RU2791879C1 - Crispr-cas12 system for detecting the exou gene encoding the type 3 secretion system exotoxin, pseudomonas aeruginosa, at ultra-low concentrations - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular, to a guide RNA molecule of the CRISPR-Cas system, a ribonucleoprotein complex of the CRISPR-Cas system containing the guide RNA molecule, and to kits containing a ribonucleoprotein complex of the CRISPR-Cas system and specific oligonucleotides.

EFFECT: invention provides the in vitro detection of single copies of the exoU gene of Pseudomonas aeruginosa.

10 cl, 9 dwg, 6 tbl, 5 ex

Description

The invention relates to the field of genetic engineering and biotechnology, and in particular to the number of guide RNA tools that can be used in CRISPR-Cas 12 systems as part of ribonucleoprotein complexes for the detection (detection, detection) of the exoU gene encoding the exotoxin of the third type secretion system, Pseudomonas aeruginosa .

EFFECT: invention allows in vitro detection of single copies of Pseudomonas aeruginosa exoU gene.

The guide RNAs described in this application can be used to detect the exoU gene encoding the exotoxin of the third type secretion system, Pseudomonas aeruginosa, after specific amplification of the DNA fragment of the exoU gene. Amplification in this case can be carried out in various ways, including polymerase chain reaction (PCR); loop isothermal amplification (LAMP); helicase-dependent amplification (HDA); recombinase-mediated amplification (RPA); strand displacement amplification (SDA); nucleic acid sequence based amplification (NASBA); transcription-mediated amplification (TMA); nicking enzyme mediated amplification (NEAR); circular amplification (RCA) and many other types of amplification.

The guide RNAs described in this application can be used to develop highly sensitive and high-tech next-generation diagnostic systems based on CRISPR technologies to combat the spread of antibiotic-resistant bacterial pathogens and strains that cause the most severe infection with a high risk of death.

To solve the epidemiological problems of deciphering outbreaks of infectious diseases, identifying and identifying the pathogen, as well as detecting specific bacterial genes, it is necessary to develop and introduce modern technologies of molecular epidemiology into the practice of surveillance and monitoring services. One of such technologies is the use of genetic editing elements of the CRISPR-Cas system. This technology is developing quite effectively in relation to the creation of drugs for the treatment of certain diseases, despite a number of difficulties associated with the occurrence of unforeseen mutations. With in-depth studies in the field of application of the CRISPR-Cas system, it was found that it can be used for subtle diagnostic procedures in identifying the pathogen/s of infection in humans, as well as their genotyping.

In 2018, it was shown that one of the enzymes of the CRISPR system, Cas12, after recognizing its target DNA target, begins to nonspecifically hydrolyze single-stranded and double-stranded DNA. This property of Cas12 can be used as an indicator of the presence of a specific target, such as the genome of a virus or bacteria. The researchers used this discovery to create a nucleic acid detection technology platform known as DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter). For the first time, DETECTR was used to detect and genotype the human papillomavirus (HPV). The proposed platform combines Cas12a nuclease, its HPV nucleic acid-specific guide RNA, and a fluorescent reporter molecule. DETECTR technology is used to detect target DNA after pre-amplification [J.S. Chen, E. Ma, L.B. Harrington, M. Da Costa, X. Tian, J.M. Palefsky, J.A. Doudna, CRISPR-Cas 12a target binding unleashes indiscriminate single-stranded DNase activity, Science 360(6387) (2018) 436-439].

An equally important application of the CRISPR-Cas system is the identification of bacterial pathogens and the detection of specific bacterial genes. For example, using the SHERLOCK platform, it was possible to correctly genotype a number of strains of Escherichia coli and Pseudomonas aeruginosa with low cross-reactivity. In addition, the SHERLOCK platform was used to differentiate clinical isolates of Klebsiella pneumoniae with two different antibiotic resistance genes, which opens up significant prospects for the creation of multiplex systems for the simultaneous identification of bacterial pathogens and detection of antibiotic resistance genes in them.

In this regard, the task of developing new effective methods for detecting antibiotic resistance genes and genes encoding exotoxins in bacterial pathogens based on genetic technologies such as CRISPR-Cas is extremely urgent.

Also known from the prior art are solutions aimed at developing and obtaining guide RNAs for regulating the composition of microbial populations using programmable nucleases, including CRISPR-Cas (patent US 10760065), sensitization and/or elimination of target pathogenic bacteria and disinfection (CN patents). 110029121, EP 3798304, WO 2019067621). According to patent RU 25313143, a solution is known for creating approaches based on CRISPR-Cas technologies for genotyping microorganisms.

In addition, there are scientific articles describing the use of the CRISPR-Cas genome directed genome editing system to identify and study host genes involved in Pseudomonas aeruginosa exotoxin ExoU cytotoxicity [V. Deruelle, S. Bouillot, V. Job et al., The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis, Nat Commun 12 (2021) 4024, https://doi.org/10.1038/s41467-021 -24337-9; S. Bagayoko, S.A. Leon-Icaza, M. Pinilla et al., Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology, PLoS pathogens 17(9) (2021) e1009927, https://doi.org/10.1371/iournal .ppat.10099271.

The closest analogues of the proposed solution are inventions according to patents RU 2743861, RU 2734520, RU 2745637, aimed at obtaining guide RNAs intended for the development of highly sensitive and high-tech new generation diagnostic systems for detecting the blaVIM-2 Pseudomonas aeruginosa antibiotic resistance gene based on CRISPR technologies. However, in order to expand the range of such diagnostic systems and improve methods for diagnosing infectious diseases, it is necessary to develop new methods for the detection of Pseudomonas aeruginosa, which have genetic markers other than blaVIM-2.

Based on the foregoing, a technical problem arises, which consists in the need to develop and obtain guide RNAs to detect single copies of the exoU gene encoding the exotoxin of the third type secretion system, Pseudomonas aeruginosa, in vitro.

The proposed technology is promising for a variety of applications, including DNA/RNA quantification, fast multiplex expression detection, and other types of sensitive detection, such as detection of sample contamination with nucleic acids. CRISPR-Cas based technology is a feature rich, error tolerant DNA detection technology suitable for rapid diagnosis, including infectious diseases, genotyping of infectious agents, and detection of antibiotic resistance and exotoxin-coding genes of bacterial pathogens.

The application of the proposed technology makes it possible to create a new generation of diagnostic systems that will have the following properties:

• high sensitivity;

• the possibility of carrying out diagnostics at the patient's bedside;

• the possibility of carrying out diagnostics in the field without the use of specialized high-tech equipment;

• speed and simplicity of analysis;

• reduced cost of analysis;

• no need to equip the diagnostic laboratory with expensive equipment;

• no need to isolate the nucleic acids of the pathogen.

The invention relates to new tools - guide RNAs that can be used in CRISPR-Cas12 systems for ultrasensitive detection, identification, detection or detection of the exoU gene encoding the exotoxin of the third type secretion system, Pseudomonas aeruginosa, in biological samples.

The technical objective of the proposed invention is the development of new tools - guide RNAs that can be used in CRISPR-Cas12 systems with Cas12 proteins, for example, LbCpf1 from Lachnospiraceae, for ultrasensitive detection of the Pseudomonas aeruginosa exoU gene.

In the implementation of the present invention, according to the set of essential features given in the claims, an unexpected technical result is achieved - the possibility of ultrasensitive detection of the Pseudomonas aeruginosa exoU gene down to single copies of DNA in one reaction. The invention has no commercially available analogues and is highly efficient in detecting the Pseudomonas aeruginosa exoU gene.

The technical result is achieved due to:

• development of guide RNA molecules that can be used in CRISPR-Cas12 systems for ultra-sensitive detection of the Pseudomonas aeruginosa exoU gene, where these guide RNAs are selected from the sequences of SEQ ID NO: 1-3, are able to bind to highly conserved target regions of the Pseudomonas aeruginosa exoU gene contain an RNA hairpin that is recognized by the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae, allowing detection of single copies of the Pseudomonas aeruginosa exoU gene.

• the use of RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae, obtained according to the method developed by the authors earlier (RF Patent No. 2707542, priority date 03/28/2019), to create ribonucleoprotein complexes (RPC) of the CRISPR-Cas system suitable for detecting the Pseudomonas exoU gene aeruginosa at ultra low concentrations (single copies).

• development of a set of specific oligonucleotides selected from SEQ ID NO: 4-7 for preliminary amplification of the Pseudomonas aeruginosa exoU gene fragment.

• optimization of conditions for preliminary amplification of the Pseudomonas aeruginosa exoU gene fragment.

• determining the conditions for ultrasensitive detection of the Pseudomonas aeruginosa exoU gene and establishing the sequence of the method steps.

A guide RNA molecule of the CRISPR-Cas system has been proposed, which is capable of binding to target highly conserved regions of the Pseudomonas aeruginosa exoU gene. The nucleotide sequence disclosed in this application guide RNA is selected from the sequences of SEQ ID NO: 1-3.

The guide RNA molecule contains a hairpin RNA, which is recognized by the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae.

The guide RNA molecule ensures the detection of single copies of the Pseudomonas aeruginosa exoU gene.

The guide RNAs of the present invention correspond to highly conserved fragments of the Pseudomonas aeruginosa exoU gene. Most preferred are guide RNAs recognized by the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae, characterized by having or containing a nucleotide sequence selected from:

• SEQIDNO: 1;

• SEQ ID NO: 2;

• SEQ ID NO: 3;

• or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 identical to any of them %, 94%, 95%, 96%, 97%, 98% or 99%,

• or complementary to any of them,

• or hybridizing with any of them under stringent conditions.

According to the proposed invention, ribonucleoprotein complexes (RPCs) are obtained, consisting of at least one guide RNA and an RNA-guided DNA endonuclease of the CRISPR-Cas LbCpf1 system from Lachnospiraceae, suitable for use to detect the exoU gene of Pseudomonas aeruginosa in ultra-low concentrations (single copies).

Ribonucleoprotein complex of the CRISPR-Cas system for detection of the Pseudomonas aeruginosa exoU gene, formed from the RNA-guided DNA endonuclease LbCpf1 from Lachnospiraceae, and at least one guide RNA.

The proposed ribonucleoprotein complex of the CRISPR-Cas system is suitable for detecting single copies of the Pseudomonas aeruginosa exoU gene.

PKK preparations are solutions containing a guide RNA selected from SEQ ID NO: 1-3 combined with a CRISPR-Cas system protein (LbCpf1 from Lachnospiraceae) or freeze-dried PKK.

The resulting guide RNAs can be used as part of a Pseudomonas aeruginosa exoU gene detection kit, with instructions for use.

Pseudomonas aeruginosa exoU gene detection kit may contain a ribonucleoprotein complex and instructions for use.

At the same time, at least one guide RNA in the kit can be complexed with a protein of the CRISPR-Cas system (LbCpf1 from Lachnospiraceae) in one container or separately in different containers.

The kit may further include components for pre-amplifying highly conserved Pseudomonas aeruginosa exoU gene fragments, including one or more specific oligonucleotides selected from SEQ ID NOs: 4-7.

Specific oligonucleotides for pre-amplification of the Pseudomonas aeruginosa exoU gene fragment according to the present invention correspond to a highly conserved region of the Pseudomonas aeruginosa exoU gene. Most preferred are oligonucleotides characterized by having or containing a nucleotide sequence selected from:

• SEQ ID NO: 4;

• SEQ ID NO: 5;

• SEQ ID NO: 6;

• SEQ ID NO: 7;

• or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 identical to any of them %, 94%, 95%, 96%, 97%, 98% or 99%,

• or complementary to any of them,

or hybridizing with any of them under stringent conditions.

The proposed technology makes it possible to identify single copies of the Pseudomonas aeruginosa exoU gene in patient biological samples taken from fluid and/or tissue suspected to contain Pseudomonas aeruginosa. The biological sample may be a sample of blood, blood serum or plasma, blood cells, saliva, sputum, lymphoid tissues, hematopoietic tissues and other biological materials from a patient, which can be used to test for the presence of the Pseudomonas aeruginosa exoU gene.

Brief description of the drawings

Fig. Fig. 1. Visualization of the amplified exoU-1 fragment of the Pseudomonas aeruginosa exoU gene (144 bp) after preliminary amplification using oligonucleotides For 1104 and Rev 1104 using agarose gel electrophoresis, where numbers 1-9 indicate:

1 Product from amplification of 20,000 fg of pGEM-T-exoU-1 model matrix;

2 - The product obtained during the amplification of 2000 fg of the pGEM-T-exoU-1 model matrix;

3 Product obtained from amplification of 200 fg of pGEM-T-exoU-1 model matrix;

4 - The product obtained during the amplification of 20 fg of the model matrix pGEM-T-exoU-1;

5 Product obtained during the amplification of 2 fg of the pGEM-T-exoU-1 model matrix;

6 - The product obtained during the amplification of 0.2 fg of the model matrix pGEM-T-exoU-1;

7 - The product obtained during the amplification of 0.02 fg of the model matrix pGEM-T-exoU-1;

8 Product obtained from amplification of 0.002 fg of pGEM-T-exoU-1 model matrix;

9 - negative control, not containing the pGEM-T-pGEM-T-exoU-1 model matrix;

M - molecular weight standards: from bottom to top 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000 bp (GeneRuler 100 bp Plus, Thermo Fisher Scientific, USA).

Fig. Fig. 2. Visualization of the amplified exoU-2 fragment of the Pseudomonas aeruginosa exoU gene (125 bp) after preliminary amplification using oligonucleotides For 2018/1982 and Rev 2018/1982 using agarose gel electrophoresis, where numbers 1-9 indicate:

1 Product from amplification of 20,000 fg of pGEM-T-exoU-2 model template;

2 - The product obtained during the amplification of 2000 fg of the model matrix pGEM-T-exoU-2;

3 Product obtained from amplification of 200 fg of pGEM-T-exoU-2 model matrix;

4 - The product obtained during the amplification of 20 fg of the model matrix pGEM-T-exoU-2;

5 - The product obtained during the amplification of 2 fg of the model matrix pGEM-T-exoU-2;

6 - The product obtained during the amplification of 0.2 fg of the pGEM-T-exoU-2 model matrix;

7 - The product obtained during the amplification of 0.02 fg of the model matrix pGEM-T-exoU-2;

8 Product obtained from the amplification of 0.002 fg of the pGEM-T-exoU-2 model template;

9 - negative control, not containing the pGEM-T-pGEM-T-exoU-2 model matrix;

M - molecular weight standards: from bottom to top 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000 bp (GeneRuler 100 bp Plus, Thermo Fisher Scientific, USA).

Fig. Fig. 3. Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa exoU-1 target treated with a ribonucleoportein complex containing exoU crRNA guide RNA No. LbCpf1 complexes from Lachnospiraceae containing guide RNA crRNA exoU No. 1104, where:

- the values of the normalized fluorescent signal generated by the dye are indicated along the vertical;

- fluorescence time is indicated horizontally, in minutes;

- curves 1-9 show the number of DNA copies per reaction, namely:

1 -58,900 copies/reaction;

2,5890,000 copies/reaction;

3 - 589,000 copies/reaction;

4 - 5,890 copies/reaction;

5 - 589 copies/reaction;

6 - 58.9 copies/reaction;

7 - 5.89 copies/reaction;

8 - 1.77 copies/reaction;

9 - mQ (marker of molecular weights).

Fig. Fig. 4. Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa exoU-2 target treated with a ribonucleoportein complex containing exoU crRNA #1982 guide RNA and LbCpf1 protein, on which the detection of the PGEM-T-EXOU-2 model template using ribonucleoproteins is visualized by means of a graph. LbCpf1 complexes from Lachnospiraceae containing guide RNA crRNA exoU No. 1982, where:

- the values of the normalized fluorescent signal generated by the dye are indicated along the vertical;

- fluorescence time is indicated horizontally, in minutes;

- curves 1-9 show the number of DNA copies per reaction, namely:

10 5,890 copies/reaction;

11 -5 890,000 copies/reaction;

12 - 58,900 copies/reaction;

13 - 589,000 copies/reaction;

14,589 copies/reaction;

15 - 58.9 copies/reaction; 16-5.89 copies/reaction;

17 - 1.77 copies/reaction;

18 - mQ (marker of molecular weights).

Fig. Fig. 5. Real-time fluorescence profile for a pre-amplified Pseudomonas aeruginosa exoU-2 target treated with a ribonucleoportein complex containing exoU crRNA guide RNA No. 2018 and LbCpf1 protein, on which the detection of the PGEM-T-EXOU-2 model template using ribonucleoproteins is visualized by means of a graph. LbCpf1 complexes from Lachnospiraceae containing guide RNA crRNA exoU No. 2018, where:

- the values of the normalized fluorescent signal generated by the dye are indicated along the vertical;

- fluorescence time is indicated horizontally, in minutes;

- curves 1-9 show the number of DNA copies per reaction, namely:

19 - 5 890,000 copies/reaction;

20,589,000 copies/reaction;

21 - 589 copies/reaction; 22-5,890 copies/reaction;

23 - 58.9 copies/reaction;

24 - 1.77 copies/reaction;

25 - 58,900 copies/reaction;

26 - 5.89 copies/reaction;

27 - mQ (molecular weight marker).

Fig. 6. FIG. 6. End-point fluorescence values (60 assay cycle, 60 minutes) for pre-amplified Pseudomonas aeruginosa exoU-1 and exoU-2 targets treated with ribonucleoportein complexes containing guide RNAs crRNA exoU #1104, crRNA exoU #1982, crRNA exoU #2018 , where the graph visualizes the detection of the exoU-2 gene of Pseudomonas aeruginosa using ribonucleoprotein complexes LbCpf1 from Lachnospiraceae, while:

- the values of the normalized fluorescent signal generated by the dye are indicated along the vertical;

- the number of copies of the model matrix in the reaction is indicated horizontally;

визуализирована эффективность выявления единичных копий гена exoU Pseudomonas aeruginosa, содержащегося в составе модельных матриц, с использованием crRNA exoU №1104;- column

the efficiency of detecting single copies of the Pseudomonas aeruginosa exoU gene contained in model matrices was visualized using exoU no. 1104 crRNA;

- the column visualizes the efficiency of detecting single copies of the Pseudomonas aeruginosa exoU gene contained in the model matrices using exoU no. 1982 crRNA;

визуализирована эффективность выявления единичных копий гена exoU Pseudomonas aeruginosa, содержащегося в составе модельных матриц, с использованием crRNA exoU №2018.- column

The efficiency of detecting single copies of the Pseudomonas aeruginosa exoU gene contained in model matrices was visualized using exoU no. 2018 crRNA.

Fig. Fig. 7. Visualization of the exoU-1 fragment of the Pseudomonas aeruginosa exoU gene amplified from clinical samples (144 bp) after preliminary amplification using oligonucleotides For 1104 and Rev 1104 using agarose gel electrophoresis, where each sample was assigned an identifier - an alphanumeric meaning.

Fig.8. Visualization of the Pseudomonas aeruginosa exoU-2 gene fragment amplified from clinical samples (125 bp) after preliminary amplification using oligonucleotides For 2018/1982 and Rev 2018/1982 using agarose gel electrophoresis, where each sample was assigned an identifier -letter- numeric value.

Fig. Fig. 9. Fluorescence values at the end point (60 assay cycle, 60 minutes) for Pseudomonas aeruginosa exoU-1 and exoU-2 targets pre-amplified from clinical specimens treated with ribonucleoportein complexes containing guide RNAs crRNA exoU No. 1104, crRNA exoU No. 1982 , exoU crRNA No. 2018, where the detection of the Pseudomonas aeruginosa exoU gene using LbCpf1 ribonucleoprotein complexes from Lachnospiraceae is visualized by means of a graph in a limited panel of clinical samples, while:

- the values of the normalized fluorescent signal generated by the dye are indicated along the vertical;

- the sample identifiers are indicated horizontally;

показана эффективность выявления гена exoU Pseudomonas aeruginosa, содержащегося в составе препаратов ДНК, выделенных из клинических образцов, с использованием crRNA exoU №1104;- column

the efficiency of detecting the Pseudomonas aeruginosa exoU gene contained in DNA preparations isolated from clinical specimens using exoU no. 1104 crRNA was shown;

- the column shows the efficiency of detecting the Pseudomonas aeruginosa exoU gene contained in DNA preparations isolated from clinical samples using exoU no. 1982 crRNA;

показана эффективность выявления гена exoU Pseudomonas aeruginosa, содержащегося в составе препаратов ДНК, выделенных из клинических образцов, с использованием crRNA exoU №2018.- column

The efficiency of detecting the Pseudomonas aeruginosa exoU gene contained in DNA preparations isolated from clinical specimens using exoU no. 2018 crRNA was shown.

EXAMPLES OF CARRYING OUT THE INVENTION

EXAMPLE 1: SELECTION OF TARGET SEQUENCES IN THE EXOU PSEUDOMONAS AERUGINOSA PSEUDOMONAS AERUGINOSA GENE TO GENERATE GUIDE RNA

To select target sequences in the exoU gene of Pseudomonas aeruginosa to create guide RNAs, modern algorithms for in silico analysis of nucleotide sequences and programs that are in the public domain, including Benchling (https://www.benchling.com/molecular-biology/), were used. A list of regions in the exoU gene of Pseudomonas aeruginosa was compiled with a theoretically calculated probability of their splitting in highly conserved regions (Table 1). Guide RNAs specifically recognizing the highly conserved region of the Pseudomonas aeruginosa exoU gene are unique sequences of SEQ ID NOs: 1-3.

EXAMPLE 2: PREPARATION OF MATERIAL FOR EXOU PSEUDOMONAS AERUGINOSA GENE DETECTION BY PRE-AMPLIFICATION METHOD

Preparation of the material for the detection of the Pseudomonas aeruginosa exoU gene was carried out by the method of preliminary amplification. The following plasmid DNAs were used as model templates for the Pseudomonas aeruginosa exoU gene of a biological sample:

1) pGEM-T-exoU-1 containing a fragment of the Pseudomonas aeruginosa exoU gene from 1053 to 1196 bp. (size 144 bp), and

2) pGEM-T-exoU-2 containing a fragment of the Pseudomonas aeruginosa exoU gene from 1934 to 2058 bp. (size 125 bp)

Pre-amplification of the regions corresponding to fragments of the Pseudomonas aeruginosa exoU gene was carried out using specific oligonucleotides with SEQIDNO: 4 and SEQIDNO: 5 to obtain an exoU-1 fragment and SEQ ID NO: 6 and SEQ ID NO: 7 to obtain an exoU-2 fragment, shown in Table 2.

PCR products encoding exoU-1 and exoU-2 fragments of the exoU gene of Pseudomonas aeruginosa were obtained in an amplification reaction using PCR mixture-2 blue (Federal Budgetary Institution of Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides For 1104 and Rev 1104, For 2018/ 1982 and Rev 2018/1982, respectively (GenTerra, Russia). The size of the amplified exoU-1 fragment was 144 bp, exoU-2 - 125 bp (Table 3).

Temperature profile of amplification for obtaining PCR products of Pseudomonas aeruginosa exoU gene fragments:

1. denaturation: 95°C for 3 minutes;

2. 40 cycles of amplification:

95°С - 15 sec,

55°С - 45 sec,

72°C - 30 sec;

3. final elongation: 72°C for 5 minutes.

During the preparation of the material for the detection of the exoU gene of Pseudomonas aeruginosa by the method of preliminary amplification, the model matrices pGEM-T-exoU-1 and pGEM-T-exoU-2 were titrated by preparing serial dilutions (Table 4).

To evaluate the efficiency of pre-amplification, the obtained Pseudomonas aeruginosa exoU gene fragments were visualized by agarose gel electrophoresis (FIGS. 1, 2).

The material prepared by the described method was used for experiments on the detection of the Pseudomonas aeruginosa exoU gene using LbCpf1 ribonucleoprotein complexes from Lachnospiraceae containing guide RNAs crRNA exoU No. 1104, crRNA exoU No. 1982, crRNA exoU No. 2018, without preliminary purification.

EXAMPLE 3: PRODUCTION OF GUIDE RNA AND CREATION OF RIBONUCLEOPROTEIN COMPLEXES TO DETECT THE EXOU GENE OF PSEUDOMONAS AERUGINOSA

To obtain guide RNAs for the detection of the exoU gene of Pseudomonas aeruginosa, a set of specific oligonucleotides was developed, shown in Table 5. The preparation of guide RNAs was carried out in several stages:

1. Obtaining a PCR product using a set of specific oligonucleotides (Table 5) encoding a guide RNA capable of binding to a target highly conserved region of the Pseudomonas aeruginosa exoU gene containing an RNA hairpin that is recognized by the RNA-guided DNA endonuclease LbCpf1 from Lachno sp irac eae;

2. Purification of a PCR product encoding a guide RNA specific for the Pseudomonas aeruginosa exoU gene fragment;

3. synthesis of a guide RNA specific to the Pseudomonas aeruginosa exoU gene fragment;

4. Purification of the guide RNA specific to the Pseudomonas aeruginosa exoU gene fragment.

The PCR product encoding guide RNA crRNA exoU No. 1104 was obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and crRNA 1104 (GenTerra, Russia). The size of the amplified fragment encoding exoU crRNA No. 1104 was 62 bp.

The PCR product encoding the guide RNA crRNA exoU No. 1982 was obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and crRNA 1982 (GenTerra, Russia). The size of the amplified fragment encoding exoU no. 1982 crRNA was 62 bp.

The PCR product encoding the guide RNA crRNA exoU No. 2018 was obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) and specific oligonucleotides T7rg and crRNA 2018 (GenTerra, Russia). The size of the amplified fragment encoding exoU no. 2018 crRNA was 62 bp.

Amplification temperature profile for obtaining PCR products encoding guide RNAs:

1. denaturation: 95°C for 3 minutes;

2. 35 amplification cycles:

95°С - 15 sec,

55°С - 45 sec,

72°C - 30 sec;

3. final elongation: 72°C for 5 minutes.

PCR products encoding guide RNAs specific for Pseudomonas aeruginosa exoU gene fragments were visualized by agarose gel electrophoresis.

PCR products encoding guide RNAs specific for Pseudomonas aeruginosa exoU gene fragments were purified using a commercially available ISOLATE II PCR and Gel Kit (BioLine, USA) according to the manufacturer's instructions. Purified PCR products encoding guide RNAs specific for Pseudomonas aeruginosa exoU gene fragments were used as a template for guide RNA synthesis.

Synthesis of guide RNAs specific to Pseudomonas aeruginosa exoU gene fragments was carried out by in vitro transcription using commercially available reagent kits (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB, USA) according to the manufacturer's instructions. The reaction products of in vitro transcription were reprecipitated from the reaction mixture by adding sodium chloride to a final concentration of 400 mM and an equal volume of isopropyl alcohol. Such modifications of the manufacturer's protocol introduced by the authors allow increasing the yield of the reaction product and obtaining the desired concentration of the final guide RNA preparation.

The creation of a ready-made ribonucleoprotein complex containing a protein of the CRISPR-Cas LbCpf1 family from Lachnospiraceae and guide RNA was carried out by the authors according to a standard protocol with some modifications [C. Anders, M. Jinek, In vitro enzymology of Cas9, Methods Enzymol. 546 (2014) 1-20, https://doi.org/10.1016/B978-0-12-801185-0.00001-51.

Immediately before combining with the Cas protein, the guide RNA preparation (250 ng) was heated at 90°C for 5 minutes and allowed to cool slowly to room temperature (incubation at room temperature for at least 10 minutes). Such heating is necessary for the formation of the correct hairpin conformation contained in the guide RNA. Many manufacturers of commercial Cas protein preparations skip this step when preparing the ribonucleoprotein complex.

To form the finished ribonucleoprotein complex, 250 ng of the LbCpf1 Cas protein from Lachnospiraceae and the prepared guide RNA were mixed and incubated for 15 minutes at room temperature. The ribonucleoprotein complex obtained in this way is ready for detection of the Pseudomonas aeruginosa exoU gene.

EXAMPLE 4: DETECTION OF SINGLE COPIES OF THE EXOU PSEUDOMONAS AERUGINOSA GENE USING CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES ON THE EXAMPLE OF A MODEL MATRIX

The pre-amplified material obtained by the method described in Example 2 was used as a template for Pseudomonas aeruginosa exoU gene detection using CRISPR-Cas ribonucleoprotein complexes obtained by the method described in Example 3.

To detect the exoU gene of Pseudomonas aeruginosa using CRISPR-Cas ribonucleoprotein complexes, a reaction mixture was prepared containing the following components:

• 10 x buffer (100 mM TrisHCl pH 8.0, 1 M NaCl);

• 50 mM MgC12 (final concentration in reaction mixture 10 mM);

• 250 ng ribonucleoprotein complex (LbCpf1 from Lachnospiraceae and guide RNAs crRNA exoU #1104, crRNA exoU #1982, crRNA exoU #2018);

• 10 pmol fluorescent probe (6FAM-TTATT-BHQ1);

• Target (preliminarily amplified Pseudomonas aeruginosa exoU gene fragments);

• mQ water.

The reaction mixtures containing all the necessary components were placed in a QuantStudio 5 amplifier (Thermo Fisher Scientific, USA) and the following reaction parameters were set:

30-60 cycles:

37°С - 35 sec,

37°C - 25 sec, fluorescence recording.

First of all, experiments were carried out to detect single copies of the exoU gene of Pseudomonas aeruginosa using CRISPR-Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae, using model templates as a target - plasmid DNA pGEM-T-exoU-1 containing in its composition of the exoU gene fragment of Pseudomonas aeruginosa from 1053 to 1196 b.p. 144 bp in size, and plasmid DNA pGEM-T-exoU-2, containing in its composition a fragment of the Pseudomonas aeruginosa exoU gene from 1934 to 2058 bp. size 125 p.

CRISPR-Cas ribonucleoprotein complexes have been shown to be able to detect single copies of the Pseudomonas aeruginosa exoU gene. Typical results of the analysis are shown using examples of real-time fluorescence profiles for pre-amplified Pseudomonas aeruginosa exoU-1 and exoU-2 targets treated with ribonucleoportein complexes containing exoU no. 1104 crRNA, exoU no. 1982 crRNA, exoU no. LbCpf1, in Fig. 3, Fig. 4 and FIG. 5, respectively. It should be noted that, on average, already at the 18th cycle of analysis (18 minutes), the value of the signal obtained during the detection of single copies (1.77 copies/reaction) of the Pseudomonas aeruginosa exoU gene exceeded the “noise” value (nonspecific fluorescence of the control sample, not containing the target) by at least five times, and by the 30th cycle (30 minutes of analysis) by 10 or more times.

In the course of the work, the efficiency of detecting single copies of the Pseudomonas aeruginosa exoU gene contained in model matrices was evaluated using various guide RNAs. It was shown that CRISPR-Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae and guide RNAs detect single copies of the exoU gene of Pseudomonas aeruginosa with different efficiency, and they can be arranged in the following order of decreasing activity: crRNA exoU no. 2018 > crRNA exoU no. 1104 ≥ crRNA exoU #1982 (Fig. 6).

EXAMPLE 5: DETECTION OF THE EXOU GENE OF PSEUDOMONAS AERUGINOSA USING CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES IN A LIMITED PANEL OF CLINICAL SPECIMENS

The developed guide RNAs were tested on a limited panel of clinical samples (17 pcs.) containing Pseudomonas aeruginosa carrying the exoU gene (previously confirmed by next generation sequencing).

To detect single copies of the Pseudomonas aeruginosa exoU gene using CRISPR-Cas ribonucleoprotein complexes, preliminary amplification of fragments of this gene was carried out. For preliminary amplification, DNA was isolated from 17 biological samples obtained from patients using a commercially available DNeasy Blood & Tissue Kit (QIAGEN, USA) according to the manufacturer's instructions.

PCR products encoding Pseudomonas aeruginosa exoU gene fragments were obtained in an amplification reaction using PCR mixture-2 blue (Central Research Institute of Epidemiology of Rospotrebnadzor, Russia) as described in Example 2, using the described temperature profile and duration of the amplification reaction. The resulting products were visualized by agarose gel electrophoresis (FIGS. 7, 8).

The material obtained in this way was used as a template for the detection of single copies of the Pseudomonas aeruginosa exoU gene using CRISPR-Cas ribonucleoprotein complexes obtained by the method described in Example 3.

Detection of single copies of the Pseudomonas aeruginosa exoU gene using CRISPR-Cas ribonucleoprotein complexes was carried out as described in Example 4.

In the course of the analysis, it was shown that CRISPR-Cas ribonucleoprotein complexes have the ability to detect the Pseudomonas aeruginosa exoU gene in DNA preparations isolated from clinical samples. At the same time, on average, already at the 20th cycle (20 minutes) of the analysis, the signal value exceeded the “noise” value (nonspecific fluorescence of the control sample that did not contain the target) by more than 5 times, and by the 35th cycle (35 minutes) of the analysis - by more than 10 times. times (Table 6).

Typical assay results are shown with examples of endpoint fluorescence values (60 assay cycle, 60 minutes) for pre-amplified Pseudomonas aeruginosa exoU-1 and exoU-2 targets (17 independent clinical specimens) treated with ribonucleoportein complexes containing exoU crRNA guide RNAs. No. 1104, exoU crRNA No. 1982, exoU crRNA No. 2018 and LbCpf1 protein, in Fig. 9.

Efficiency of detecting the Pseudomonas aeruginosa exoU gene contained in DNA preparations isolated from clinical samples using various guide RNAs in the composition of CRISPR-Cas ribonucleoprotein complexes formed on the basis of LbCpf1 from Lachnospiraceae, estimated by the ratio of signal values to the value of "noise" during detection , can be presented in the following descending order: crRNA exoU #2018 > crRNA exoU #1982 > crRNA exoU #1104 (Table 6).

Thus, the developed guide RNAs allow ultrasensitive detection of single copies of the exoU gene encoding the exotoxin of the third type secretion system, Pseudomonas aeruginosa, and are able to detect it in DNA preparations isolated from clinical samples after preliminary amplification in the CRISPR-Cas ribonucleoprotein complexes.

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Sequence listing

<110> FBUN Central Research Institute of Epidemiology of Rospotrebnadzor

<120>

<160> NUMBER OF SEQ ID NO: NOS: 7

<210> SEQ ID NO: NO 1

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 1 (crRNA exoU #1104):

aau uuc uac uaa gug uag aua cag cgg gcc acu gcg gcg c 40

<210> SEQ ID NO: NO 2

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 2 (crRNA exoU #1982):

aau uuc uac uaa gug uag auc cga aac gca gga agc cuc g 40

<210> SEQ ID NO: NO 3

<211> 40

<212>RNA

<213> artificial

<400> SEQUENCE 3 (crRNA exoU #2018):

aau uuc uac uaa gug uag aug gca aac ccu uga guu cga c 40

<210> SEQ ID NO: NO 4

<211> 20

<212> DNA

<213> artificial

<400> SEQUENCE 4 (For 1104):

gcc ggt ccc tga gat gat cg 20

<210> SEQ ID NO: NO 5

<211> 20

<212> DNA

<213> artificial

<400> SEQUENCE 5 (Rev 1104):

acg acc cag ccc ttg agc gc 20

<210> SEQ ID NO: NO 6

<211> 20

<212> DNA

<213> artificial

<400> SEQUENCE 6 (For 2018/1982):

cgg aaa tca atc aag cgc tg 20

<210> SEQ ID NO: NO 7

<211> 21

<212> DNA

<213> artificial

<400> SEQUENCE 7 (Rev 2018/1982):

gaa ctc ctt att ccg cca agc 21

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Claims (19)

1. A CRISPR-Cas guide RNA molecule capable of binding to target highly conserved regions of the exoU gene encoding the Pseudomonas aeruginosa secretion system exotoxin of the third type, where the nucleotide sequence of the guide RNA is selected from the sequences of SEQ ID NO: 1-3. 2. The guide RNA molecule according to claim 1, characterized in that it contains an RNA hairpin, which is recognized by the RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae. 3. A guide RNA molecule according to claim 1 or 2, characterized in that it ensures the detection of single copies of the Pseudomonas aeruginosa exoU gene. 4. The ribonucleoprotein complex of the CRISPR-Cas system for detecting the exoU gene of Pseudomonas aeruginosa, formed from the RNA-directed DNA endonuclease LbCpf1 from Lachnospiraceae and at least one guide RNA according to paragraphs. 1-3. 5. Ribonucleoprotein complex of the CRISPR-Cas system according to claim 4, suitable for detecting single copies of the Pseudomonas aeruginosa exoU gene. 6. Pseudomonas aeruginosa exoU gene detection kit containing (i) (a) a guide RNA molecule of the CRISPR-Cas system according to any one of claims 1 to 3 and a guided DNA endonuclease LbCpf1 from Lachnospiraceae; or (i) (b) a ribonucleoprotein complex according to any one of paragraphs. 4, 5; And (ii) instructions for use. 7. Pseudomonas aeruginosa exoU gene detection kit containing (i) (a) a guide RNA molecule of the CRISPR-Cas system according to any one of claims 1 to 3 and a guided DNA endonuclease LbCpf1 from Lachnospiraceae; or (i) (b) a ribonucleoprotein complex according to any one of paragraphs. 4, 5; And (ii) one or more specific oligonucleotides selected from SEQ ID NO: 4-7, And (iii) instructions for use.

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