Method for detecting minority genetic variants

Abstract

The present invention relates to a method for detecting a first nucleic acid sequence in a biological sample, which comprises a second nucleic acid sequence competing in the detection of said first sequence.

Description

[0001] METHOD FOR DETECTING MINORITY GENETIC VARIANTS

[0002] The present invention relates to a method for detecting a first nucleic acid sequence in a biological sample, which comprises a second nucleic acid sequence potentially competing in the detection of such first nucleic acid sequence.

[0003] The presence of cell-free DNA (cfDNA) was first identified in samples of human peripheral blood in 1948 (Mandel and Metais, (1948) “Les acides nucleiques du plasma sanguin chez 1’homme” Biologie 3- 4:241-243). Right from its identification, cell-free DNA rapidly became the focus of intense interest for research into bio-markers with clinical applications and in various different research areas.

[0004] One of the greatest strengths of the application of cell-free DNA as a bio-marker is associated with the possibility to analyze it using non-invasive sampling, such as for example a blood sample.

[0005] Cell-free DNA can originate from various different mechanisms and can be for example released: by cells, as a communication and adjustment mechanism of the immune system (Korabecna et al., (2020) Cell-free DNA in plasma as an essential immune system regulator. Ski Rep 10, 17478 DOI: https: / / DOI.org / 10.1038 / s41598-020-74288-2); as a consequence of apoptosis and necrosis of the cells (Ranucci R (2019) Cell-Free DNA: Applications in Different Diseases. Methods Mol Biol. 1909:3-12. DOI: 10.1007 / 978-1-4939-8973-7 1); from the fetus, during embryonic development in the maternal blood flow (Ranucci (2019); Bronkhorst et al., (2022) New Perspectives on the Importance of Cell-Free DNA Biology. Diagnostics, 12(9):2147. DOI: 10.3390 / diagnosticsl2092147); from pathogens during the development of infections (Blauwkamp et al., (2019) Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol 4, 663-674 DOI: https: / / DOI.org / 10.1038 / s41564-018-0349-6); from the human microbiome, as well as from assimilated environmental DNA (Bronkhorst et al., 2022). Cell-free DNA often is in highly fragmented form, with fragment dimensions of approximately 160-200bp, and is present not only in the peripheral blood of patients, but also in various different types of biological samples, such as for example saliva, sputum, urine, feces, cerebrospinal liquid and, in general, in samples referred to as “liquid biopsies”.

[0006] The variety of possible cell-free DNA derivations makes possible a wide spectrum of application thereof via the selection of specific markers. The scopes of application therefore vary from oncology to prenatal diagnosis, from pathogen identification to monitoring of transplanted organs, from the study and control of cardiovascular and chronic diseases to the control of the presence of contaminants through searching for assimilated environmental DNA (Bronkhorst et al., 2022).

[0007] In addition to the variety of possible sources of cell-free DNA, another advantage of its application is associated with the duration of its half-life, generally limited over time. This aspect enables, especially in the clinical setting, non-invasive and continuous monitoring over time of the conditions of the patient under observation. For example, the application of circulating tumor DNA (ctDNA) is well known and widespread in the phases of diagnosis, prognosis and choice / monitoring of therapy in cancer patients.

[0008] Generally, the quantity of cell-free DNA is very low in the blood flow in healthy individuals. However, the concentration of cfDNA in blood samples tends to increase during the development of tumors, especially in the advanced stage, during the gestation period in maternal blood, and in the presence of cardiovascular and chronic diseases.

[0009] Together with cell-free DNA, in recent years, particular interest has been directed, especially in oncology, to the study and identification of biomarkers in the DNA present in extracellular vesicles (EVs).

[0010] It has been observed that EVs are particularly stable in plasma and they can be considered a promising marker, for example in the diagnosis of patients with tumors at the initial stage (Fernando et al., WL (2017) New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes. PLoS ONE 12(8): e0183915 DOI: https: / / DOI.org / 10.1371 / joumal.pone.0183915).

[0011] Furthermore, EVs have been shown to be crucial bio-markers in prognoses. In fact, the study of EV content can be of great help in early identification of the formation of premetastatic niches, since the tumoral cells have shown a greater activity of secreting exosomes with altered content (Chin and Wang, (2016) Cancer Tills the Premetastatic Field: Mechanistic Basis and Clinical Implications. Clin Cancer Res. 2016 Aug l;22(15):3725-33. DOI: 10.1158 / 1078-0432.CCR-16-0028).

[0012] In recent years, various highly-sensitive and specific methods have been developed for detecting cfDNA and its specific bio-markers, including “BEAMing Safe-Sequencing System” (BEAMing Safe-SeqS), “Tagged- Amplicon deep Sequencing” (TamSeq), “Cancer Individual Profiling by deep Sequencing” (CAPP-Seq), digital PCR for detecting single-nucleotide mutations in cfDNA, whole-genome sequencing (WGS) for establishing changes in the number of copies, or the application of CRISPR-Cas systems to cut a target of interest in a highly specific manner.

[0013] However, the above mentioned techniques require a prior knowledge of the sequence of interest, together with the need for ultra- deep sequencing in order to be able to identify the first sequence of interest with statistical significance and be able to distinguish it from the second sequence. The need for ultra-deep sequencing limits the repeatability of analysis on samples of the same origin, for example from the same patient, owing to the long analysis times and high costs.

[0014] In principle, technologies can be divided into targeted approaches, aimed at detecting mutations in a set of predefined genes [for example, mutations in the EGFR gene are important for the response of patients with non- small-cell lung cancer (NSCLC) to blocking by tyrosine kinase inhibitors (TKI)] or non-targeted approaches (for example, comparative genomic hybridization on arrays, WGS or exome sequencing), which entail screening of the whole genome. Usually, targeted approaches have greater analytical sensitivity than non-targeted approaches, but considerable efforts are being made to improve the detection limitations of the latter.

[0015] Such limitations are mainly caused by the very nature of cfDNA (short half-life, low concentrations, and high fragmentation levels) which requires a great deal of time and highly optimized workflows, as well as an ultra-deep sequencing for a significant analysis of the markers of interest.

[0016] In light of the foregoing, it is necessary to improve the reliability of cfDNA analysis techniques in terms of sensitivity and specificity, as well as to identify protocols that can become standard and more accessible both in terms of applicability and of times and costs.

[0017] The aim of the present invention is to provide a method for detecting a rare genetic variant in a biological sample which overcomes the limitations of the known art.

[0018] Within this aim, an object of the invention is to provide a method that makes it possible to detect a specific rare genetic variant in a context of nonmutated sequences, so reducing the need for ultra-deep sequencing with respect to the known art.

[0019] Another object of the invention is to provide a method that makes it possible to detect an exogenous DNA sequence in a biological sample.

[0020] Another object of the present invention is to provide a method that makes it possible to detect a DNA sequence of fetal or embryonic origin in a biological sample.

[0021] Another object of the invention is to provide a method that is highly reliable, and relatively easy to execute.

[0022] This aim and these and other objects which will become better apparent hereinafter are achieved by a method for identifying a first nucleic acid sequence in a biological sample, said method comprising the following steps:

[0023] (i) isolating nucleic acids from the biological sample, said nucleic acids comprising:

[0024] (a) a first nucleic acid sequence, and

[0025] (b) a second nucleic acid sequence;

[0026] (i-a) optionally reverse-transcribing RNA present in the nucleic acids;

[0027] (ii) optionally fragmenting DNA obtained in step (i) or step (i-a), obtaining DNA fragments with a length comprised between 100 and 10,000 base pairs;

[0028] (iii) circularizing DNA fragments present in the DNA isolated in step (i) or in the DNA obtained in step (ii), obtaining a mixture of circularized DNAs comprising circular DNA comprising the "first sequence" and circular DNA comprising the "second sequence";

[0029] (iv) placing the mixture of circularized DNAs obtained in step (iii) in contact with a CRISPR system characterized by an sgRNA chosen from an sgRNA that is complementary to the "second sequence" of DNA and an sgRNA comprising from 1 to 3 nucleotides that are not complementary to the "second sequence" of DNA, thus obtaining a mixture of DNA comprising circular DNA comprising the "first sequence" and linear DNA comprising the "second sequence";

[0030] (v) selectively amplifying the circular DNA obtained in step (iv) by rolling circle amplification (RCA), thus obtaining an amplification product comprising a plurality of copies of the first DNA sequence;

[0031] (vi) identifying the first DNA sequence in the amplification product obtained in step (v).

[0032] In the present invention the term “first sequence” means a genetic sequence selected from the group constituted by a genetic sequence of tumoral origin, a genetic sequence of fetal origin, a genetic sequence of embryonic origin, a genetic sequence of bacterial origin, a genetic sequence of viral origin, a genetic sequence of plant origin, a genetic sequence of animal origin.

[0033] In the present invention, the term “second sequence” means any genetic sequence that potentially competes in the detection of the marker of interest (“first sequence”).

[0034] The method of the invention entails isolating the nucleic acids from the biological sample with methods known to the person skilled in the art such as, for example, extraction of nucleic acids with phenol-chloroform, extraction of nucleic acids using magnetic beads, and extraction of nucleic acids using silica membrane columns.

[0035] The biological sample is selected from the group consisting of blood, saliva, sputum, urine, feces, cerebrospinal fluid, and liquid biopsies.

[0036] Some applications require cell-free biological samples. In such an eventuality, if cell samples are used, the method includes a step of removing the cellular portion. For example, if the method is applied to the detection of cfDNA of tumoral origin, the presence of cells would entail an excessive presence of DNA of the “second sequence”, so compromising the sensitivity and specificity of the method.

[0037] In a preferred embodiment the “first sequence” is a DNA sequence.

[0038] Isolated nucleic acids comprise the sequence of interest (“first sequence”) often in the presence of a great excess of one or more sequences (“second sequence”) potentially competing in the detection of the “first sequence”. For example, for a liquid biopsy in oncology, the “first sequence” is mutated DNA of tumoral origin present in the sample together with an excess of non-mutated DNA (“second sequence”). In the event a contamination is detected, the “first sequence” is exogenous DNA present in the sample together with an excess of endogenous DNA (“second sequence”). In the field of prenatal diagnosis, the “first sequence” is fetal or embryonic DNA in a sample constituted predominantly by maternal DNA.

[0039] The circulating DNA extracted in step (i) of the method is generally sufficiently fragmented that it can be circularized as described in step (iii) of the method of the invention.

[0040] In an embodiment, particularly when the DNA is extracted from EVs, the method of the invention further comprises the optional step of (ii) fragmenting DNA obtained in step (i) or step (i-a), obtaining DNA fragments with a length comprised between 100 and 10,000 base pairs. Such fragmentation can be obtained with chemical or physical methods known to the person skilled in the art, such as for example sonication and tagmentation.

[0041] The method of the invention then comprises a step of (iii) circularizing DNA fragments present in the DNA isolated in step (i) or in the DNA obtained in step (ii), obtaining a mixture of circularized DNAs comprising circular DNA comprising the "first sequence" and circular DNA comprising the "second sequence”. Circularization of the fragments is done with methods known to the person skilled in the art (“Application of Circular Ligase to Provide Template for Rolling Circle Amplification of Low Amounts of Fragmented DNA”. Ada N. Nunez, MSFS, Mark F. Kavlick, BS, James M. Robertson, PHD, CFSRU, and Bruce Budowle, PHD, FBI Laboratory, FBI Academy, Quantico, VA 22135).

[0042] In an embodiment the circularization of step (iii) is obtained by means of the following steps: a. repairing the ends of the DNA fragments; b. adding a nucleotide to the 3' end of each fragment; c. adding an adapter comprising: cl. a nucleotide complementary to the nucleotide added in step b. to one end of the adapter; c2. a restriction enzyme recognition sequence "a"; c3. a sequence "0" comprised between 15 and 35 base pairs, characterized in that it does not form secondary structures or homodimers. c4. an arbitrary sequence "y" comprising 150 to 1000 base pairs; d. adding a ligase, thus obtaining the formation of a mixture of circularized DNAs comprising:

[0043] - circular DNA comprising the adapter and the "first sequence"; and

[0044] - circular DNA comprising the adapter and the "second sequence".

[0045] The function of the “y” sequence of the adapter is to support facilitation of circularization via a reduction in conformational entropy. This is a synthetic sequence designed so that under reaction conditions it will not form hybrids with the “first sequence”, the “second sequence” or the sgRNA of the CRISPR systems used.

[0046] In step (iv) the mixture of circularized DNAs obtained in step (iii) is brought into contact with a CRISPR system characterized by an sgRNA that is substantially complementary to the “second sequence” of DNA. This system makes it possible to specifically cut the “second sequence” which is thus linearized, while the circular DNA that comprises the “first sequence” remains intact. For the purposes of the specificity of the method it is therefore critically important to minimize hybridization of the sgRNA to the “first sequence”. This can be achieved using various different approaches known to the person skilled in the art. For example, the specificity can be optimized by conducting the reaction under conditions of low salinity and / or by using CRISPR systems characterized by sgRNA designed to maximize discrimination during hybridization. In a preferred embodiment, particularly when the first and the second sequence differ by a single nucleotide, the specificity can be optimized by using CRISPR systems characterized by “destabilized” sgRNA, i.e. sgRNA that comprise from 1 to 3 bases that are not complementary to the second sequence, preferably chosen from CG residues.

[0047] In a preferred embodiment, the CRISPR system is a CRISPR-Cas9 system. The high specificity of the CRISPR system makes it possible to simultaneously degrade multiple sequences using several CRISPR systems characterized by respectively complementary sgRNA, simultaneously in multiplex mode. In this manner the method of the invention makes it possible to simultaneously linearize several sequences (“second sequence”) which potentially compete with one or more sequences of interest (“first sequence”).

[0048] In an embodiment, the “second sequence” is chosen from the group consisting of the sequences from SEQ ID NO: 1 to SEQ ID NO:2I023.

[0049] The method of the invention advantageously combines CRISPR technology to linearize the second sequence, with a step of (v) selectively amplifying the circular DNA obtained in step (iv) by rolling circle amplification, thus obtaining an amplification product comprising a plurality of copies of the first DNA sequence; the combination of steps (iv) and (v) results in an extremely selective amplification of the one or more markers of interest (“first sequence”). The method then entails (vi) identifying the first DNA sequence in the amplification product obtained in step (v).

[0050] Advantageously, the selective amplification of the non-linearized sequences in step (iv) using RCA can be obtained with a single primer complementary to the sequence “P” introduced to the circular template in step (iii) of the method. The method therefore makes it possible to investigate in a multiplexed manner multiple “first” sequences without needing to use specific primers for the amplification step.

[0051] The identification in step (vi) can be done using various methods such as for example real-time PCR, illumina sequencing, or nanopore sequencing.

[0052] In a preferred embodiment, identification of the first sequence in step (vi) is obtained by sequencing the amplification product obtained in step (v). Advantageously, with respect to the known art, by virtue of the selective amplification of the marker of interest, the identification does not require ultra-deep sequencing.

[0053] The DNA extracted in step (i) and optionally fragmented in step (ii) can be subjected to one or more steps of enrichment with one or more gene sequences of interest before proceeding with the circularization in step (iii).

[0054] In a preferred embodiment the method of the invention further comprises, prior to step (iii), the step of (ii)a capturing one or both of the "first sequence" and the "second sequence" by means of a probe complementary thereto.

[0055] Alternatively, or in addition, in another embodiment the method of the invention comprises, prior to step (iii), the step of (ii)b amplifying one or both of the "first sequence" and the "second sequence". The amplification in step (ii)b is preferably obtained by PCR.

[0056] The method of the invention therefore makes it possible to detect and identify rare genetic variants in a biological sample. Advantageously, the method does not require knowledge of the sequence of the variation of interest. The combination of CRISPR systems, designed to cut potentially competing sequences, with RCA amplification makes it possible to amplify the rare variant with exquisite specificity, resulting in a simplification of the procedure with respect to the methods currently used and with a reduced need for ultra-deep sequencing to identify the variants.

[0057] The disclosures in Italian Patent Application No. 102022000027141 from which this application claims priority are incorporated herein by reference.

Claims

CLAIMS1. A method for identifying a first nucleic acid sequence in a biological sample, said method comprising the following steps:(i) isolating nucleic acids from the biological sample, said nucleic acids comprising:(a) a first nucleic acid sequence, and(b) a second nucleic acid sequence;(i-a) optionally reverse-transcribing RNA present in the nucleic acids;(ii) optionally fragmenting DNA obtained in step (i) or step (i-a), obtaining DNA fragments with a length comprised between 100 and 10,000 base pairs;(iii) circularizing DNA fragments present in the DNA isolated in step (i) or in the DNA obtained in step (ii), obtaining a mixture of circularized DNAs comprising circular DNA comprising the "first sequence" and circular DNA comprising the "second sequence";(iv) placing the mixture of circularized DNAs obtained in step (iii) in contact with a CRISPR system characterized by an sgRNA chosen from an sgRNA that is complementary to the "second sequence" of DNA and an sgRNA comprising from 1 to 3 nucleotides that are not complementary to the "second sequence" of DNA, thus obtaining a mixture of DNA comprising circular DNA comprising the "first sequence" and linear DNA comprising the "second sequence";(v) selectively amplifying the circular DNA obtained in step (iv) by rolling circle amplification, thus obtaining an amplification product comprising a plurality of copies of the first DNA sequence;(vi) identifying the first DNA sequence in the amplification product obtained in step (v).

2. The method according to claim 1, wherein the biological sample is selected from a cell-free biological sample and a biological sample from which the cellular portion has been removed.

3. The method according to claim 1 or 2, wherein the identification of the first sequence in step (vi) is obtained by sequencing the amplification product obtained in step (v).

4. The method according to any one of the preceding claims, wherein said "first sequence" is a DNA sequence.

5. The method according to any one of the preceding claims, further comprising, prior to step (iii), the step of (ii)a capturing one or both of the "first sequence" and the "second sequence" by means of a probe complementary thereto.

6. The method according to any one of the preceding claims, further comprising, prior to step (iii), the step of (ii)b amplifying one or both of the "first sequence" and the "second sequence".

7. The method according to claim 6, wherein said amplification is obtained by PCR.

8. The method according to any one of the preceding claims, wherein step (iii) comprises the following steps: a. repairing the ends of the DNA fragments; b. adding a nucleotide to the 3' end of each fragment; c. adding an adapter comprising: cl. a nucleotide complementary to the nucleotide added in step b. to one end of said adapter; c2. a restriction enzyme recognition sequence "a"; c3. a sequence "0" comprised between 15 and 35 base pairs, characterized in that it does not form secondary structures or homodimers. c4. an arbitrary sequence "y" comprising 150 to 1000 base pairs; d. adding a ligase, thus obtaining the formation of a mixture of circularized DNAs comprising:- circular DNA comprising the adapter and the "first sequence";and- circular DNA comprising the adapter and the "second sequence".

9. The method according to any one of the preceding claims, wherein said CRISPR system is a CRISPR-Cas9 system.

10. The method according to any one of preceding claims, wherein said "second sequence" is chosen from the group consisting of SEQ ID NO: 1 to SEQ ID NO:21023.

11. The method according to any one of the preceding claims, wherein said biological sample is selected from the group constituted by blood, saliva, sputum, urine, feces, cerebrospinal fluid, and liquid biopsies.

12. The method according to any one of the preceding claims, wherein said "first sequence" is selected from the group constituted by a genetic sequence of tumoral origin, a genetic sequence of fetal origin, a genetic sequence of embryonic origin, a genetic sequence of bacterial origin, a genetic sequence of viral origin, a genetic sequence of plant origin, a genetic sequence of animal origin.