Systems and methods for mapping single guide RNA crispr libraries in plants

Abstract

The present invention relates to system and methods for robust mapping of single guide RNAs (sgRNAs) in plants, the methods enable the association of specific sgRNA sequences with individual plants.

Description

[0001] SYSTEMS AND METHODS FOR MAPPING SINGLE GUIDE RNA CRISPR

[0002] LIBRARIES IN PLANTS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to systems and methods for robust mapping of single guide RNAs (sgRNAs) in plants, the methods enabling the association of specific sgRNA sequences with individual plants at a large-scale mapping.

[0005] BACKGROUND OF THE INVENTION

[0006] CRISPR-based genetic screens are instrumental tools in modern genetic research, particularly in the realm of plant breeding. These screens operate by inducing targeted mutations in specific genes, often resulting in knockout mutations that disable the gene's function. This process allows exploring gene functions in a highly controlled manner, providing valuable insights into the roles of genes in various biological processes. At the core of CRISPR-based genetic screens is the concept of a CRISPR library. This library comprises a collection of different single guide RNAs (sgRNAs) designed to target specific genes or gene families of interest. Each sgRNA within the library is capable of guiding the CRISPR endonuclease protein to a particular gene, where it induces mutations that disrupt gene function. The versatility of CRISPR libraries makes them invaluable in plant breeding programs. For instance, if researchers aim to study genes associated with promoting drought resistance in plants, they can compile a list of candidate genes involved in this process. Subsequently, a CRISPR library can be designed with sgRNAs targeting these specific genes. By bulk transformation of the CRISPR library into a population of plants, each plant is genetically edited in one or several of the targeted genes. Through comprehensive screening and analysis, plants exhibiting desired drought- related phenotypes can be identified, and analyzed for the presence of sgRNA(s). This approach allows for targeted genetic modifications that can ultimately contribute to the development of drought-resistant crop varieties.

[0007] However, since the library is transformed as a bulk into plants, a major shortcoming of these screens is tracking the CRISPR constructs within the transformed plant library and mapping them in the field as part of the breeding process. Therefore, only plants with significant phenotypes are selected for “regular” Sanger sgRNA sequencing and further research, leaving all other plants out of the analyses, primarily due to the high costs and resources.

[0008] There is a need in the field of plant breeding programs for efficient, high throughput technologies for mapping sgRNAs CRISPR libraries.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides efficient and robust methods and systems for mapping single guide RNA (sgRNA) libraries, preferably to be used in plants. The methods described herein have shown to be highly efficient in plant genetic, enabling precise identification and manipulation of genetic traits.

[0011] The methods described herein comprise the step of PCR amplification of DNA segments containing sgRNAs using combinations of PCR primers having unique barcodes. The amplification step is followed by pooling the PCR products and deep sequencing for a large-scale tagging of plants, in terms of the ability to associate an sgRNA or multiple sgRNAs with individual plants. In some embodiments, the methods described herein comprise providing DNA samples from plants that were transformed with different sgRNAs.

[0012] The sgRNA mapping CRISPR-GuideMap system described herein, enables to reveal the identity of the inserted sgRNA in each and every plant within the transformed CRISPR library. All plants in the library may be sequenced, allowing one to identify which sgRNA is present in each plant, regardless of whether or not it has a phenotype of interest at first sight. This immensely improves the robustness and the potential of a breeding process, as it increases the amount of obtainable data from the plant library and enhances the system's overall utility by enabling more complete utilization of its capabilities.

[0013] The present invention is based, in part, on the unexpected results demonstrating the ability of the systems and methods of the invention to associate specific sgRNA(s) with specific plants in an efficient and accurate way. In a single step of deep sequencing employing unique barcodes as described herein, it is possible to map sgRNAs in many plants including the possibility of identifying and associating multiple sgRNAs with individual plant. The ability to detect specific sgRNAs in individual plants within a population, as well as to identify multiple sgRNAs in a single plant when present, is a unique capability that cannot be achieved using Sanger sequencing.

[0014] Advantageously, the methods described herein support high-throughput experimentation, allowing a large number of plant samples to be processed and analyzed simultaneously, making it possible to extract substantial amounts of data from population of plants in an efficient and cost-effective manner. The methods described herein significantly reduce both the time and resources required for large-scale studies, offering a powerful tool for research and development, particularly in the field of plant breeding.

[0015] According to certain aspects, the present invention provides a method for mapping one or more single guide RNAs (sgRNAs) in a population of plants, the method comprising:

[0016] (i) amplifying a plurality of DNA segments from a plurality of DNA samples of a population of plants, wherein each of the DNA samples comprises at least one polynucleotide encoding a single sgRNA or multiple sgRNAs and wherein the amplification comprises a PCR reaction using a forward primer and a reverse primer each having a DNA sequence complementary to a common sequence located upstream or downstream of the polynucleotides encoding the sgRNA, and wherein each of the forward and reverse primers further comprises at least one DNA sequence barcode, further wherein each DNA sample is amplified with a unique combination of barcodes;

[0017] (ii) pooling of the PCR reaction products; and

[0018] (iii) sequencing the pooled products using deep sequencing; thereby mapping the sgRNAs in the population of plants.

[0019] According to some embodiments, step (iii) further comprises analysis of the sequences to associate one or more specific sgRNA(s) with individual plants.

[0020] According to some embodiments, each DNA sample is amplified with forward and reverse primers, each having a different barcode. According to other embodiments, each DNA sample is amplified with forward and reverse primers having the same barcode, wherein each DNA sample assigned a unique barcode.

[0021] According to some embodiments, the PCR reaction products are amplicons of 100- 400 bp. According to certain embodiments, the amplicons are of 100-200 base pairs. According to certain embodiments, the method comprises the generation of amplicons of about 150 base pairs.

[0022] According to some embodiments, the method comprises a step of transforming the plants with a library of polynucleotides each encoding at least one sgRNA molecule. According to certain embodiments, the library comprises polynucleotides each encoding a single sgRNA molecule. The transformation step is performed before step (i) of the method. According to some embodiments, the transformation is a stable transformation. According to other embodiments, the transformation is transient. According to some embodiments, the transformation is performed using Agrobacterium-mediated transformation or particle bombardment.

[0023] According to some embodiments, the polynucleotides encode a single sgRNA.

[0024] The common DNA sequences located upstream and downstream the polynucleotide encoding the sgRNA are used for the amplification step.

[0025] According to some embodiments, the library comprises at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, 2000, 3000, 4000, 5000 or more polynucleotides encoding unique sgRNAs. Each possibility represents a separate embodiment of the invention.

[0026] According to some embodiments, the sgRNAs target a certain family of genes. According to some embodiments, the sgRNAs target genes of a certain pathway within a plant. According to some embodiments, the sgRNAs target genes encoding proteins that perform a similar function. According to some embodiments, the sgRNAs target different sequences within the same gene. According to other embodiments, the sgRNAs target random sequences. According to some embodiments, the family of genes comprises at least 10, 20, 30, 40, 50, 100, 200, 300 or more different genes. According to some embodiments, the family of genes comprises 5-100, 10-50, 50-100, 50-200, or 200-400 different genes. According to some embodiments, each of the barcodes has a length of between 1- 15 nucleic acids. According to some embodiments, each of the barcodes has a length of between 6-8 nucleic acids. According to some embodiments, each of the barcodes has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleic acids. Each possibility represents a separate embodiment of the invention.

[0027] According to some embodiments, the common sequence located upstream or downstream of the polynucleotide encoding the sgRNA comprises at least 10, 15, 20, 25, 30 or more nucleotides.

[0028] According to some embodiments, the common sequence located upstream or downstream of the polynucleotide encoding the sgRNA comprises 5-25, 10-15, 15-25, or 20-25 nucleotides. According to certain exemplary embodiments, the common sequence located upstream or downstream of the polynucleotide encoding the sgRNA comprises about 20 nucleotides.

[0029] According to some embodiments, the common sequences are 17-25 bp in length. According to some embodiments, the common sequences are designed to be specific, in that they do not occur naturally within the plant genomic DNA. According to some embodiments, the common sequences are complementary to primers having a GC content of 40-60%. According to some embodiments, the common sequences are complementary to primers having a GC content of 45-55%. According to some embodiments, the common sequences are complementary to primers having Melting Temperature (Tm) of 55-65°C. According to some embodiments, the common sequences are complementary to primers having minimal or no secondary structures such as hairpins, self-dimers, or cross-dimers.

[0030] According to some embodiments, the primers used for the PCR reaction further comprise Unique Molecular Identifiers (UMI) for the identification of specific plant populations.

[0031] According to some embodiments, the method comprises amplifying of at least 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more individual DNA segments. Each possibility represents a separate embodiment of the invention.

[0032] According to some embodiments, the method is capable of identifying the presence of a plurality of different sgRNAs within an individual plant. According to some embodiments, the method is capable of identifying the presence of at least two, at least three, at least four, or at least five different sgRNAs within an individual plant. Each possibility represents a separate embodiment of the invention.

[0033] According to some embodiments, the PCR reaction comprises more than 5, 10, 15, 20, 25, 30, 35 or 40 cycles. According to some embodiments, the PCR reaction comprises between 10 and 15 cycles, between 15 and 20 cycles, between 20 and 25 cycles, between 25 and 30 cycles between 30 and 35 cycles, or between 20 and 25 cycles. Each possibility represents a separate embodiment of the invention.

[0034] According to some embodiments, the PCR reaction is performed using a proofreading polymerase.

[0035] The deep sequencing and / or analysis may be performed using any method as is known in the art. According to some embodiments, the sequencing is performed using a next generation sequencing (NGS) method. According to some embodiments, the sequencing is performed using a next generation sequencing (NGS) method based on the Illumina sequencing platform.

[0036] According to certain exemplary embodiments, the sequencing is deep sequencing using PE150 (paired-end 150) or PE250 (paired-end 250).

[0037] According to some embodiments, the method further comprises a step of adding to the PCR product a next generation sequencing region or adapter. According to certain embodiments, this step is performed prior to step (iii).

[0038] According to some embodiments, at least one plant in the population of plants further comprises a nucleic acid sequence encoding an RNA-guided DNA endonuclease enzyme. According to some embodiments, the endonuclease is selected from the group consisting of caspase 9 (Cas9), Cpfl, Casl2, or other Cas proteins. According to certain exemplary embodiments, the endonuclease is Cas9.

[0039] According to some embodiments, the sgRNAs target genes. According to some embodiments, the sgRNAs target coding sequences. According to some embodiments, the sgRNAs target non-coding sequences. According to certain embodiments, the sgRNAs target genetic regulatory elements. According to certain embodiments, the sgRNAs target at least one of a promoter, a terminator, an enhancer, a silencer or a combination thereof. According to additional embodiments, the sgRNAs target promoters. According to other embodiments, the sgRNAs target a combination of different genetic elements, such as coding regions, promoters and / or any other regulatory elements (e.g. enhances, repressors).

[0040] According to some embodiments, the population of plants comprises at least one sgRNA targeting more than one gene.

[0041] According to some embodiments, each of the polynucleotides encodes a single sgRNA. According to additional embodiments, each of the polynucleotides encodes multiple sgRNA multiplexed together. According to additional embodiments, the polynucleotides encode multiple different sgRNAs multiplexed together. According to other embodiments, the polynucleotides encode multiple identical sgRNAs multiplexed together.

[0042] It is to be understood that the methods disclosed herein are compatible with plants comprising sgRNA - CAS complexes used for generating gene knockout as well as other CRISPR applications, including, but not limited to, CRISPR activation, CRISPR inhibition / repression, prime-editing / knock-inn, and CRISPR epigenetics.

[0043] The plants to be used in the method of the present invention can be wild type plants as well as plant cultivars, the later can be hybrid lines or inbred lines. According to certain embodiments, the plants are monocot plants. According to other embodiments, the plants are dicot plants. According to certain embodiments, the plants are of the same species.

[0044] According to some embodiments, the plant is selected from the group consisting of a wild plant, a crop plant, and a model plant.

[0045] According to certain embodiments, the crop plant is selected from the group consisting of tomato, rice, maize, wheat, barley, potato, eggplant, sesame, soybean, sunflower, canola, sugarcane, alfalfa, millet, Leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, and cotton. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the crop plant is tomato. According to certain further exemplary embodiments, the crop plant is rice.

[0046] According to some embodiments, the model plant is Arabidopsis.

[0047] According to certain embodiments, the plant is a genetically modified plant. According to certain embodiments, the plant is a non-transgenic plant.

[0048] Since most genome-editing techniques can leave behind minimal traces of DNA alterations evident in a small number of nucleotides as compared to transgenic plants, crop plants created through gene editing could avoid the stringent regulation procedures commonly associated with genetically modified (GM) crop development, and are typically defined as non-transgenic crop plants

[0049] According to some embodiments, the sgRNAs target candidate genes for examining an agricultural trait selected from the group consisting of yield, harvest index, growth rate, biomass, plant vigor, root system, leaf color, rosette size, plant height, flowering time, photosynthetic capacity, nitrogen use efficiency, biotic stress resistance, abiotic stress resistance and any combination thereof. Each possibility represents a separate embodiment of the invention.

[0050] According to an additional aspect, the present invention provides a kit for mapping single guide RNAs (sgRNAs) in a population of plants, wherein each plant comprises at least one polynucleotide encoding a single sgRNA or multiple sgRNAs, the kit comprising a plurality of forward and reverse primer pairs, wherein each of the primers comprises a barcode and a sequence complementary to a common sequence located upstream or downstream of the polynucleotides encoding sgRNA; and instructional material for the use of the kit.

[0051] According to some embodiments, the kit comprises a plurality of DNA constructs for preparing a sgRNA library, the DNA construct have common sequences complementary to the forward and reverse primers. The common upstream DNA sequences are distinct from the common downstream DNA sequences.

[0052] According to some embodiments, the kit comprises means for PCR reaction. According to certain embodiments, the kit comprises proofreading polymerase, reaction buffer, dNTPs, and / or Taq polymerase.

[0053] According to some embodiments, the kit comprises means for next generation sequencing.

[0054] According to certain aspects, the present invention provides a system for mapping single guide RNAs (sgRNAs) in a population of plants, the system comprises plant DNA constructs designed for incorporating and expression of sgRNAs, wherein the incorporating sites are flanked with common sequences, and the system further comprises PCR primers complementary to the common sequences, each having unique 5’ overhangs (barcodes).

[0055] It is to be understood that any combination of each of the aspects and the embodiments disclosed herein is explicitly encompassed within the disclosure of the present invention.

[0056] Other objects, features and advantages of the present invention will become clear from the following description and drawings.

[0057] BRIEF DESCRIPTION OF THE FIGURES

[0058] Figures 1A-1G. CRISPR-GuideMap tool and analysis. Fig 1A - Graphic representation of barcode primer design. Figs IB- ID - Graphs showing the distribution of unique sgRNAs in plants and the frequency of occurrence in (Fig. IB) tomato, (Fig. 1C) Arabidopsis, and (Fig. ID) rice. Figs. 1E-1G - Graphs showing the number of sgRNAs per plant across transformed plants in tomato (Fig. IE), Arabidopsis (Fig. IF), and rice (Fig. 1G).

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention discloses compositions and methods for robust mapping of single guide RNAs (sgRNAs) in plants that were transformed with a library of sgRNAs, the methods enabling the association of specific sgRNA sequences with individual plants. Advantageously, the methods described herein enable in-planta high throughput screening of plants for identifying sgRNAs in individual plants.

[0061] The CRISPR-GuideMap sgRNA mapping described herein involves individually amplifying the sgRNA region of each plant comprising the same with a combination of primers with unique 5’ overhangs (Fig. la). These about 8-nucleotide long overhangs serve as barcodes for the sample. Each sample has a unique combination of 2 barcodes that allows the identification of the individual plant source from which a given sgRNA sequence was harbored. All individual PCR amplification products are pooled together (hundreds or thousands of pooled plants) and undergo deep sequencing, for example, by using PE 150 (paired-end 150) or PE250, in which each individual amplicon is sequenced from both 5’ and 3’ ends creating an overlapping region for initial verification. By using n unique forward primers and n unique reverse primers, an nA2 combinations of barcodes can be obtained. For example, a library of 1024 samples will require 32 forward primers and 32 reverse primers. Computational post-processing of the raw data reveals the number and identity of the harbored sgRNA in every plant.

[0062] The CRISPR-GuideMap tool is compatible with all types of CRISPR libraries: CRISPR libraries of a single sgRNA or multiple sgRNA (multiplexed sgRNAs). In addition, the tool fits CRISPR knockouts of all types of CAS enzymes (CAS9, CAS 12), CRISPR activation, CRISPR inhibition, CRISPR methylation, CRISPR to promoters or other regulatory regions, cell-type specific CRISPR and more.

[0063] Definitions

[0064] As used herein, the term "a plurality" refers "at least two", typically more than two.

[0065] As used herein, the term “about” when combined with a value refers to ± 10% of the reference value.

[0066] As used herein the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of such compounds. It should be noted that the term “and” or the term “or” are generally employed in their sense including “and / or” unless the context clearly dictates otherwise.

[0067] According to certain aspects, the present invention provides a method for mapping one or more single guide RNAs (sgRNAs) in a population of eukaryotic organisms, the method comprising:

[0068] (i) amplifying a plurality of DNA segments from a plurality of DNA samples of a population of eukaryotic organisms, wherein each of the DNA samples comprises at least one polynucleotide encoding a single sgRNAs and wherein the amplification comprises a PCR reaction using a forward primer and a reverse primer each having a DNA sequence complementary to a common sequence located upstream or downstream of the polynucleotides encoding the sgRNA, and wherein each of the forward and reverse primers further comprises at least one DNA sequence barcode, further wherein each DNA sample is amplified with a unique combination of barcodes;

[0069] (ii) pooling of the PCR reaction products; and

[0070] (iii) sequencing the pooled products using deep sequencing; thereby mapping the sgRNAs in the population.

[0071] According to preferred embodiments, the population of eukaryotic organisms is of plants.

[0072] The term “common sequence” when describing a region upstream or downstream of the sgRNA, refers to a sequence that is shared among DNA segments encoding the different sgRNAs within the same region (upstream or downstream), but the upstream common sequence is distinct from the downstream common sequence. Each plant within the population has the same common sequence upstream or downstream to its unique sgRNA.

[0073] According to some embodiments, the barcodes have a length of between 1-15 nucleic acids. According to some embodiments, the barcodes have a length of between 4- 10, 6-8, 5-9, 6-10, or 8-12 nucleic acids. According to some embodiments, the barcodes have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleic acids. Each possibility represents a separate embodiment of the invention. According to some embodiments, the barcodes have a length of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleic acids. According to certain exemplary embodiments, the barcodes have a length of 8 nucleic acids.

[0074] The primers' barcodes are a domain that uniquely identifies the sample source of the nucleic acid being sequenced to enable sample multiplexing by marking every molecule from a given sample (e.g. from a single plant) with a specific barcode or "tag”.

[0075] The term “library” as used herein refers to a collection of similarly sized DNA fragments, a collection that includes several different items. The term “CRISPR library" is used herein to describe a collection of constructs comprising polynucleotides encoding sgRNAs and optionally, additional means for CRISPR, such as nucleic acids encoding an RNA-guided DNA endonuclease enzyme.

[0076] Clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems are known in the art and can be engineered for directed genome editing. Cas genes encode RNA-guided DNA endonuclease enzymes capable of introducing a double strand break in a double helical nucleic acid sequence. The Cas enzyme can be directed to make the double stranded break at a target site within a gene using the single guide RNA (sgRNA) and tracer cellular machinery.

[0077] The terms "single guide RNA", "sgRNA" and "gRNA" are used herein interchangeably and refer to a piece of RNA that function as guides for RNA- or DNA- targeting enzymes, which they form complexes with. The targeting specificity of the CRISPR / Cas system is determined by a short sequence (e.g., 20-nt) at the 5' end of the gRNA. The desired target sequence must precede the protospacer adjacent motif (PAM). After base pairing of the gRNA to the target, Cas mediates a double strand break about 3-nucleotides (nt) upstream of PAM. In some embodiments, the unique sgRNA used herein comprises a targeting sequence designed to match only one target site within the plurality of DNA samples.

[0078] The term “sgRNA library” refers to the DNA constructs comprising the DNA encoding sgRNAs and common sequences as described herein.

[0079] The term sgRNA, as described in the application, is context-dependent as is known in the field. For example, identification of an sgRNA in a plant is carried out by identifying the polynucleotide encoding it. Similarly, the common DNA sequences upstream or downstream to the sgRNA refer to sequences adjacent to the polynucleotides encoding the sgRNA.

[0080] According to some embodiments, the plant further comprises polynucleotide encoding a Cas enzyme. A Cas enzyme can be from any appropriate species (e.g., an archaea or bacterial species). For example, a Cas enzyme can be from Streptococcus pyogenes, Pseudomonas aeruginosa, or Escherichia coli. In some cases, a Cas enzyme can be a type I (e.g., type IA, IB, IC, ID, IE, or IF), type II (e.g., IIA, IIB, or IIC), or type III (e.g., IIIA or IIIB) Cas enzyme. The encoded Cas enzyme can be any appropriate homolog or Cas fragment in which the enzymatic function (i.e., the ability to introduce a sequence-specific double strand break in a double helical nucleic acid sequence) is retained. In some embodiments, a Cas enzyme is a Streptococcus pyogenes Cas9 enzyme. In some cases, a Cas enzyme can be codon optimized for expression in particular cells, such as dicot or monocot plant cells. The Cas enzyme can further be a protospacer- adjacent motif (PAM) edited variant, including, for example, the Cas9 enzyme variants SpG and SpRY.

[0081] According to some embodiments, the polynucleotides encoding a single sgRNA.

[0082] Each of the polynucleotides encoding different sgRNA comprises common upstream and downstream DNA sequences that are used for the amplification step.

[0083] According to some embodiments, the library comprises at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000 or more polynucleotides encoding unique sgRNAs. Each possibility represents a separate embodiment of the invention.

[0084] According to some embodiments, the sgRNAs target a certain family of genes. According to some embodiments, the sgRNAs target genes of a certain pathway within a plant. According to some embodiments, the sgRNAs target genes encoding proteins that perform a similar function. According to some embodiments, the sgRNA target different sequences within the same gene. According to other embodiments, the sgRNA target random sequences. According to some embodiments, the family of genes comprises at least 10, 20, 30, 40, 50, 100, 200, 300 or more different genes. According to some embodiments, the family of genes comprises 5-100, 10-50, 50-100, 50-200, or 200-400 different genes.

[0085] According to some embodiments, the common sequence located upstream or downstream of the polynucleotide encoding the sgRNA comprises at least 5, 10, 12, 14, 15, 18, 20, 25, 30 or more nucleotides. Each possibility represents a separate embodiment of the invention.

[0086] According to some embodiments, the common sequence located upstream or downstream of the polynucleotide encoding the sgRNA comprises 5-25, 10-15, 15-25, or 20-25 nucleotides. According to certain exemplary embodiments, the common sequence located upstream or downstream of the polynucleotide encoding the sgRNA comprises about 20 nucleotides.

[0087] The common sequences are designed for efficient PCR amplification process as is known in the art. According to some embodiments, the common sequences are 17-25 bp in length. According to some embodiments, the common sequences are designed to be specific, in that they do not occur naturally within the plant genomic DNA. According to some embodiments, the common sequences are complementary to primers having a GC content of 35-70%. According to some embodiments, the common sequences are complementary to primers having a GC content of 40-60%. According to some embodiments, the common sequences are complementary to primers having a GC content of 40-65%. According to some embodiments, the common sequences are complementary to primers having a GC content of 45-65%. According to some embodiments, the common sequences are complementary to primers having Melting Temperature (Tm) of 55-65°C. According to some embodiments, the common sequences are complementary to primers having Melting Temperature (Tm) of 50-65°C, 53-63°C. or 55-65°C. According to some embodiments, the common sequences are complementary to primers having Melting Temperature (Tm) of about 58°C. According to some embodiments, the common sequences are complementary to primers having minimal or no secondary structures such as hairpins, self-dimers, or cross-dimers.

[0088] According to some embodiments, the primers used for the PCR reaction further comprises Unique Molecular Identifiers (UMI) for the identification of specific plant populations. According to some embodiments, the UMIs comprises 5-15, 4-14, 6-12, or 8-10 nucleic acids in length. The incorporation of unique molecular identifiers (UMIs) facilitates the screening of multiple plant populations and enhances the robustness and analytical capabilities of the methods described herein.

[0089] According to some embodiments, the method is capable of identifying the presence of a plurality of different sgRNAs within an individual plant. According to some embodiments, the method is capable of identifying the presence of at least two, at least three, at least four, or at least five different sgRNAs within an individual plant. Each possibility represents a separate embodiment of the invention.

[0090] According to some embodiments, the PCR reaction comprises more than 5, 10, 15, 20, 25, 30, 35 or 40 cycles. According to some embodiments, the amplification step is a PCR reaction comprises between 10 and 15 cycles, between 15 and 20 cycles, between 20 and 25 cycles, between 25 and 30 cycles between 30 and 35 cycles, or between 20 and 25 cycles. According to some embodiments, the PCR reaction comprises between 15 and 25 cycles, between 20 and 30 cycles, between 25 and 35 cycles, between 30 and 40 cycles between 35 and 45 cycles, or between 20 and 25 cycles. Each possibility represents a separate embodiment of the invention.

[0091] According to some embodiments, the PCR reaction is performed using a proofreading DNA polymerase. Non limiting examples of DNA polymerases are Kapa HiFi Hot Start DNA Polymerase (Roche), NEB Q5 Hot Start DNA Polymerase (NEB), PrimeStar GXL Hot Start DNA Polymerase (Takara) and High Fidelity Hot Start DNA Polymerase (Qiagen). According to certain embodiments, the PCR reaction is performed using the Rapid Taq Master Mix by Vazyme.

[0092] The methods of the invention disclose the production of PCR reaction products for in depth sequencing followed by computational analysis. Acceptable methods for next generation sequencing (NGS), including polynucleotide adapters and hybridization blockers, are known in the art.

[0093] The commonly used NGS workflows implement the steps of library preparation, including an adapter addition or ligation, surface attachment, and in-situ amplification.

[0094] The adapters suitable for NGS in some embodiments, are incorporated during the steps of amplification. These procedures are more efficient than the addition of adapters using ligation from both sides. According to certain embodiments, this step is performed prior to step (iii).

[0095] “Sequencing" refers to reading a sequence of nucleotides out of a DNA library to produce a set of sequencing reads which can be processed by a bioinformatics computer in a bioinformatics workflow. High throughput sequencing (HTS) or next-generation- sequencing (NGS) refers to real time sequencing of multiple sequences in parallel, typically between 50 and a few thousand base pairs per sequence. Exemplary NGS technologies include those from Illumina, Ion Torrent Systems, Oxford Nanopore Technologies, Complete Genomics, Pacific Biosciences, BGI, and others. Depending on the actual technology, NGS sequencing may require sample preparation with sequencing adapters or primers to facilitate further sequencing steps, as well as amplification steps so that multiple instances of a single parent molecule are sequenced, for instance with PCR amplification prior to delivery to flow cell in the case of sequencing by synthesis. The NGS protocol will vary depending on the particular NGS sequencing system employed. Detailed protocols for sequencing an NGS library, e.g., which may include further amplification (e.g., solid-phase amplification), sequencing the amplicons, and analyzing the sequencing data are available from the manufacturer of the NGS sequencing system employed.

[0096] According to certain embodiments, the NGS adapters are added to the library in a separate step. According to some embodiments, the NGS is directly employed on the PCR products. According to certain embodiments, the NGS workflows comprises steps of DNA fragmentation, DNA end-repair, surface attachment, and in-situ amplification. Fragmentation can be done, for instance, by mechanical shearing, sonification, enzymatic fragmentation and other methods. After fragmentation, the DNA pieces may be end repaired to ensure that each molecule possesses blunt ends. To improve ligation efficiency, an adenine may be added to each of the 3' blunt ends of the fragmented DNA, enabling DNA fragments to be ligated to adapters with complementary dT-overhangs. These methods result in a "DNA-adapter product" that is compatible with a nextgeneration sequencing workflow.

[0097] According to certain exemplary embodiments, the sequencing is deep sequencing using PE150 (paired-end 150) or PE250 (paired-end 250).

[0098] According to some embodiments, the method further comprises a step of obtaining DNA sample from a plant tissue. The plant tissue may include, but is not limited to, leaf, root, stem, flower, seed, or fruit tissue. The sample may be processed to extract genomic DNA using any method known in the art, for example, the CTAB-based protocol, optionally supplemented with additives such as polyvinylpyrrolidone (PVP) to remove polyphenols and RNase to eliminate RNA contaminants. Following DNA extraction, the sample is subjected to polymerase chain reaction (PCR) amplification using primers that comprise nucleotide barcode sequences, allowing for the unique labeling of each sample.

[0099] According to some embodiments, the plants further comprise a nucleic acid sequence encoding an RNA-guided DNA endonuclease enzyme. According to some embodiments, the endonuclease is selected from the group consisting of Caspase (Cas) endonuclease, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN). According to certain exemplary embodiments, the endonuclease is selected from the group consisting of caspase 9 (Cas9), Cpfl, Casl2, additional Cas proteins, or other DNA editing proteins. According to certain exemplary embodiments, the endonuclease is Cas9.

[0100] According to some embodiments, the sgRNAs target genes. According to some embodiments, the sgRNAs target coding sequences. According to some embodiments, the sgRNAs target non-coding sequences. According to certain embodiments, the sgRNAs target genetic regulatory elements. According to certain embodiments, the sgRNAs target at least one of a promoter, terminator, enhancer or silencer. According to additional embodiments, the sgRNAs target promoters. According to other embodiments, the sgRNAs target a combination of different genetic elements, such as coding regions, promoters and / or any other regulatory elements (e.g. enhances, repressors).

[0101] According to some embodiments, at least one plant comprises at least one sgRNA targeting more than one gene.

[0102] According to some embodiments, the polynucleotides encode a single sgRNA. According to additional embodiments, at least one of the DNA samples comprises a polynucleotide encoding multiple sgRNA multiplexed together. According to additional embodiments, at least one of the DNA samples comprises a polynucleotide encoding multiple different sgRNAs multiplexed together. According to other embodiments, each of the polynucleotides encode multiple identical sgRNAs multiplexed together.

[0103] It is to be understood that the methods disclosed herein are compatible with plants comprising sgRNA - Cas complexes used for generating gene knockout as well as other CRISPR applications, such as CRISPR activation, CRISPR inhibition / repression, prime- editing / knock-inn, and CRISPR epigenetics.

[0104] The term “plant” is used herein in its broadest sense. It includes, but is not limited to, any species of woody, herbaceous, perennial, or annual plant. The plants to be used in the method of the present invention can be wild type plants as well as plant cultivars, the latter can be hybrid lines or inbred lines. According to certain embodiments, the plants are monocot plants. According to other embodiments, the plants are dicot plants. According to certain embodiments, the plants are of the same species.

[0105] According to some embodiments, the plant is selected from the group consisting of a wild plant, a crop plant, and a model plant. According to certain embodiments, the crop plant is selected from the group consisting of tomato, rice, maize, wheat, barley, potato, eggplant, sesame, soybean, sunflower, canola, sugarcane, alfalfa, millet, Leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, and cotton. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the crop plant is tomato or rice.

[0106] According to some embodiments, the model plant is Arabidopsis.

[0107] According to certain embodiments, the plant is a genetically modified plant. According to certain alternative embodiments, the plant is a non-genetically modified plant.

[0108] According to some embodiments, the sgRNAs target candidate genes for examining an agricultural trait selected from the group consisting of yield, harvest index, growth rate, biomass, plant vigor, root system, leaf color, rosette size, plant height, flowering time, photosynthetic capacity, nitrogen use efficiency, biotic stress resistance, abiotic stress resistance and any combination thereof. Each possibility represents a separate embodiment of the invention.

[0109] According to an additional aspect, the present invention provides a kit for mapping single guide RNAs (sgRNAs) in a population of plants, the kit comprises a plurality of forward and reverse primer pairs, each of the primers comprises a barcode and a sequence complementary to a common sequence located upstream or downstream of the polynucleotides encoding sgRNA; and instructional material for the use of the kit.

[0110] According to some embodiments, the kit comprises a plurality of DNA constructs for preparing a sgRNA library, the DNA construct have common sequences complementary to the forward and reverse primers.

[0111] According to some embodiments, the kit comprises means for PCR reaction. According to certain embodiments, the kit comprises proofreading polymerase, reaction buffer, dNTPs, and / or Taq polymerase.

[0112] According to some embodiments, the kit comprises means for next generation sequencing.

[0113] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.

[0114] EXAMPLES

[0115] Methodology

[0116] Generation of a tomato CRISPR library of -250 plants: The plants were transformed in bulk with hundreds of polynucleotides encoding different sgRNAs and therefore, it was not known which sgRNA(s) is in each plant. To address this challenge, the CRISPR-GuideMap was developed.

[0117] CRISPR-GuideMap Steps:

[0118] • DNA was extracted from -250 TO transgenic CRISPR library plants (TO generation) and the sequence surrounding the sgRNA insertion was amplified using PCR. Primers with no overhangs were tested to verify sufficient amplification of transgenic TO plants from the library (Forward 5'- cacatcgcttagataagaaaacg-3' (SEQ ID NO: 1), Reverse 5'- cctaggtaatgccaactttgtac- 3' (SEQ ID NO: 2). PCR settings: annealing 54°, elongation 5 sec, 30 cycles, X2 Rapid Taq Master Mix by Vazyme.

[0119] • 64 barcode sequences were designed, each of 8 bp in length. These barcodes are error-prone and to ensure optimized PCR amplification, specific criteria were followed, including 40-60% G / C content, no consecutive triplets and / or avoiding self-complementarity. 32 of the sequences were designated to the 5' end of the Forward primers and the remaining 32 to the 5' end of the Reverse primers.

[0120] • When amplifying samples, each sample is amplified using a unique combination of barcode primers (using the same PCR parameters provided above). The length of the amplified fragment including the two 8 bp barcodes should be 222 bp in our plasmid, but could vary based on individual design. Once samples were amplified and individually verified via gel electrophoresis, the individual PCR products were pooled together and run on a gel using electrophoresis. The band was then cleaned from the gel using “Nucleospin Gel and PCR Clean-up Kit” by Macherey and Nagel, and a sample was sent for Paired End (PE-150) deep sequencing by Novogene.

[0121] • The analysis of deep sequencing PE 150 data commenced with rigorous quality checks to ensure the reliability of the reads. Each pair of reads was examined for an overlapping region of 78 bp, encompassing the sgRNA. Reads with discrepancies in this region were discarded. Additionally, reads containing barcode sequences at the 5’ end of reads that did not match the predefined list were removed from the dataset. Non-variable regions in the sequencing were scrutinized, and reads with mismatches, insertions, or deletions were removed as well. Once this process of filtering for high-quality reads was completed, pairs of reads were assigned to their respective plant. Once the origin plant was identified, the corresponding sgRNA sequence was assigned to that plant.

[0122] • To reduce noise, we categorized reads per plant based on their abundance and conducted a careful examination for adequate representation. Plants with the most abundant read accounting for less than 20% of all reads or with limited occurrences (<750 reads) were classified as having insufficient sequencing results. Plants where the most abundant read was at least three times more frequent than the next most abundant read, were identified as containing a single sgRNA. Subsequently, for the remaining plants, we analyzed the reads in order of abundance. When we encountered a read that was more than double the abundance of the following read, we determined the number of sgRNAs present in the plant, resulting in 2, 3, or in rare cases 4 sgRNAs.

[0123] Results

[0124] CRISPR-GuideMap results for mapping 250 TO transgenic plants in the field.

[0125] The sequence flanking the sgRNA insertion was amplified using PCR (Fig. 1A). 17 plants (6.7%) exhibited no PCR amplification, likely because of low DNA extraction or unsuccessful PCR amplification. Among the remaining plants (236), we found that 186 (78%) had a single sgRNA, 41 (17%) had 2 sgRNAs, and 9 plants (4%) harbored 3 or more sgRNAs (Fig. 1B-1E). These findings are in accordance with existing literature1. The barcoding-seq plants revealed the presence of a total of 146 unique sgRNAs among the 253 samples. The majority of these sgRNAs were observed relatively few times, which was crucial for achieving widespread sgRNA distribution throughout the library. However, we identified three sgRNAs that were significantly overrepresented, appearing 11, 24, and 37 times, respectively, across our sample set (Fig. IB, IE).

[0126] Summary

[0127] Identifying the specific sgRNA a transgenic plant encompasses is a crucial and frequent procedure comprised of DNA extraction, amplification of the sgRNA-containing region, and finally either Sanger or next-generation sequencing. However, during the process of Agrobacterium-mediated transformation occasionally more than one T-DNA insertion occurs. While such an event might bear less significance in the context of transforming a solitary construct, its ramifications are a considerable worry when applied to a comprehensive library transformation. When the plant harbors more than one sgRNA, two main challenges arise. Firstly, traditional Sanger sequencing approaches fall short of effectively separating the two sgRNAs present. Secondly, the attempt to correlate phenotypic traits with their underlying genetics is substantially more complicated due to another set of target genes whose mutations may be the true cause of a phenotype.

[0128] CRISPR-GuideMap enabled us to comprehensively map the CRISPR library in a crop field. The large-scale analysis of over 250 samples showed that 79% of plants showcased the presence of a solitary sgRNA, while the remaining samples bore two or more sgRNAs. This pattern of distribution aligned with prior research findings, corroborating the prevalence of multiple sgRNA insertions1,2. As opposed to Sanger sequencing, Deep sequencing is comprised of thousands of individual reads, enabling differentiation between various sgRNAs even within plants that harbored multiple such insertions. Despite this ability to distinguish between coexisting sgRNAs, our research leaned toward favoring the characterization of lines with a single sgRNA insertion. This preference stemmed from the desire to streamline analyses and optimize the possibility of decisively matching observed traits to specific genetic modifications.

[0129] To further demonstrate the versatility and wide applicability of the CRISPR- GuideMap system, we have successfully implemented it in tomato, Arabidopsis thaliana (Fig. 1C, IF), and rice (Fig. ID, 1G) with similar results, representing both dicot and monocot model and crop species. Importantly, each one of the libraries contained hundreds of sgRNAs across hundreds of independent plants. The libraries contained single sgRNA and multiplex sgRNAs (more than one sgRNA on one vector). These results highlight CRISPR-GuideMap as a broadly compatible tool for diverse plant systems and support its utility in a wide range of genetic screens and breeding efforts. Its integration into both research and agricultural pipelines across plant types reinforces its potential as a useful, large-scale technology.

[0130] To summarize, CRISPR-GuideMap offers a range of practical applications in genetic research and plant breeding:

[0131] 1. Reveal Library Distribution and Variance: CRISPR-GuideMap unveils the true distribution and variance of transformed libraries, offering a comprehensive overlook of the potential genetic variance generated.

[0132] 2. Identify Multiple sgRNAs in Single Plants: CRISPR-GuideMap enables the identification of plants harboring multiple sgRNAs, providing crucial insights into their genetic makeup that cannot be obtained through simple Sanger sequencing.

[0133] 3. Verify Genotype-Phenotype Causality: By identifying other plants within the transformed library with either the same or different sgRNAs targeting the same gene(s) of interest, genotype-phenotype causality can efficiently be verified. This example alone can significantly expedite research, potentially saving close to a year of work.

[0134] 4. Expand Homology Research: Expand homology research by searching for transformed plants with sgRNAs targeting neighboring genes on the phylogenetic tree. This approach enhances understanding of genetic relationships and evolutionary patterns.

[0135] 5. Design Specific High-Definition Screens: CRISPR-GuideMap allows for the design of specific high-definition screens targeting subgroups of plants and specific sets of genes. For instance, if the initial library targeted all transporters in Arabidopsis, researchers can conduct a more precise screen targeting only plants with sgRNAs targeting a specific group of transporters or genes involved in transporting specific molecules such as nutrients or phytohormones. This tailored approach enhances the efficiency and accuracy of genetic screenings and analyses.

[0136] 6. Reverse Genetics: CRISPR-GuideMap generates a complete map of available mutants within the field. Thus, the seeds of any one of the mutants can be provided upon request, opening a new commercial opportunity for selling genetic variation to customers.

[0137] Overall, CRISPR-GuideMap revolutionizes genetic research by offering unprecedented insights into library genetic compositions and distribution, leading to increased versatility and robustness of CRISPR libraries. This, together with other features such as an expedited verification process of phenotype-genotype correlation, and homology-based research, CRISPR-GuideMap paves the way for accelerated discoveries and advancements in plant biology and breeding.

[0138] References

[0139] 1. De Buck, S., Podevin, N., Nolf, J., Jacobs, A. & Depicker, A. The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell. Plant Journal 60, 134-145 (2009).

[0140] 2. Frary, A. & Hamilton, C. M. Efficiency and Stability of High Molecular Weight DNA Transformation: An Analysis in Tomato. Transgenic Research vol. 10 (2001).

[0141] 3. Hamady, M., Walker, J. J., Harris, J. K., Gold, N. J. & Knight, R. Error-correcting barcoded primers for pyro sequencing hundreds of samples in multiplex. Nat Methods 5, 235-237 (2008).

[0142] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

CLAIMS1. A method for mapping one or more single guide RNAs (sgRNAs) in a population of plants, the method comprising:(i) amplifying a plurality of DNA segments from a plurality of DNA samples of a population of plants, wherein each of the DNA samples comprises at least one polynucleotide encoding a single or multiple sgRNAs and wherein the amplification comprises a PCR reaction using a forward primer and a reverse primer each having a DNA sequence complementary to a common sequence located upstream or downstream of the polynucleotides encoding the sgRNA, and wherein each of the forward and reverse primers further comprises at least one DNA sequence barcode, further wherein each DNA sample is amplified with a unique combination of barcodes;(ii) pooling of the PCR reaction products; and(iii) sequencing the pooled products using deep sequencing; thereby mapping the sgRNAs in the population of plants.

2. The method of claim 1, wherein each DNA sample is amplified with forward and reverse primers each comprising a different barcode.

3. The method of any one of claims 1 or 2, the PCR reaction products are amplicons of 100-400 base pairs.

4. The method of any one of the preceding claims, wherein step (iii) further comprises analysis of the sequences to associate one or more specific sgRNA(s) with individual plants.

5. The method of any one of the preceding claims, comprising a step of transforming the plants with a library of polynucleotides each encoding at least one sgRNA molecule.

6. The method of claim 5, wherein the library comprises at least 50 polynucleotides encoding unique sgRNAs.

7. The method of claim 6, wherein the library comprises unique sgRNAs targetinga certain family of genes, targeting genes of a certain pathway within a plant, and / or targeting different sequences within the same gene.

8. The method of any one of the preceding claims, wherein each of the barcodes has a length of between 1-15 nucleic acids.

9. The method of any one of the preceding claims, comprising amplifying of at least 20 or more individual DNA segments.

10. The method of any one of the preceding claims, wherein the method is capable of identifying the presence of one or more different sgRNA within an individual plant.

11. The method of any one of the preceding claims, wherein the method is capable of identifying the presence of a plurality of different sgRNAs within an individual plant.

12. The method of any one of the preceding claims, wherein the common sequence located upstream or downstream of the polynucleotide encoding the sgRNA comprises at least 10 or more nucleotides.

13. The method of any one of the preceding claims, wherein the common sequence located upstream of the polynucleotide encoding the sgRNA and the common sequence located downstream of said polynucleotide are distinct.

14. The method of any one of the preceding claims, wherein the common sequence does not occur naturally within the plant genomic DNA, complementary to a primer having a GC content of 40-60%, and / or complementary to a primer having Melting Temperature (Tm) of 55-65°C.

15. The method of any one of the preceding claims, wherein the sequencing is performed using a next generation sequencing (NGS) method based on the Illumina sequencing platform.

16. The method of claim 15, wherein the sequencing is deep sequencing using PE 150 (paired-end 150) or PE250.

17. The method of any one of the preceding claims, wherein the plants further comprise a nucleic acid sequence encoding an RNA-guided DNA endonucleaseenzyme.

18. The method of claim 17, wherein the endonuclease is selected from the group consisting of caspase 9 (Cas9), Cpfl, Casl2, or other Cas proteins.

19. The method of any one of the preceding claims, wherein the sgRNAs target candidate genes for examining an agricultural trait selected from the group consisting of yield, harvest index, growth rate, biomass, plant vigor, root system, leaf color, rosette size, plant height, flowering time, photosynthetic capacity, nitrogen use efficiency, biotic stress resistance, abiotic stress resistance and any combination thereof.

20. The method of any one of the preceding claims, wherein the sgRNAs target coding regions, non-coding regions, regulatory elements or any combinations thereof.

21. The method of any one of the preceding claims, wherein each of the polynucleotides encodes a single sgRNA.

22. The method of any one of claims 1-20, wherein at least one of the DNA samples comprises a polynucleotide encoding multiple sgRNAs multiplexed together.

23. The method of claim 22 wherein each of the polynucleotides encodes multiple different sgRNA multiplexed together.

24. The method of any one of the preceding claims, wherein the plants comprising sgRNA - CAS complexes used for generating gene knockout, CRISPR activation, CRISPR inhibition / repression, prime-editing / knock-inn, CRISPR epigenetics, and / or combinations thereof.

25. The method of any one of the preceding claims, wherein the plant is selected from the group consisting of a wild plant, a crop plant, and a model plant.

26. The method of claim 25, wherein the crop plant is selected from the group consisting of tomato, rice, maize, wheat, barley, potato, eggplant, sesame, soybean, sunflower, canola, sugarcane, alfalfa, millet, Leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, and cotton.

27. The method of claim 25, wherein the model plant is Arabidopsis.

28. A kit for mapping single guide RNAs (sgRNAs) in a population of plants, wherein each plant comprises at least one polynucleotide encoding a single sgRNA or multiple sgRNAs, the kit comprising a plurality of forward and reverse primer pairs, wherein each of the primers comprises a barcode and a sequence complementary to a common sequence located upstream or downstream of the polynucleotides encoding sgRNA; and instructional material for the use of the kit.

29. The kit of claim 28, wherein when the kit comprises a plurality of DNA constructs for preparing an sgRNA library, the DNA constructs have common sequences complementary to the forward and reverse primers.

30. The kit of claim 29, further comprising means for PCR reaction.

31. The kit of claim 30, comprising proofreading polymerase, reaction buffer, dNTPs, and / or Taq polymerase.

32. A system for mapping single guide RNAs (sgRNAs) in a population of plants, the system comprises plant DNA constructs designed for incorporating and expression of sgRNAs, wherein the incorporating sites are flanked with common sequences, and the system further comprises PCR primers complementary to the common sequences, each having unique 5’ overhangs (barcodes).

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