe. Two-dimensional ¹H-¹³C heteronuclear single quantum coherence (HSQC) spectra were collected using a Bruker standard pulse sequence ('hsqcetgpsi2'). The central DMSO solvent peaks (5H/5C = 2.49/39.5 ppm) were used for chemical shift calibration. Volume integration of cross peaks in HSQC spectra was carried out using Bruker' s TopSpin 2.1 software. Aromatic regions of the NMR spectra of wild-type of pv4cll-26 are shown in FIGURE 9. A semiquantitative analysis of hydroxycinnamates and interlinkages is shown in Table 5 below.

Table 5

[70] NMR analysis provided structural information of lignins including the abundance of hydroxycinnamates and relative distribution of interunit linkages. The content of

hydroxycinnamates (pCA and FA) showed various levels of changes. Compared to control, ferulate (FA) in pv4cll-25, -26, and -29 increased 2, 5 and 2% respectively. For /?-coumarate (pCA), the increases were 1, 26, and 10%. The composition of interunit linkages, β-Ο-4 (C-0 bond), β-5 (C-C bond), and β- β (C-C bond) were analyzed regarding lignin polymer structure. The β-Ο-4 (C-0 bond) was slightly increased in pv4cll-25 and pv4cll-26 plants and was not changed in pv4cll-29. The β-5 (C-C bond) was slightly decreased in pv4cll-25 and pv4cll-26 plants but was not changed in pv4cll-29. The β- β (C-C bond) was barely detectable in control and mutant plants. Thus, the mutants did not show significant change in lignin polymer structure.

[71] The polysaccharide compositions of the cell wall were measured for the mutants and control. Results for the analysis are shown in Table 6 below.

Table 6

[72] The contents of glucose and xylose were slightly increased in pv4cll-25 and pv4clI-26, while pv4cll-29 showed no change. The contents of arabinose and galactose were not changed in the pv4cll plants.

[73] Biomass saccharification effiency was analyzed by enzymatic hydrolysis without acid pretreatment. Dried, Wiley-milled switchgrass was analyzed for sugar-release efficiency. About 250 mg of smaples (oven-dry weight) was loaded in 50 mM citrate buffer solution (pH 4.8) with Novozymes Ctec2 (70 mg protein per g-biomass). Sugar release was conducted at 50 °C with 200 rpm in an incubator shaker. Liquid hydrolysate was periodically collected at 0, 6, 12, 24, 48, and 72 hours, and enzymes in the hydrolysate were deactivated in boiling water before carbohydrate analysis. Released sugars in each hydrolysate were measured using a Dionex ICS- 3000 ion chromatography system. The results for glucose release and xylose release in shown in FIGURES 1 OA and 10B.

[74] As shown in FIGURES 10A-10B, more enzymatic sugar (glucose/xylose) was released in mutants compared to the control at different timepoints. At 72 hours, the pv4cll mutant lines produced more glucose in the range of 7-11% and more xylose from 23-32%. [75] Switchgrass internode samples from Rl stage tillers of a control plant and both pv4cll-25 and pv4cll-26 were also collected in the greenhouse and immediately frozen in liquid nitrogen. The middle portions of internode two were cut into 30 μιη sections with a Leica CM 1850 cryostat (Leica Microsystems Inc., Buffalo Grove, Illinois) at -25 °C. The cryosections were transferred to glass slides, thawed, stained, and covered with coverslips. Lignin was stained with 0.5% aqueous safranin-0 (Sigma, St. Louis, Missouri) (w/v) dissolved in 50% ethanol or 0.5% toluidine blue-0 (Sgima, St. Louis, Missouri) (w/v) dissolved in 50% ethanol. These stains increased the contrast of cell walls for bright field microscopy and at the same time reduced their autofluorescence when viewed under UV illumination. Photographs were taken using a Nikon Optiphot-2 microscope system with NIS-Elements F3.0 (Nikon Instruments Inc., Laguna Hills, California).

[76] As shown in FIGURE 11, the primary cell wall embodied thin and bright green lines, while the secondary cell wall had thick and dark green bands in a cross section of a switchgrass internode. Secondary cell wall was observed in the outer parenchyma cells and was mainly distributed in the interfascicular region and the vascular sheath, which were composed of sclerenchyma cells as shown in FIGURES 12A-12F. Compared to the control, pv4cll-25 and pv4cll-26 showed thinner secondary cell wall in the sclerenchyma and in the parenchyma cells as observed in FIGURES 12A-12F. At the seedling and vegetative stages, the pv4cll-26 plant showed normal green color in the leaf and the stem. However, at the reproductive stage, the plant exhibited purple color in the stem (without staining) while the leaf color had no change as shown in FIGURES 13A-13E.

[77] The foregoing results demonstrate homologous mutation of the four Pv4CLl alleles without any mutation of Pv4CL2 or Pv4CL3 at a rate of 10% (4/39) after Agrobacterium- mediated transformation. Each mutant plant had its own mutation pattern which occurred at a single target site. The presence of four different mutation patterns is reasonable given that switchgrass has four alleles for each gene. Tetra-allelic mutations were observed, with all four alleles simultaneously knocked out which is significant for outcrossing or vegetatively propogated polyploidy species. By targeting a 4CL homolog with preferential expression in lignified tissues, mutant plants were produced with reduced lignin content and improved sugar release. [78] The foregoing description of specific embodiments of the present disclosure has been presented for purpose of illustration and description. The exemplary embodiments were chosen and described in order to best explain the principles of the disclosure and its practical application, to thereby enable others skilled in the art to best utilize the subject matter and various embodiments with various modifications are suited to the particular use contemplated.

Claims

What is claimed is:

1. A method for selectively disrupting a target gene without disrupting other genes in a plant, comprising:

selecting a target gene in a plant; analyzing at least a portion of the nucleotide sequence of the target gene by comparison of said nucleotide sequence of said target gene to the nucleotide sequence of the genome of the plant to determine a target sequence that is specific to the target gene; designing a guide RNA having a nucleotide sequence complementary to the target sequence and a scaffold sequence for Cas9 binding for CRISPR-mediated disruption of the target gene; and transforming at least one cell of said plant type with one or more nucleic acid molecules encoding CRISPR associated protein 9 (Cas9) and said guide RNA.

2. The method of claim 1, further comprising:

generating a plant from said at least one cell of said plant type having a disruption in said target gene.

3. The method of claim 2, further comprising:

generating a seed from said plant generated from said at least once cell of said plant type having a disruption in said target gene.

4. The method of claim 1, wherein said plant has a polyploid genome.

5. The method of claim 1, wherein said plant is switchgrass.

6. The method of claim 1, wherein said at least one cell is selected from a callus, a protoplast, and a plant.

7. The plant produced by the method of any one of claims 1-6.

8. A seed produced by the plant of claim 7.

9. A method for selectively disrupting a target gene without disrupting other genes in a plant, comprising: selecting a target family of genes in a plant; selecting a target tissue in said plant; collecting samples of said target tissue from said plant; measuring the mRNA expression of each gene of said target family of genes in said target tissue; selecting one target gene from said target family genes that is preferentially expressed at a higher level than the other genes in said target family of genes in said target tissue; analyzing at least a portion of the nucleotide sequence of the target gene by comparison of said nucleotide sequence of said target gene to the nucleotide sequence of the genome of the plant to determine a target sequence that is specific to the target gene; designing a guide RNA having a nucleotide sequence complementary to the target sequence and a scaffold sequence for Cas9 binding for CRISPR-mediated disruption of the target gene; and transforming at least one cell of said plant type with one or more nucleic acid molecules encoding CRISPR associated protein 9 (Cas9) and said guide RNA.

10. The method of claim 9, further comprising:

generating a plant from said at least one cell of said plant type having a disruption in said target gene.

11. The method of claim 10, further comprising:

generating a seed from said plant generated from said at least once cell of said plant type having a disruption in said target gene.

12. The method of claim 9, wherein said plant has a polyploid genome.

13. The method of claim 9, wherein said plant is switchgrass.

14. The method of claim 9, wherein said at least one cell is selected from a callus, a protoplast, and a plant.

15. The plant produced by the method of any one of claims 9-14.

16. A seed produced by the plant of claim 15.

17. A method for reducing lignin in a switchgrass, comprising:

designing a guide RNA for Pv4CLl;

transforming at least one switchgrass cell with one or more nucleic acid molecules encoding CRISPR associated protein 9 (Cas9) and said guide RNA;

generating a plant from said at least one switchgrass cell having a disruption in said Pv4CLl gene.

18. The method of claim 17, wheein said plant has a disruption in all four alleles of said Pv4CLl gene.