RU2785923C1 - METHOD FOR DETERMINING THE GENOME-WIDE LOCALISATION AND NUMBER OF COPIES OF T-DNA IN THE GENOME OF TRANSFORMED PLANTS USING Cas9-TARGETED NANOPORE SEQUENCING - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: presented is a method for determining the genome-wide localisation and number of copies of T-DNA in the genome of transformed plants using Cas9-targeted nanopore sequencing. The method includes the following stages: isolating the high molecular weight fraction of the DNA of individual transgenic plants (HMW DNA), pooling the DNA of individual transgenic plants; selecting gRNAs specific to the T-DNA region and synthesising gRNAs in vitro on a double-stranded DNA matrix molecule; assembling Cas9/gRNA ribonucleoprotein complexes; performing Cas9-targeted nanopore sequencing; identifying the T-DNA insertion sites in the plant pool using the NanoCasTE approach; selecting specific primers for each T-DNA insertion into regions flanking the insertion site; and genotyping individual plants.

EFFECT: new method for finding the location and number of copies of T-DNA in the genome of plants and creating markers for genotyping transformed plants using Cas9-targeted nanopore sequencing.

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Description

The technical field to which the invention belongs

The invention relates to the field of biotechnology and genetic engineering of plants, and in particular to a method for determining the genomic localization and copy number of Transgenic DNA (T-DNA) in the genome of transformed plants.

Prior Art

The place of genomic localization and the number of T-DNA copies in the genome of transformed plants are the most important characteristics for the further use of transgenic plants. Ignoring these characteristics can lead to unstable inheritance and expression of the transgene, phenotypic abnormalities not related to the functioning of T-DNA, as well as genetic cleavage and loss of the transgene in subsequent generations. In addition, rapid and accurate determination of the zygosity of the introduced sequences, as well as the structure of the loci they occupy, makes it possible to reduce the number of generations required to obtain lines with stable expression from the initial transformants. In the case of plants, this is most relevant, since the onset of the generative period in them can take from several months to tens of years. Modern transformation methods do not allow one to control the number of copies of inserted genes, their orientation, and location in the genome. In view of this, the presence of repeated, truncated, or rearranged copies of the introduced sequence can be observed in the genome of transformed plants, and the regions encoding native genes can also be disrupted. Molecular analysis of the introduced gene and the area of its integration allows the detection and study of a significant proportion of undesirable effects. The latter can be mediated, for example, by inserting the gene into an unfavorable location in the genome (native reading frame or region of low expression). In addition, when using the transformation method using Agrobacterium tumefaciens, integration of two or more copies of the transgene into one locus is often observed, which can lead to silencing of the transgene expression.

Various methods have been developed to determine the location of the transgene in the genome and the number of copies. Currently, for the molecular analysis of introduced alleles, such methods as Southern blotting (Southern E.M., et. al., 1975), quantitative PCR (qPCR) ((Ingham, D.J. et. al., 2001)), thermal asymmetric interlaced PCR (TAIL-PCR) (Liu Y.G., et. al., 2007), as well as digital drop PCR (ddPCR) (Collier, R. et. al., 2017) Southern blotting is one of the most accurate methods for determining the number of copies of alleles introduced into the genome. Compared to PCR-based methods, the disadvantages of Southern blotting are laboriousness and complexity, and often the impossibility of automation. In addition, the use of this method to determine the copy number of T-DNA in plants with a large genome is practically impossible ((Southern E.M., et. al., 1975)).

The qPCR method in the analysis of transformed plants involves a relative assessment of the amount of the original template (in this case, the sequence integrated into the genome), based on the fluorescence of an intercalating dye or a reporter DNA probe. Using qPCR, it is possible to determine the number of copies of introduced alleles, however, deviations in amplification efficiency, as well as the exponential nature of the increase in the amount of the original product, are factors that limit accuracy, especially with small differences in copy number. In addition, since qPCR allows quantification of the introduced gene sequence, it is necessary to somehow resort to progeny segregation analysis to confirm zygosity (Ingham, D.J. et. al., 2001).

Thermal asymmetric interlaced PCR can be used both to determine the number of copies and to localize inserted copies in the genome. This method involves the use of insert-specific primers used in conjunction with arbitrary degenerate primers. The resulting PCR products are subject to cloning, sequencing, and, as a rule, contain a part of the introduced gene sequence together with the genome flanking it. The main limitation of TAIL-PCR is the lack of amplification in the analysis of some of the insertions of the introduced gene, which often leads to underestimated results when counting the number of its copies. Also, the insertion of T-DNA into a region with repeated sequences can lead to artifacts (Liu Y.G., et. al., 2007).

Digital drip PCR also makes it possible to analyze the number of copies of alleles introduced into the genome. Like qPCR, ddPCR uses an intercalating dye or specific DNA probes, but the polymerase chain reaction itself proceeds independently in multiple microvolumes (droplets) into which the reaction mixture is divided. The analysis of single DNA molecules, implemented with ddPCR, allows achieving an accuracy comparable to Southern blotting, but this method does not allow determining the localization of the introduced genes (Collier, R. et. al., 2017).

A separate group of methods is associated with whole genome sequencing of transgenic plants using NGS methods followed by bioinformatic analysis of T-DNA insertion sites. Although this method is very informative, it has a number of disadvantages, namely: (1) the need for whole genome sequencing with high coverage (>10x), which is not economically viable when working with plants with a large genome and when analyzing a large number of plants; (2) dependence of the method on the initial assembly of the genome.

Thus, today there is no universal approach that allows one to determine the T-DNA insertion sites in different plant species with large and small genome sizes without resorting to costly whole genome sequencing.

The essence of the invention

The objective of the invention is to develop a new method for determining the location and number of copies of T-DNA in the plant genome, devoid of the limitations of the prior art and the creation of markers for genotyping transformed plants using Cas9-targeted nanopore sequencing.

To solve this problem, a method is proposed, which is based on targeted nanopore sequencing using Cas9/gRNA ribonucleoprotein complexes.

At the first stage, the high molecular weight fraction of DNA (HMW DNA) is isolated from individual transgenic plants by the CTAB method (Kirov I. et. al., 2021). The quantity and quality of the extracted DNA is assessed on spectrophotometers: NanoDrop One UV-Vis Spectrophotometer (Thermo Scientific, USA) and Quantus Fluorometer (Promega, USA). Only pure DNA is suitable for further studies (A260/A280 ~ 1.8 and A260/A230 ~ 2.0 according to NanoDrop data) with practically no differences in concentrations obtained using Nanodrop and Quantus devices.

In the next step, DNA from individual plants is pooled in equal amounts to produce at least 10 µg. DNA in 60 µl. nuclease-free water. Enrichment of the combined sample with long fragments is carried out using the Short Read Eliminator Kit (Circulomics, USA) according to the method recommended by the manufacturer.

Next, there is a bioinformatic selection of gRNA using the FlashFry program (McKenna A. et. al., 2018) specific for the T-DNA region and gRNA synthesis in vitro on a double-stranded DNA template molecule containing the sequence of the T7 promoter and the sequence obtained by combining two oligonucleotides, specific and universal, complementary to T-DNA borders. For the assembly of ribonucleoprotein (RNP) complexes, 50 ng of gRNA and 20 pmol of Cas9 nuclease (Biolabmix, Novosibirsk, Russia) are used.

Preparation of the DNA library for Cas9-targeted nanopore sequencing on the MinION device (Oxford Nanopore Technologies, UK) is carried out according to the protocols described in the article by Kirov I. et. al., 2021. First, the analyzed DNA is dephosphorylated by the Quick CIP enzyme (New England Biolabs, UK), then the dephosphorylated DNA is cleaved using Cas9/gRNA ribonucleoprotein complexes. Then protein adapters for sequencing ((Oxford Nanopore Technologies, UK) are ligated to the ends of the DNA free from the Cas9/gRNA ribonucleoprotein complexes using the SQK-LSK109 kit (Oxford Nanopore Technologies, UK). Sequencing is performed on a MinION instrument (Oxford Nanopore Technologies, UK). ), with flow cells R9.4.1 or R10.3.

The NanoCasTE approach developed by us (https://github.com/Kirovez/NanoCasTE) is used to identify T-DNA insertion sites in the analyzed plants. Insertions confirmed by 2 or more reads are used for analysis.

For genotyping individual plants, from the studied pooled DNA sample, using the Primer3 program, two sets of specific primers are selected for each insertion: one pair of primers flanking the insertion site for detection of variants without insertion and the second pair of primers with one primer (R) per site 35S promoter (universal primer to detect all insertions) and (F) primer on the flanking region. Primer specificity was assessed using the Primer-BLAST program.

Thus, the method described above for determining the genomic localization and copy number of T-DNA in the genome of transformed plants using Cas9-targeted nanopore sequencing makes it possible to determine the T-DNA insertion sites in different plant species with large and small genome sizes without resorting to expensive genome-wide sequencing and time-consuming Southern blotting.

Brief description of the drawings

Figure 1. Schematic of the pCambia vector carrying the coding sequence for GAG-eGFP for transformation.

Figure 2. Schematic of the pCambia vector carrying the coding sequence for eGFP-GAG for transformation.

Figure 3. High molecular weight DNA concentration measurements on Qubit 4 and Nanodrop Onec.

Figure 4. Data on chromosomal localization and the number of indicative reads, obtained by the proposed method, according to T-DNA insertions in a mixture of 5 A. thaliana transformant plants.

Figure 5. Example of detected T-DNA insertions in A. thaliana.

Example 1

Determination of genomic localization and T-DNA copy number in a pool of A. thaliana plants transformed with the pCambia vector with the target GAG gene using Cas9-targeted nanopore sequencing.

Plants - transformants of Arabidopsis thaliana generation T1 carrying the T-DNA insert with the target GAG gene were chosen as the object. Plants were obtained by transformation using the floral-dip method, followed by selection of transformants on a selective medium containing hygromycin (Kirov I. et. al., 2021). Plants were transformed with one of two pCambia vectors differing in the position of the reading frame for the eGFP protein relative to GAG: GAG-eGFP (FIG. 1) or eGFP-GAG (FIG. 2).

Seeds obtained from Arabidopsis thaliana Columbia ecotype plants transformed by the agrobacterial method were sown in pots (6 cm × 6 cm) with soil. Plants were grown in a light chamber for a month at a temperature of 20±1°C. and long day conditions (16 hours of light/8 hours of darkness). For the isolation of high molecular weight DNA, healthy developed leaf blades of plants were selected. Isolation of high molecular weight DNA was carried out in a fume hood using the CTAB method. Measurement of the concentration of high molecular weight DNA was performed using a Nanodrop Onec spectrophotometer and a Qubit 4 fluorimeter (Fig. 3).

The isolated high molecular weight DNA of five samples was mixed in equal proportions in 60 µl of nuclease-free water. Enrichment in long fragments was performed using the Short Read Eliminator Kit (Circulomics, USA).

Three gRNAs specific to the T-DNA region were designed for sequencing.

Guide RNAs (gRNAs) were obtained by in vitro transcription on a double-stranded DNA template molecule containing the T7 promoter sequence and obtained by combining two oligonucleotides, the specific TgagF1-2, TgagR1, and the universal CRISPR R:

TgagF1

TgagF2

TgagR1

CRISPR R

T7 F

T7R

For transcription, we used the T7-transcription kit for high-performance in vitro RNA synthesis (Biolabmix, Novosibirsk, Russia) in accordance with the manufacturer's recommendations. Analysis of the concentration and quality of the obtained gRNAs was carried out using a Nanodrop OneC (Thermo Scientific, USA), Qubit 4 (Thermo Scientific, USA) spectrophotometer and separation in a 2% agarose gel. 50 ng of each gRNA was used to assemble ribonucleoprotein (RNP) complexes. Guide RNAs were mixed in an equimolar ratio of 1:1 in 11 µl of RNase-free water. To remove secondary structures, the gRNA mixture was denatured at 95°C for 3 minutes, then placed on ice. RNP complexes were assembled by adding 1 µl of Cas9 nuclease and 3 µl of reaction buffer (Biolabmix, Novosibirsk, Russia) to 11 µl of the gRNA mixture, followed by incubation at room temperature for 30 minutes. At the end of the time, the tube containing the RNP complexes was placed on ice. Genomic DNA was preliminarily dephosphorylated using the Quick CIP enzyme (New England Biolabs, UK) followed by incubation at 37°C for 30 minutes. Enzyme inactivation was carried out at 80°C for 2 minutes. DNA cutting with RNP complexes was performed by combining 40 µl of dephosphorylated DNA, 15 µl of a mixture of RNP complexes, 1.5 µl of dATP (10 mM), and 1 µl of Encyclo polymerase (Evrogen, Moscow, Russia). The mixture was incubated at 37°C for 30 minutes, then 10 minutes at 72°C. Next, libraries were prepared using the SQK-LSK109 kit (Oxford Nanopore Technologies, UK).

The resulting DNA library was sequenced using the MinION instrument with cell R9.4. The sequencing time was about 24 hours and more than 78395 reads were collected with N50 ~1600 bp. For the detection of T-DNA insertions, we used the NanoCasTE approach developed by us (https://github.com/Kirovez/NanoCasTE). To determine the number of insertions, insertion detection was carried out, confirmed by 2 or more reads. After reaching the number of 56,000 reads, the number of insertions did not change and reached 7. Thus, T-DNA insertions in a mixture of samples from 5 plants were successfully identified by this method. Insertions were localized on 5 chromosomes of A. thaliana. The list of insertions identified as a result of the analysis is shown in FIG. 4. Detected insertions were confirmed by 2 or more reads (Fig. 5).

For genotyping of individual plants, two sets of specific primers were selected for each insertion: one pair of primers flanking the insertion site for detection of variants without insertion and the second pair of primers with one primer (R) per region of the 35S promoter (universal primer for detection of all insertions) and F primer on the flanking area. Primers were selected using the Primer3 program and tested for specificity using Primer-BLAST. Primers from the first group amplified fragments up to 700 bp. The list of primers is given below:

At the next stage, PCR was carried out with a combination of primers and DNA from individual plants. As a result, all 7 insertions were validated and data on chromosomal localization and the number of T-DNA loci in each of the analyzed plants were obtained.

The work on the creation of the present invention was financed from the funds of Agreement No. 075-15-2019-1667 dated October 31, 2019 on the provision of grants from the federal budget in the form of subsidies in accordance with paragraph 4 of Article 78.1 of the Budget Code of the Russian Federation for state support creation and development of the world-class center for genomic research "Kurchatov Genomic Center" within the framework of the federal project "Development of scientific and scientific-industrial cooperation" of the national project "Science".

Bibliography

Southern E.M., Edwin Mellor. Detection of specific sequences among DNA fragments separated by gel electrophoresis // J mol biol. - 1975. - T. 98. - No. 3. - C. 503-517.

Ingham, D.J., Beer, S., Money, S., & Hansen, G. Quantitative real-time PCR assay for determining transgene copy number in transformed plants // Biotechniques. - 2001. - T. 31. - No. 1. - C. 132-140.

Liu Y.G., Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences // Biotechniques. - 2007. - T. 43. - No. 5. - C. 649-656.

Collier, R., Dasgupta, K., Xing, Y.P., Hernandez, V.T., Shao, M., Rohozinski, D., … & Thilmony / Accurate measurement of transgene copy number in crop plants using droplet digital PCR // The Plant Journal. - 2017. - T. 90. - No. 5. - C. 1014-1025.

Ilya Kirov, Pavel Merkulov, Sofya Gvaramiya, Roman Komakhin, Murad Omarov, Maxim Dudnikov, Alina Kocheshkova, Alexander Soloviev, Gennady Karlov, Mikhail Divashuk (2021). Illuminating the transposon insertion landscape in plants using Cas9-targeted Nanopore sequencing and a novel pipeline. bioRxiv - 2021.

McKenna A, Shendure J. FlashFry: a fast and flexible tool for large-scale CRISPR target design. BMC biology. Dec 2018; 16(1):1-6.

Claims (9)

A method for determining the genomic localization and copy number of T-DNA in the genome of transformed plants using Cas9-targeted nanopore sequencing, which includes: isolation of the high molecular weight DNA fraction of individual transgenic plants (HMW DNA), pooling DNA samples from individual transgenic plants; selection of gRNA specific to the T-DNA region and in vitro synthesis of gRNA on a double-stranded DNA template molecule, assembly of ribonucleoprotein Cas9/gRNA complexes, Cas9-targeted nanopore sequencing, identification of T-DNA insertion sites in analyzed plants using the NanoCasTE approach, selection of specific primers for each T-DNA insertion to the regions flanking the insertion site, genotyping of individual plants.

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