TECHNICAL FIELD

The present invention relates to a method for detecting CpG methylation in a DNA target sequence and kit for employing this method. The method and kit can be used in the diagnosis of cancer.

BACKGROUND OF THE INVENTION

Cancer starts with a genetic change in the genome that results in the downregulation of genes involved in “normal” cell behaviour. This genetic change can either be induced by a change in the DNA code (mutation) or epigenetic alterations, such as cytosine methylation of so-called promotor CpG islands. Hypermethylation of CpG islands has shown to be a critical hallmark in many cancer cells (Ehrlich et al. Oncogene. 2002 Aug 12;21(35):5400-13). More specifically, the loss of expression of genes occurs about ten times more often by hypermethylation of CpG islands than by (point) mutations (Beggs et al. J Pathol . 2013 Apr;229(5):697-704).

There is a desire for accurate and rapid detection of epigenetic CpG methylation involved in tumour development. CRISPR/Cas systems are increasingly being investigated for their use as molecular diagnostic tool, including in detection of cancer biomarkers. However, epigenetic alterations such as CpG methylation can generally not be differentiated by Cas proteins and therefore CRISPR sensing methods are generally considered unsuitable for this purpose. Li et al. (ACS Synth. Biol. 2019, 8, 10, 2228-2237) proposed the use of bisulfite conversion to allow detection of CpG methylation by CRISPR sensing. However bisulfute conversion is laborious, and may damage the DNA, and does not allow easy targeting of specific sequences.

Another challenge in the field of using CRISPR sensing in detection of CpG methylation status is that its limit of detection (LOD), which is in the fM range or higher, is typically far away from the concentrations of DNA found in liquid biopsies such as urine (typically sub fM). Preamplification has been used to solve this problem, but since all commonly available preamplification techniques used for CRISPR sensing will result in a loss of epigenetic modifications, other methods need to be exploited to lower the LOD of CRISPR/Cas assays while retaining their methylation selectivity. Another general disadvantage relating to digital CRISPR sensing methods is their binary outcome: The assays tell the user only whether the target is present or not, but no information about the properties of the target sequence is obtained. For example, using (pre-) amplification methods in digital CRISPR assays results in a loss of methylation information. In these assays, the end-point fluorescence of individual droplets cannot be related to the CpG methylation of the single target sequence present in the droplet at the start of the experiment. There is a need for a method that allows measuring the methylation percentage at a single CpG site, allowing for higher diagnostic or prognostic value.

It is an objective of the present invention to overcome one or more of the above or other problems.

DESCRIPTION OF THE INVENTION

In an aspect, the present disclosure provides for an amplification-free in vitro diagnostic method to discriminate single CpG site methylation in DNA using methylation-sensitive restriction enzymes (MSREs) followed for example by Cas12a-assisted sensing. The method allows drastically lowering the detection limit of methylation-sensitive CRISPR sensing by employing portioning techniques as used in digital droplet assays. The present method combines absolute quantification of target sequence concentration with individual dynamic profiling of the positive droplets. This allows for simultaneously determining the target sequence’s total concentration and the methylation percentage of a single CpG site on this target sequence at sub fM concentrations.

Specifically, the present disclosure provides for a method for determining CpG methylation of at least one target nucleic acid sequence in a liquid sample, wherein the method comprises: a) providing a liquid sample comprising multiple molecules (e.g. copies or occurrences) of the at least one (target) nucleic acid sequence, preferably at least 2, 5, 10, 100, 500, 1000, 10000, 100000 of said molecules; b) dividing the liquid sample into a plurality of liquid partitions, such as at least 2, 5, 10, 100, 500, 1000, 10000, 100000 of said liquid partitions; c) contacting the liquid sample of step a) (or the liquid partitions of step b)) with at least one methylation-sensitive restriction enzyme (MSRE); d) determining the proportion of molecules of the at least one (target) nucleic acid sequence comprising at least one methylated CpG site by detecting the proportion of liquid partitions comprising non-(MSRE)restricted molecules of the at least one (target) nucleic acid sequence and/or detecting the proportion of liquid partitions comprising fragments of molecules of the at least one (target) nucleic acid sequence. The method according to the present disclosure preferably does not comprise a DNA amplification step, and/or for example does not comprise a polymerase chain reaction (PCR) amplification step.

The present disclosure also provides for a kit comprising:

- a device for dividing a liquid sample into a plurality of liquid partitions, preferably a microfluidic device;

- at least one MSRE; and

- at least one activatable protein which is activated in the presence of the at least one target nucleic acid sequence (e.g. entire or non-MSRE restricted molecules of the at least one target nucleic acid sequence (and not (or less) activated in the presence of MSRE restricted fragments thereof) or vice versa.

The present disclosure can be performed with a single device, i.e. not requiring a separate device for e.g. sequencing or determining bisulfite conversion to determine degree of methylation.

The kit may be used for determining CpG methylation of at least one target nucleic acid sequence in a liquid sample and/or for determining concentration of the at least one target nucleic acid sequence in a liquid sample.

In step a) of the method, a liquid sample is provided comprising (molecules of the) at least one (target) nucleic acid sequence. The target nucleic acid sequence is preferably comprised in a tissue sample and/or body fluid sample (e.g. blood, blood plasma or urine) obtained from a subject or (cancer) patient, such as selected from the group consisting of a tissue biopsy, urine, blood, saliva, serum, blood plasma, interstitial fluid, synovial fluid, transudate, pus, breast milk, synovial fluid, and semen, more preferably urine. Accordingly, the kit according to the present disclosure may be in combination with (molecules of) the at least one (target) nucleic acid sequence and/or the kit according to the present disclosure may be in combination with said tissue sample and/or body fluid sample.

The liquid sample may comprise the at least one target nucleic acid sequence in a concentration of between 1x10' ¹⁹ M and 1 x10 ^-9 M, preferably between 1x10' ¹⁸ M and 1 x10' ¹ ° M, more preferably between 1x10' ¹⁹ M and 1 x10 ^-14 or between 1x10' ¹⁹ M and 1 x10 ^-16 or between 1x10' ¹⁸ M and 1 x10 ^-16 . The at least one (target) nucleic acid sequence may be at least one double-stranded nucleic acid sequence and/or at least one single stranded nucleic acid sequence. The tern “target” means that the nucleic acid sequence is of (particular) interest, for example it comprises a cancer biomarker. The term “molecules or copies” refers to individual or separate occurrences or sequences of the target nucleic acid sequence. In an embodiment, the target nucleic acid sequence is single-stranded ribonucleic acid (RNA). In an embodiment, the target nucleic acid is double-stranded RNA. The RNA as disclosed herein encompasses messenger RNA (mRNA), transfer RNA (tRNA) and/or ribosomal RNA (rRNA). In an embodiment, the target nucleic acid is single-stranded deoxyribonucleic acid (DNA). In an embodiment, the target nucleic acid is double-stranded DNA. The DNA as disclosed herein encompasses circulating tumour DNA (ctDNA) and cell free DNA (cfDNA). The at least one target nucleic acid sequence as according to the present disclosure may have a length of between 10 bp and 10 kb, preferably between 20 bp and 1 kb.

The target nucleic acid sequence as disclosed herein preferably comprises a biomarker (in disease), more preferably a cancer biomarker. The term “biomarker” in the context of the current disclosure means a marker which presence or absence (e.g. in a body tissue or a body fluid) indicates a normal or abnormal process, or indicates a condition or disease (e.g. cancer). In addition or alternatively, a biomarker (level) may indicate efficacy of treatment of a disease or condition (e.g. cancer).

The target nucleic acid sequence preferably comprises one or more sites which can undergo (or has undergone) post-translational modification, preferably one or more of (hyper)methylation, glycosylation, acetylation, neddylation, phosphorylation, prenylation, SUMOylation, and ubiquitination, and wherein said post-translational modification is associated with disease and/or is a biomarker according to the present disclosure. Preferably, the target nucleic acid can undergo (or has undergone) CpG methylation (i.e. methylated cytosines in CpG dinucleotide).

The term “biomarker” in the context of the current disclosure encompasses molecular markers and signature molecules. In an embodiment, the biomarker represents epigenetic changes and/or post-translational modifications associated with the development of disease, preferably cancer. One of these epigenetic changes includes DNA (hyper)methylation. The target nucleic acid sequence according to the present disclosure may comprise at least one, at least two or at least three CpG methylation site(s). The present method may be used to discriminate single CpG site methylation among molecules or copies of the at least one target nucleic acid sequence. The target nucleic acid sequence of the present disclosure is preferably associated with development, progression and/or treatment of disease, preferably cancer. The cancer in the context of the current invention preferably is one or more of breast cancer, lung cancer, bronchus cancer, prostate cancer, colon cancer, rectum cancer, melanoma, bladder cancer, non-Hodgkin lymphoma, kidney cancer, renal pelvis cancer, endometrial cancer, leukaemia, pancreatic cancer, thyroid cancer, and liver cancer. Accordingly, the method and/or kit according to the present disclosure may be used in the diagnosis of cancer.

The “subject” as disclosed herein may be any mammal, preferably a human. The subject may be healthy or diseased. The subject can be a patient, preferably a cancer patient. The method of the present disclosure is preferably for in vitro or ex vivo use. Preferably, the method and/or kit of the present disclosure is not for practicing on the human or animal body. For example, a tissue or fluid of a subject is obtained (comprising the one or more target nucleic acid sequence, biomarker), whereafter the detection is performed outside of the body, such as in the laboratory. Preferably, the tissue or fluid is not introduced (back) into the subject.

In step b) of the method, the liquid sample is divided into a plurality of (separate) liquid partitions. The plurality of liquid partitions may relate to at least 10, 100, 1000, 10000 liquid partitions. Accordingly, the device for dividing a liquid sample into a plurality of liquid partitions as comprised in the kit according to the present disclosure is capable of providing multiple partitions, such as at least 10, 20, 100, 1000, 10000 separated partitions. Accordingly, the present disclosure provides a corral-type sensor, e.g. where each corral acts as an individual sensor e.g. for different targets. Subsets or said partitions may even serve to detect a different target nucleic acid sequence (e.g. biomarker) and/or comprise different activatable proteins and/or supplemented with different (crRNA) sequences complementary to the target nucleic acid sequence. The use of multiple partitions can accommodate a marker panel, wherein the level of expression of each marker or the overall marker profile can be linked to presence of disease and/or disease status.

In addition or alternatively, the liquid partitions may have a volume (on average) of between 10-100000 fl_, preferably between 50-10000 fl_, preferably between 100 - 5000 fl_, more preferably between 100-1500 fL Liquid partition size or volume is preferably determined by measuring the average radius of the partitions or droplets directly after generation.

Preferably step b) or the device obtains or is capable of obtaining an (average) concentration of the at least one target nucleic acid sequence in the plurality of liquid partitions of between 1x10 ^-19 M and 1 x10 ^-9 M, preferably between 1x10 ^-18 M and 1 x10 ^-10 M, more preferably between 1x10 ^-19 M and 1 x10 ^-14 or between 1x10 ^-19 M and 1 x10 ^-16 or between 1x10 ^-18 M and 1 x10’ ¹⁶ .

Step b) preferably divides the liquid sample into a plurality of liquid partitions such that at least 75% (or at least 90%) of the plurality of liquid partitions contain between 0-3 molecules or copies of the at least one target nucleic acid sequence or fragments thereof, preferably at least 75% (or at least 90%, 100%) of the plurality of liquid partitions contain 0 or 1 molecule or copy of the at least one target nucleic acid sequence or fragment thereof. Preferably between 30-80%, preferably between 40-70% of the plurality of liquid partitions contains 1 molecule or copy of the at least one target nucleic acid sequence or fragment thereof.

Dividing the liquid sample into a plurality of liquid partitions may be performed by one or more of flow distribution, filtration, and oil-water partitioning, preferably my means of a microfluidic device. The flow distribution may involve a polar and an apolar phase.

Preferably, dividing the liquid sample into a plurality of liquid partitions is not based on methylation status of the molecules of the target nucleic acid sequences, Preferably, dividing the liquid sample into a plurality of liquid partitions is performed without discriminating for methylation status.

In a preferred embodiment, the plurality of separate partitions are formed by physical and/or hydrophobic enclosures thereby preventing exchange of the at least one activatable protein and/or the one or more reporter constructs between the at least two partitions. The enclosure preferably extends in a vertical orientation from the a horizontally-orientated surface. The enclosure may provide a physical and/or hydrophilic/hydrophobic barrier to prevent exchange of the at least one activatable protein and/or the one or more reporter constructs between the at least two partitions. Hydrophilic means the moiety or surface attracts water and/or does not repel water and/or comprises polar (surface) molecule(s) (or comprises (surface) molecules having a solubility in water of above 1000, 100, 10, 5, 1 mg/l). Hydrophobic means repelling water and/or not attracting water and/or comprising non-polar (surface) molecules (or comprises (surface) molecules having a solubility in water of below 1000, 100, 10, 5, 1 mg/l).

In step c) of the method, the liquid sample of step a), and/or the liquid partitions of step b, is/are contacted with at least one methylation-sensitive restriction enzyme (MSRE). This can allow fragmentation of non-methylated (molecules of) the at least one target nucleic acid sequence (e.g. not comprising at least one, or two, methylated CpG site(s)). MSREs, as their name implies, typically are not able to cleave (restrict) methylated nucleic acid sequences (e.g. to cleave methylated-cytosine residues), thereby leaving methylated nucleic acid sequences intact. Most MSREs cleave DNA at specific unmethylated-cytosine residues. MSREs are well-known to the skilled person. The at least one MSRE of the present disclosure may for example be one or more of Aat II, Acc II, Aor13H I, Aor51H I, BspT104 I, BssH II, Cfr10 I, Cla I, Cpo I, Eco52 I, Hae II, Hap II, Hha I, Mlu I, Nae I, Not I, Nru I, Nsb I, PmaC I, Psp1406 I, Pvu I, Sac II, Sal I, Sma I, and SnaB I.

In a preferred embodiment of the present disclosure, step c) (and/or step d)) is performed at a temperature of between 20-60 °C and/or in presence of one or more of dithiothreitol (preferably 0.1 - 5 mM), tris(2 carboxyethyl)phosphine (preferably 0.1-4 mM), MgCI2 (preferably 0.5 - 40 mM), MnCI2 (preferably 0.5 - 40 mM), polyethylene glycol (preferably 0.5 - 40 w/v), NaCI (preferably 1-100 mM), Tris-HCI (preferably 0.5 - 40 mM), and a serum protein (preferably 10 - 500 ug/ml). Concentration are relative to the liquid medium wherein step c) and/or step d) is performed.

Accordingly it is preferred that the kit according to the present disclosure is in combination with or comprises a buffer comprising one or more of dithiothreitol (preferably 0.1 - 5 mM), tris(2 carboxyethyl)phosphine (preferably 0.1-4 mM), MgCh (preferably 0.5 - 40 mM), MnCh (preferably 0.5 - 40 mM), polyethylene glycol (preferably 0.5 - 40 w/v), NaCI (preferably 1- 100 mM), Tris-HCI (preferably 0.5 - 40 mM), and a serum protein (preferably 10 - 500 ug/ml). Concentrations are relative to the total buffer.

In step d) of the method according to the present disclosure, the proportion (or percentage) is determined of molecules of the at least one (target) nucleic acid sequence comprising at least one methylated CpG site, preferably by detecting the proportion of liquid partitions comprising non-(MSRE)restricted molecules of the at least one (target) nucleic acid sequence and/or detecting the proportion of liquid partitions comprising (MSRE)restricted molecules, i.e. fragments of molecules of the at least one (target) nucleic acid sequence.

Alternatively, the proportion is determined of molecules of the at least one (target) nucleic acid sequence comprising at least one non-methvlated CpG site by detecting the proportion of liquid partitions comprising non-(MSRE)restricted molecules of the at least one (target) nucleic acid sequence and/or detecting the proportion of liquid partitions comprising fragments of molecules of the at least one (target) nucleic acid sequence. In a preferred embodiment, step d) is directed to determining the proportion of methylation of a single CpG methylation site of (molecules of) the at least one target nucleic acid sequence and/or the proportion is determined of molecules of the at least one (target) nucleic acid sequence comprising one (non)methylated CpG site.

In a particularly preferred embodiment, step d) additionally involves determining the concentration of the at least one target nucleic acid sequence in the liquid sample by detecting the number of molecules of the at least one target nucleic acid sequence in the plurality of liquid partitions, preferably detecting the ratio between presence and absence of molecules of the at least one target nucleic acid sequence in the plurality of liquid partitions.

By tuning the volume of the plurality of partitions to the expected concentration range of the at least one target nucleic acid sequence to be measured, one can for example ensure that each partition contains either one or no target molecule. The target molecules are randomly distributed over the partitions. Accordingly, Poisson statistics can be used to calculate the initial concentration of the at least one target nucleic acid sequence in the liquid sample from the fraction of partitions containing a target nucleic acid sequence. Accordingly, step d) of determining the concentration preferably involves Poisson’s distribution statistics.

Hence, it is preferred that the partitions, or at least 50, 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% of the partitions, contain either one or zero molecule of the at least one target nucleic acid sequence. This allows to more precisely quantify low concentrations of target nucleic acid sequences.

In an advantageous embodiment of the method of the present disclosure, step d) is performed by: i. providing the plurality of liquid partitions with at least one activatable protein which is activated in the presence of (molecule(s) of) the at least one target nucleic acid sequence, e.g. entire or non-MSRE restricted molecules of the at least one target nucleic acid sequence (and not (or less) activated in the presence of MSRE restricted fragments thereof) or vice versa; and ii. detecting activation of the at least one activatable protein in the plurality of liquid partitions.

In an embodiment, the activatable protein undergoes a “conformational change”, meaning a change of the protein's tertiary structure in response to external factors (e.g. pH, temperature, solute concentration) or after association with a (target) ligand or nucleic acid. The conformational change preferably leads to enzymatic activity, such as cleavage activity as disclosed herein. Preferably, the at least one activatable protein is (capable of being) differentially activated in the presence of entire or non-MSRE restricted molecules versus in the presence of fragment(s) of (molecule(s) of) the at least one target nucleic acid sequence. For example, the at least one activatable protein is less (or more) activated in the presence of (only) MSRE restricted molecules or fragments of) the at least one target nucleic acid sequence than it is in the presence of entire or non-MSRE restricted molecules of the at least one target nucleic acid sequence. The skilled person knows how to arrange for this, for example by choosing at least one MSRE that has a recognition site (comprising methylation site) in the at least one target nucleic acid sequence downstream or upstream the activatable protein’s recognition sequence in the at least one target nucleic acid sequence. Preferably, the at least one MSRE has a recognition site (comprising methylation site) in the at least one target nucleic acid sequence wherein said MSRE recognition site is located between a recognition sequence of a first activatable protein and a recognition sequence of a second activatable protein. This reduces, upon MSRE restriction, the chance that both fragments are encapsulated and therefore the number of activated protein(s) in the partition is decreased (by 50%, or more if more activatable proteins are used). In other words, the at least one activatable protein(s) may have at least two recognition sites in the at least one target nucleic acid sequence (e.g. by being supplemented by at least two RNA sequences complementary to the first and second recognition site respectively), wherein the at least one target nucleic acid sequence has an MSRE recognition site comprising a CpG methylation site in between these at least two recognition sites of the at least one activatable protein.

More preferably, the at least one MSRE has a recognition site (comprising CpG methylation site) in the at least one target nucleic acid sequence which interferes with (e.g. is comprised in, or overlaps with) the activatable protein’s recognition sequence in the at least one target nucleic acid sequence.

In an embodiment, the activatable protein provides, upon activation, (increased) cleavage activity of nucleic acid, preferably single-stranded nucleic acid (ssRNA, ssDNA), wherein the cleavage may be cis- (i.e. cleavage of the target nucleic acid sequence) and/or transcleavage (i.e. cleavage of non-target nucleic acid sequence). The conformational change preferably leads to a detectable signal, such as provided by the one or more reporter constructs as disclosed herein.

In a preferred embodiment, activation of the activatable protein provides said protein of transcleavage activity and preferably the trans-cleavage activity cleaves single or double stranded nucleic acids (in the vicinity) thereby providing a detectable signal. In a particularly preferred embodiment, the at least one activatable protein is supplemented with at least one, two, or three RNA sequence(s) complementary to at least one, two, or three recognition sites, respectively, on the at least one target nucleic acid sequence, e.g. CRISPR (crRNA), meaning that the RNA sequence(s) and their recognition sequences can undergo base pairing.

For example, the activatable protein may be a ribonucleotide protein (RNP), e.g. a (CRISPR/) Cas protein supplemented with an RNA sequence (e.g. crRNA) which is preferably complementary to the at least one target nucleic acid sequence as disclosed herein. The Cas protein and/or the RNP preferably (further) comprises or is supplemented with a guide RNA (gRNA) which recognizes the at least one target nucleic acid sequence and directs the Cas protein to make double-strand breaks in nucleic acids.

The term “activation” (of a protein) preferably means that said protein undergoes a conformational change and/or obtains increased (enzymatic) activity, such as cleavage activity. In an embodiment, a Cas protein or RNP is activated following base-pairing of the at least one target nucleic acid sequence and the complementary RNA sequence it is supplemented with.

In an embodiment, the RNP undergoes a structural change upon hybridization between the crRNA and a complementary target nucleic acid sequence; this structural change may reveal the catalytic site of the protein and unleashes the enzymatic activity such as trans-cleavage activity of the protein (e.g. Cas protein such as Cas12, Cas 12a). In a preferred embodiment, activation of the activatable protein provides said protein of trans-cleavage activity.

In a preferred embodiment, the activatable protein is a Cas (effector) protein. The (CRISPR/) Cas protein may be of the class I, class II and/or class III, and/or may be chosen from the group consisting Cas 1-14. In an embodiment, the Cas protein is Cas12, e.g. Cas12a (such as FnCas12a , Cas12a, or LbCas12a) or Cas 12b. In an embodiment, the Cas protein is Cas13, e.g. Cas 13a (i.e. C2c2), Cas13b, Cas13c or Cas13d. In an embodiment, the Cas protein is Cas14, e.g. Cas 14a, Cas 14b, or Cas 14c. In an embodiment, the Cas12a is FnCas12a, LbCas12a. In an embodiment, the (CRISP/) Cas protein is an Alt-R (CRISP/) Cas protein, such as ALT-R Cas12a. In a preferred embodiment of the present disclosure, the activatable protein is a Cas12, or Cas12a (Cpf1), Alt-R CRISPR-Cas12a, LbCas12a, AsCas12a V3, AsCas12a Ultra or Cas 14.

The trans-cleavage activity varies between the different Cas12a variants. For example, lbCas12a can be used for digital droplet assay (as according to the present disclosure) exploiting droplets of e.g. 20 pm diameter. Furthermore, upon comparing three Cas12a enzymes (LbCas12a, AsCas12a V3, or AsCas12a Ultra coupled to their respective guide RNA sequence), LbCas12a may have the fastest transcleavage activity. For dual crRNA approach, the variant of Cas12a matters less than for the single crRNA approach.

Accordingly, the kit according to the present disclosure preferably comprises an activatable protein which is a Cas protein, preferably Cas12, or Cas12a, preferably supplemented with an RNA sequence complementary to at least one target nucleic acid sequence, and preferably the RNA sequence complementary to the at least one nucleic acid sequence is a CRISP RNA (crRNA). In addition or alternatively, the kit according to the present disclosure further comprises one or more reporter constructs which provide a detectable signal upon activation of the at least one activatable protein. As disclosed herein, the activation of the at least one activatable protein preferably provides said protein of trans-cleavage activity which allows cleavage of at least one single-stranded or double stranded nucleic acid in the one or more reporter constructs thereby providing a detectable signal.

Accordingly, in step b) of the method according to the present disclosure, the plurality of liquid partitions further provide for one or more reporter constructs which provide a detectable signal upon activation of the at least one activatable protein, preferably wherein activation of the at least one activatable protein provides said protein of trans-cleavage activity and wherein the trans-cleavage activity cleaves at least one single-stranded or double stranded nucleic acid in the one or more reporter constructs thereby providing a detectable signal.

As said, the reporter constructs preferably provide a detectable signal upon activation of the activatable protein, typically downstream/caused by the conformation change in the activatable protein. For example, a change in the protein's tertiary structure in response to an external factors and/or the presence and base pairing with at least one target nucleic acid sequence may lead to (increased) enzymatic activity of the activated protein, said activity inducing the reporter construct to provide (or increase) the detectable signal. In a preferred embodiment, the binding of complementary RNA of the Cas protein/RNP leads to cleavage activity by the Cas protein/RNP, thereby cleaving a nucleic acid sequence in the reporter construct. The cleavage of the nucleic acid sequence may lead to an increase in the detectable signal provided by the reporter construct(s), such as following release of a label, preferably a fluorophore (or quencher thereof). The presence and/or amount of the at least one target nucleic acid sequence and/or methylation degree thereof can be correlated to the amount of fluorescence signal. The cleavage activity is preferably trans-cleavage activity, such that any single-stranded nucleic acid sequence (in the vicinity) is cleaved. In another example, the activation of the activatable protein leads to release of a chemical moiety, which can be detected by further reacting the sample with a substrate for the chemical moiety and quantifying the chemical reaction.

Preferably, the detectable signal as provided by the reporter construct(s) is associated with the activation of the activatable protein, meaning that the signal is absent or low when said protein is not activated, but present and/or high when said protein is activated. Preferably, the signal provided by the reporter constructs is/can be correlated to the amount of target nucleic acid (and/or methylation degree thereof) and/or the activation of the activatable protein.

The term "reporter construct" as used herein means any molecule that can be used to provide a detectable (preferably quantifiable) effect such as a detectable (physical) signal. Suitable reporter constructs in the context of the current invention can be or comprise a label such as a dye (e.g. a fluorescent dye or moiety, a fluorophore), a radioactive label, a moiety allowing one or more types of bonds as disclosed herein, a hapten, a luminescent molecule or compound, a phosphorescent molecule or compound, or a (metal) nanoparticle. In addition or alternatively, a suitable reporter construct or label could be one that can provide one or more of the following signals: fluorescence, radioactivity, colorimetry, gravimetric analysis, X-ray diffraction or absorption, magnetic properties, enzymatic activity, quality characteristics, mass and charge. For example, the detectable signal as disclosed herein is one or more of fluorescence, luminescence, electrical activity, chemical activity, charge, enzymatic activity, radioactivity, colorimetry, mass, mass change, optical shift, and magnetism.

In a preferred embodiment, the one or more reporter constructs as according to the present disclosure comprise a fluorophore and a quencher of the fluorophore separated by at least one (single stranded) nucleic acid cleavable by trans-cleavage activity.

It may be advantageous in the context of the current disclosure to provide a specific number of activatable proteins and reporter constructs per partition in the case of two or more separated partitions). Preferably, there are between 1-10 activatable proteins per partition. In a preferred embodiment, there are (on average) one, two, or three activatable proteins per surface or partition. In addition or alternatively, there are between 10 and 1x10 ¹² or between 100 and 1x10 ¹² or at least 1 , 10, 100, 1x10 ³ , 1x10 ⁴ , 1x10 ⁵ , 1x10 ⁶ , 1x10 ⁷ , 1x10 ⁸ , 1x10 ⁹ , 1x10 ¹ °, or 1x10 ¹² reporter constructs per partition.

In a preferred embodiment, the number of activatable proteins and the number of reporter constructs (per surface or per partition) is in a ratio of between 1 :1 - 1 : 1000000, preferably 1 :10 - 1 :100000, more preferably 1 :200 - 1 :50000. In a particularly preferred embodiment of the present disclosure, e.g. in step d) of the present method, the detectable signal is measured at least on two or more time points, for example on 2, 3, 4, 5, 10, 20, 50, 100 time points, thereby measuring a change in detectable signal over time. Preferably the time points are separated by 0.001 - 120 seconds, preferably by 0.001 - 60 seconds, more preferably by 0.001 -1 seconds. Accordingly, the present method and/or use of the present kit allows for dynamic and/or real time measurement. The rate of increase of detectable signal may be indicative of presence, concentration and/or methylation degree (proportion) of molecules of the at least one target nucleic acid sequence.

The present invention also pertains to a method and/or kit as disclosed herein, preferably for use in the diagnosis of disease, preferably cancer. The method and/or kit can be used in vitro or ex vivo, and preferably is not practiced on the human or animal body.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

In this document and in its claims, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

In the context of the current invention, the terms ‘to increase’ and ‘increased level’ and the terms ‘to decrease’ and ‘decreased level’ refer to the ability to significantly increase or significantly decrease or to a significantly increased level or significantly decreased level. Generally, a level is increased or decreased when it is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher or lower, respectively, than the corresponding level in a control or reference. Alternatively, a level in a sample may be increased or decreased when it is statistically significantly increased or decreased compared to a level in a control or reference. The term “to reduce” may herein be used interchangeably with “to decrease”. The term “reducing” may herein be used interchangeably with “decreasing”.

The Example illustrates the different embodiments of the invention. FIGURE LEGENDS

Figure 1: Dynamic profiling of single-molecule CRISPR reactions in droplets to determine DNA copy number and CpG methylation of the targeted fragment. A) CRISPR sensing in bulk solutions makes it hard to distinguish low target concentrations. B) Due to the small volume of the droplets, Cas12a associated with a target can induce relatively high concentrations of cleavage fluorophore-quencher pairs, resulting in a (relatively high) increase in fluorescence over time for intact methylated DNA. C) non-methylated CpG sites overlapping an MSRE recognition site will be cleaved, resulting in fragments with lower trans-cleavage activity D) Because not all CpG sites will be methylated, differences in signal/time will be observed for the different droplets, enabling dynamic profiling of the methylation % of a single CpG site. Figure 2. Schematic illustration of how digital single molecule sensing dose response curves compare to bulk sensing and regular single molecule sensing.

Figure 3. Optimized CRISPR buffer for the detection of a target. Data are represented as mean ± standard error of the difference between means of three absolute and three technical replicates (n=9).

Figure 4. Optimization of the temperature of the Cas12a assay. And the corresponding signal/noise ratio. Data are represented as mean ± standard error of the difference between means of three absolute and three technical replicates (n=9).

Figure 5. Schematic representation of the PDMS microfluidic droplet generator with its main features. Different versions of the chip were used. The channel height was varied (using the same mask but changing the height of the SU-8 in the lithography process), as well as the nozzle width.

Figure 6. Representative images of droplets generated under different conditions. A) 3638 ± 236 fL droplets generated in an 8.5 pm high chip with a 10 pm wide nozzle and an 800 mbar (continuous phase): 500 mbar (discontinuous phase) applied pressure ratio. B) 1049 ± 111 fL droplets generated in an 8.5 pm high chip with a 7.5 pm wide nozzle and an 800 mbar (continuous phase): 500 mbar (discontinuous phase) applied pressure ratio. C) 1810 ± 270 fL droplets generated in a 21 pm high chip with a 7.5 pm wide nozzle and a 1000 mbar (continuous phase): 200 mbar (discontinuous phase) applied pressure ratio. D) 202 ± 20 fL droplets generated in an 8.5 pm high chip with a 7.5 pm wide nozzle and a 1000 mbar (continuous phase): 200 mbar (discontinuous phase) applied pressure ratio

Figure 7. The increase in average droplet fluorescence intensity over time for two ‘positive’ droplets containing a target sequence (indicated with asterisks) and a ‘negative’ droplet (indicated by arrow) containing no target sequence. Scale bar equals 100 pm. Figure 8. crRNA design on the target sequences and corresponding trans-cleavage activity; MAL crRNA 1-3 and the combination 1+2 and 2+3. Data are represented as mean ± standard error of the difference between means of three absolute and three technical replicates (n=9). Figure 9. crRNA design on the target sequences and corresponding trans-cleavage activity; GHSR crRNA 1-3 and the combination 1+2 and 1+3. Data are represented as mean ± standard error of the difference between means of three absolute and three technical replicates (n=9).

Figure 10. Droplets containing dual crRNA assay and 500 fM target sequence before and after heating for 2 hours at 50°C A) Brightfield image before heating showing a monodisperse droplet formation B) Fluorescence image after heating. C) Zoom-in of the fluorescence image after heating. All scale bars equal 100 pm.

EXAMPLES

Herein, a method is developed that allows simultaneously determining the target sequence’s total concentration and the methylation percentage of a single CpG site. Proof-of-concept experiments show that determining the trans-cleavage activity in droplets is possible allowing targeting of single CpG methylation in the droplets.

Methods

Oligonucleotides

All DNA oligonucleotides were synthesized by Eurofins Genomics. All crRNA fragments were Alt-R A.s. Cas12a crRNAs, synthesized by Integrated DNA Technologies (IDT, Coralville IA).

Cas12a ribonucleoprotein complex formation

Alt-R® CRISPR-Cas12a (Cpf1) Ultra (Integrated DNA technologies) and custom crRNA were mixed in a ratio of 1:2 for 30 minutes at room temperature to form RNP complexes. The assembled complex was then diluted 100 times in RNAse-free water (MACHEREY-NAGEL) and stored in the freezer at -20°C until further use.

Cas12a “bulk" assay in 384 well plates

In a total volume of 70 pl, 1x buffer, 20 nM of RNP complex, and 1000 nM of fluorophore quencher “reporter” DNA (Eurofins Genomics) were mixed with different concentrations of target dsDNA. Mass screening experiments were performed in a black polypropylene 384 well plate (Corning), where each well was filled with 20 pl of the reaction mixture, allowing triplicates of the total reaction volume. In these mass screening experiments, a MasterMixwas prepared to contain the RNP, buffer, and reporter DNA. Both the MasterMix and reaction mixture was pipetted on ice, and the reaction mixture was transferred to a pre-heated, black 384- well plate to inhibit reactions until the plate was transferred to the plate reader, which was heated at 38 °C unless otherwise stated.

Cas12a buffer optimization

To optimization of the AsCas12a Ultra mutant (IDT) reaction buffer, different buffers were prepared to compare the trans-cleavage activity of RNP in the presence of a 1 nM target concentration, a 20 nM RNP complex concentration, and a reporter concentration of 1000 nM. In Table 1, an overview of the buffers can be found. All experiments were conducted at 37 °C unless otherwise stated.

For Na+ optimization, Buffer-Na+ was used, supplemented with different concentration of Na+ (0, 10, 25, 50, 75 mM) prior to Cas12a addition. For M ²⁺ optimization, Buffer-M ²⁺ was used, supplemented with different concentration of either Mg2+ (0, 5, 10, 15, 20 mM) or Mn ²⁺ (0, 5, 10, 15, 20 mM) were added prior to Cas12a addition. For DTT or TCEP optimization Buffer-DTT was used, supplemented with different concentration of DTT or TCEP (0, 0.5, 1, 2, 4 mM) prior to Cas12aaddition. For PEG-200 optimization Buffer-PEG was used, supplemented with different percentages (v/v%) (0, 2.5, 5, 10, 20 mM) prior to Cas12a addition.

Table 1. Reaction buffers used.

Temperature optimization

Temperature optimization was performed similarly to buffer composition optimization by realtime fluorescence detection in a plate reader system. The enhanced buffer was used in combination with 1000 nM reporter and 20 nM Cas12a, with a total reaction volume of 20 pl per well. The well plate was incubated on 30- 55 °C with a 5 °C step-size in the plate-reader with fluorescence measurements taken every 5 minutes.

Microfluidic chip design and fabrication

A mask design for the microfluidic chips was used. The microfluidic chip has one sample inlet for the dispersed phase and one oil inlet for the continuous phase. A flow-focusing geometry is adopted for droplet generation, followed by a tree-shaped distribution channel network that nds in a droplet-incubation chamber. Due to the small slits that connect the incubation channel with the outlet channel that surrounds the incubation chamber on three sides, the droplets can be densely packed without too much backpressure built up in the system.

The chips were designed using CleWin software (version 5.0.12, WieWeb software). Standard, well-established lithography processes were used to create a 20 pm layer of Sll-8 on a silicon wafer. Polydimethylsiloxane (PDMS, DOWSIL 184 silicone elastomer kit) and curing agent (10 : 1 ratio) were mixed, degassed, and PDMS chips were fabricated using standard soft lithography with the prepared silicon wafer as a mold.

The PDMS was baked for at least two h at 60 °C on the mold after the PDMS chips were pealed-off, cut, and bonded to standard microscopy glass slides after 40 seconds of O2 plasma cleaning (model CUTE, Femto Science, Hwaseong-Si, South Korea). Finally, the PDMS-glass chip was baked for at least 20 minutes at 60 °C to enhance binding. Prior to droplet formation, the microfluidic chip was again plasma-cleaned, directly followed by filling the oil-inlet of the chip with Aquapel (PPG Industries, Inc., Pittsburgh, PA, USA) to make the channels (at least until the droplet-generation orifice) hydrophobic.

Droplet generation

Both chip inlets (dispersed and continuous phase) were connected to a 1.5 mL Eppendorf tube using fused silica capillaries (Polymicro Technologies, inner diameter (ID) 100 pm, outer diameter (OD) 360 pm, Molex) and Tygon tubing (ND 100-80, ID 250 pm, OD 760 pm, Saint- Gobain Performance Plastics). The flow for the inlets was controlled with a pressure pump consisting of two Flow-EZ modules (LineUp Series, Fluigent) with a p-cap connector (Fluigent). The flow was induced by applying the desired pressures to the continuous and dispersed inlet. Prior to droplet generation, the chip was filled with FC 40 oil (3M) supplemented with 2w/w% FluoSurf surfactant (Emulseo, Darwin microfluidics). Then, the dispersed phase was started, and a cooled Cas12a MasterMix was pushed through the channels at constant pressure, after which the on-chip incubation chamber was filled with microdroplets. This droplet formation took place at room temperature. Dynamic droplet imaging

The prepared droplets are incubated on the same chip using a hot plate at 50 °C. Simultaneously, fluorescence and brightfield images were taken every 5 minutes using the time-lapse function of the Invitrogen EVOS M5000. Brightfield images were used to determine the position and size of the droplets with an in-house developed MATLAB script. In this MATLABT script, droplets are found on the brightfield images using the built-in imfindcircles. The position (x,y coordinates) and corresponding radius are saved and used to determine the area under the curve for each individual droplet on the fluorescence images. The absolute fluorescence intensity that follows from these calculations is corrected for the area-specific background fluorescence: On three individual points of the chip, preferably situated in the oil evacuation channel where no droplets are present, circles varying from 2 pm to 15pm in size were used to calibrate for the increase in background fluorescence for the different droplet sizes.

Sample partitioning method for digital sensing

Digital single molecule sensing differs from bulk/analogue sensing by the type of output. In bulk sensing, the magnitude of the signal can directly be related to the amount of target molecules sensed. In digital single molecule sensing, the sample is typically partitioned in such a way that each partition contains a discrete number of target molecules (typically one or none) (Figure 2) and the original concentration can be calculated back, based on the fraction of positive droplets via partitioning statistics.

Partitioning used for digital sensing comes with several benefits, which makes it particularly interesting for DNA detection, like its superior precision, and extended dynamic range compared to bulk-sensing (depending on the number of partitions). For the CRISPR assay, main benefits are the increased local concentration of the target of interest, as well as a so called enrichment effect, that “purifies” the target of interest from interfering compounds. The last point is particularly interesting, since by statistics the chance of finding multiple fragments of non-methylated DNA in the same droplet, which potentially could result in restoring of the amplification efficiency, becomes lower.

In digital sensing several partitioning methods exist, but the most popular approaches consist of arrays of physically isolated reaction chambers or droplet emulsions where the individual partitions are separated by a continuous phase, generally oil. Both have their advantages and disadvantages. Droplet-based microfluidic devices allow for individual control of each droplet in the nanoliter to femtoliter range and are widely utilized for manufacturing, mixing, and transfer of droplets or fluids. Partitioning statistics

During the digital CRISPR experiments, the sample, including Cas12a and reporter, is randomly partitioned in thousands, if not millions of discrete volumes. To enable quantification of the sample in the droplets, it is important that each discrete volume contains zero or one molecule of interest. Since the probability that a molecule is present in a certain volume (P(X = 1)) occurs randomly and independently at a fixed average instantaneous rate A (or density), the number of occurrences of this event in a unit time, approximately obeys the Poisson Distribution (Majumdar et al. PLoS One. 2015 Mar 25; 10(3):e0118833) and the probability P(x=k) can be calculated according to the following formula:

Where A is the average occurrence number of this random events per unit time, area or volume and k is the number of occurrences. For digital quantification of partitioning based digital sensing, one should first consider the mean number of molecules per droplet with volume V is:

A(V) = C ■ V

Where C (copies/volume) is the concentration of the target in the sample of interest and V is the volume droplet. If one assumes the volume of the partitions to be constant one could calculate the concentration based on the following formula:

Where PPD is the positive chamber ratio, N(positive) is the number of positive chambers, N(chambers) is the total number of detection chambers and V is the volume of chamber.

Characterization of droplet-dynamic data

The optimal temperature for the Cas12a trans-cleavage reaction as found during the reaction condition optimization, was 50 °C. This high temperature causes evaporation of the droplets over time in the oil phase, reducing the droplet size and increasing the local concentration of molecules inside the original droplet. This means that both the evaporation ratio and the trans-cleavage activity of the activated Cas12a inside the droplet contribute to the increase in intensity as observed by fluorescence microscopy. The evaporation rate is not homogenous over the entire chip design, and depends on the position of the droplet in the assemble of droplets. Droplets on the edge of the ensemble show much higher evaporation rates than the droplets in the middle of the ensemble, who change slightly in droplet size over the experimental running time (1-2 hours).

To extract the trans-cleavage information from the fluorescence signal per droplet over time, the intensity per droplet is averaged over the calculated volume of the droplet. For all droplets where radius R < height of the channel h, the volume is calculated with the well-known formula for the volume of a sphere:

However, in the case the microchannel height is lower than the droplet size, the droplet is considered to be a discoid. The original volume of the non-squeezed droplet can be calculated from the height of the channel and the radius of the discoid R:

Results

1. Principle of Digital CRISPR Assays with Dynamic Profiling

Typically, integrating assays in microfluidic chips does not improve the limit of detection (LOD) compared to bulk measurements in well plates. Lowering of the LOD can be achieved by techniques where microfluidics enables the partition of a sample volume into thousands, if not millions, of small parts. This partitioning is known for its commercialized application, e.g. digital droplet PCR (ddPCRTM) (Quan et al. Sensors (Basel). 2018 Apr 20;18(4):1271).

These digital methods achieve absolute quantification without external standards. By tuning the volume of the parts to the expected concentration range measured, one can ensure that each part contains either one or no target molecule. The assumption is that these target molecules are randomly distributed over the droplets. Because of this assumption, Poisson statistics can be used to calculate the initial sample concentration from the fraction of droplets containing a target sequence.

In digital CRISPR sensing techniques, the ratio of droplets containing a target molecule vs. droplets containing no target molecule is determined by fluorescence end-point measurements. The collateral trans-cleavage activity of active Cas12 or Cas13 effector proteins towards a single-stranded nucleotide-fluorophore-quencher pair is used to determine whether a target is present. In the first publications exploiting digital CRISPR sensing, additional target sequence amplification was performed in the droplets simultaneously with the collateral trans-cleavage activity to enhance the signal-to-noise ratio (Park et al. Adv Sci (Weinh) . 2021 Jan 12;8(5):2003564). However, upon optimization of the effector proteinspecific reaction conditions, the trans- cleavage activity corresponding to a single target molecule was proven to result in an acceptable signal-to-noise ratio (Tian et al. ACS Nano . 2021 Jan 26; 15(1): 1167-1178). Furthermore, with the digital techniques mentioned above, the LCD could be lowered to low fM concentrations, > 1000 times lower than the (quantitative) LCD observed for CRISPR assays performed in bulk (tens of pM) (Park et al. Adv Sci (Weinh). 2021 Jan 12;8(5):2003564).

A general disadvantage of using digital CRISPR sensing methods is their binary outcome: The assays tell the user only whether the target is present or not, but no information about the properties of the target sequence is obtained. For example, using (pre-)amplification methods in digital CRISPR assays results in a loss of methylation information. In these assays, the end-point fluorescence of individual droplets cannot be related to the CpG methylation of the single target sequence present in the droplet at the start of the experiment. Unfortunately, in the current digital assays, the fluorescence intensity of these droplets is not quantified, neither for the end-point measurement nor dynamically.

As part of the current invention, it is considered that digital CISPR assays can potentially gain target-sequence information, for example, by using only the effector protein’s trans- cleavage activity for read-out.

For CpG methylation quantification, it is part of the current invention to dynamically follow Cas12a’s trans- cleavage activity. Herein, CpG methylation quantification using Cas12a includes an extra step prior to CRISPR sensing, where methylation-sensitive restriction enzymes (MSREs) are introduced. The MSREs only induce fragmentation on non-methylated CpG target sites. This fragmentation negatively affects the trans-cleavage rate of the Cas12a effector protein. So, by using a fluorophore-quencher reporter, the trans-cleavage as an increase in fluorescence can be dynamically followed and related to the methylation of a single CpG site that overlaps the target site of the MSRE.

Translating the concept of methylation-sensitive CRISPR sensing to digital CRISPR sensing means that rather than determining the ratio between positive and negative droplets, one must also discriminate between the different positive droplets. Figure 1 gives a schematic representation of the assay. By treating the target solution with an MSRE prior to droplet generation, the methylation of a single CpG site that overlaps with the specific MSRE recognition site could be determined. Because of the low expected target sequence concentrations in combination with the small droplet size, the chance of multiple fragments being present in the same droplet is extremely low (See Figure 2 and section “Sample partitioning method for digital sensing" in Methods).

Summarizing the information above, three types of droplet read-outs are possible while targeting a single CpG methylation site:

I. Negative droplets: containing no target sequence, resulting in no increase in fluorescence over time.

II. Positive droplets: contain the entire target sequence because of CpG site methylation, resulting in the fastest increase in fluorescence over time.

III. Positive droplets: contain one of the two fragments due to a nonmethylated CpG site, resulting in a slow(er) increase in fluorescence over time.

For droplet types III, the increase in fluorescence depends on the cleavage position and, therefore, the remaining length of the target sequence fragment. For example, for the MAL it is found that the MSRE recognition site selected results in a short fragment (10 bp) and a longer fragment (21 bp). This resulted in a higher trans-cleavage rate for the longer fragment in solution. The same behaviour is expected in the droplets, and therefore four different droplet intensities in a digital CRISPR methylation assay detecting this gene are expected.

By combining dynamic profiling with the advantages of digital assays, it is possible to simultaneously determine the target sequence’s total concentration and the methylation percentage of a single CpG site on this target sequence. In the following sections, proof-of- concepts are shown.

2. Proof -of -Concept AsCasl2a Buffer Optimization

The collateral cleavage activity of Cas12 and Cas13 effector proteins is a relevant parameter to consider when developing digital CRISPR sensing systems. The effector protein’s structure changes upon target sequence binding, making the catalytic site accessible. After target sequence cleavage, this catalytic site remains accessible for single-stranded (ss) nucleic acids, resulting in the collateral cleavage of all ssDNA (Cas12a) or ssRNA (Cas13) in its neighbourhood. The collateral trans-cleavage activity of Cas12a is visualized by the cleavage of ssDNA fluorophore-quencher reporters, increasing the fluorescence signal.

For digital sensing, where typically the increase in fluorescence corresponds to the transcleavage activity of a single effector protein, it is generally beneficial to have optimal reaction conditions to maximize trans-cleavage. In this study, the AsCas12a Ultra mutant (IDT) is exploited. These Cas12a proteins have a higher on-target potency than wild type As- or LbCas12a proteins. Furthermore, the AsCas12a Ultra mutant has a trans-cleavage activity that is highly correlated to the target sequence length that complements the spacer part of the CRISPR RNA (crRNA).

Several reaction conditions were systematically screened to find the optimum buffer composition for the AsCas12a Ultra mutant Figure 3, Figure 4). For optimization purposes, the effect of different reaction parameters on the trans-cleavage activity of Cas12a in bulk solutions was evaluated. The trans- cleavage was quantified by recording the increase in fluorescence over time in a 384 well plate, keeping the target sequence concentration constant at 1 nM.

An optimized buffer was determined by for the Cas12a assay, and studied the dosedependent effect of DTT, TECTP, MgCh, MnCh, PEG-200 and NaCI (see also Table 1).

The optimized buffer (Figure 3) was tested against a commonly used and commercially available buffer (NEB2.1 buffer) at the same temperature (37 °C) and at the elevated temperature of 50 °C (found to be optimal regarding the signal-to-noise ratio). As a result, (on average) 3x higher first derivatives of the increase in fluorescence over time was gained by using the optimized buffer conditions. Furthermore, with the elevated temperature of 50°C, gain another 3x gain was achieved compared to sensing at 37°C (~10x more than NEB2.1 buffer at 37°C) (Figure 4). This increase in the trans-cleavage improves the digital CRISPR assay: the larger the first time-derivative observed in bulk, the more likely one can follow the trans-cleavage of a single Cas12a protein in micro-sized droplets. Droplet Generation and Incubation on a Micro fluidic Chip

As part of the invention, it is considered that in the case of digital CRISPR sensing, partitioning the target solution into small volumes where every volume contains one or no target sequence is beneficial. Partitioning of the sample of interest is used, using a microfluidic chip for droplet generation (schematic representation in Figure 5).

Due to the presence of slit channels on three sites of the incubation chamber, both droplet generation, and droplet incubation take place on the same microfluidic chip. These slit channels allow the evacuation of the additional oil from the incubation chamber while blocking droplet exit. In this way, tight packing of the droplets is ensured.

While the overall design of the chip remained the same, chips with different nozzle sizes and channel heights were fabricated, allowing the generation of microdroplets of different sizes. The size of the generated microdroplets is essential in CRISPR digital assays. Based on Poisson statistics, the average droplet size determines the linear detection range in which quantification can be performed. Furthermore, the droplet size determines the concentration of the cleaved fluorophore-quencher pairs during the trans- cleavage reaction of the activated Cas12a protein.

This last factor, the local concentration of cleaved fluorophore-quencher pairs, is considered to be a more relevant parameter for our assay than for digital droplet PCR applications. In PCR amplification reactions in droplets, the amplification will increase local DNA concentration depending on the number of amplification cycles. However, in the digital CRISPR assay, there will be an increase in fluorescence due to the trans-cleavage activity originating from the Cas12a effector protein. Since this trans-cleavage activity is determined by the activity of a single activated Cas12a it proceeds at a linear rate, in contrast to the exponential increase in local concentration regulated by the number of temperature cycles occurring in PCR amplification.

Using microfluidic chips with varying nozzle sizes and channel heights can generate a different range of droplet sizes. The volume of the droplets can also be tuned by changing the continuous phase: discontinuous phase flow rate ratios. Figure 6 demonstrates this effect: Changing the nozzle width from 10 pm (Figure 6A) to 7.5 pm (Figure 6B) leads to droplets with a three times smaller volume while keeping the continuous phase: discontinuous phase flow rate ratio similar (applied pressures 800 mbar: 500 mbar). However, if the flow rate ratio (applied pressures 1000 mbar: 200 mbar) is increased and the nozzle width is kept the same as in Figure 6B (7.5 pm), the droplet volume can be lowered by a factor of 5 (Figure 6D). Figure 6C also shows the effect of the channel height, as 21 m high channels result in ten times larger droplets as 8.5 pm high channels with identical nozzle width (Figure 6D).

Dynamic Profiling CRISPR Reactions in Droplets

As part of the current invention, dynamic profiling of the trans- cleavage is used in combination with methylation quantification using CRISPR. It is shown that using a different Cas variant (LbCas12a), 20 pm droplets (corresponding to a volume of 4189 fL) have a sufficiently high local concentration of cleaved fluorophore-quencher to enable single DNA molecule sensing.

Using a 21 pm high microfluidic chip with a 5 pm nozzle size and a continuous phase: discontinuous phase applied pressure ratio of 300 mbar: 300 mbar, droplets of 19.6 ± 0.58 pm (n = 1188 droplets) diameter were generated. The discontinuous phase consisted of Cas12a, fluorophore-quencher reporter, and 50 fM of the target sequence in the buffer.

Directly after droplet generation, the entire chip was heated to the optimal 50°C while imaging using an epifluorescence microscope at set time intervals.

Figure 7A depicts an end-point measurement of the experiment. In these preliminary experiments, one could see a difference between the “positive” and “negative” droplets. The increase in intensity over time for three different droplets is given in Figure 7B. The two positive droplets have a similar increase in fluorescence over time, while the negative droplet shows a minimal increase in fluorescence over time.

It was observed that 0.88% of the droplets are positive (22 out of 2501 droplets). To further increase trans-cleavage activity to be visible using an appropriate setup (microscope, camera, illumination time etc.), end-point fluorescence measurements were performed to enhance the signal-to-noise ratio and further enable droplet intensity tracking over time for droplets containing a single target sequence.

3. Methods to Increase the Signal-to-Noise Ratio

Dual crRNA Approach

The current invention allows distinguishing between methylated and non-methylated single CpG sites. To facilitate this, two crRNAs targeting the same target sequence were used, which have a methylation site in between these sites.

Cas12a experiments were performed where ribonucleotide protein (RNP) complexes were formed with different combinations of crRNA sequences to test whether an improved signal- to-noise ratio in bulk could be observed. Every crRNA targets a different part of the target sequence so that multiple Cas12a proteins can bind simultaneously to the same target sequence.

Several design proposals were formulated for the selection of these crRNA sequences:

I. The sequence that the crRNA targets has an upstream PAM sequence so that Cas12a can recognize the site and bind.

II. The crRNA sequences have an MSRE recognition site in between the recognition sites of the different crRNA, enabling cleavage between these sites in the case of non-methylated CpGs.

III. The MSRE cleavage position covers only one single CpG site between the crRNA recognition sites enabling sensing with single CpG methylation sensitivity.

In this example, MAL and GHSR are targeted as proof-of-principle. These two genes are known to indicate cancer in a hypermethylated state (Bosschieter et al. PLoS One . 2018 Aug 24;13(8):e0200906). Three crRNA sequences were found on each of these genes that match design requirements I and II, of which two combinations matched requirement III and could be used to target a single CpG site methylation. Figures 8 and 9 shows the results of bulk experiments, where the dual-targeting strategy resulted in a higher trans-cleavage activity than in experiments with only one crRNA type. Improved signal-to-noise ratios are expected to be obtained using the crRNAs.

Subsequently, the same dual crRNA assay were performed in droplets. The RNPs were mixed with the two crRNAs (GHSR crRNA 1+ 2) in equal ratios, together with the fluorophore- quencher and target sequence, before droplet generation. The droplet size was determined by measuring the average radius of the droplets directly after generation. With the droplet volume obtained (1049 ± 110 fL, based on 2067 droplets, Figure 10A) and the known concentration of target sequence (500 fM), the percentage of droplets containing > 1 target sequence was predicted to be around 27%. As shown in Figure 10B, 3.2% (64/2067 droplets) having a positive signal is seen which matches the percentage of droplets that contain > 2 copies (3.6%). 3. Detection of cancer biomarkers

Herein, a method is developed that allows simultaneously determining the target sequence’s total concentration and the methylation percentage of a single CpG site. Proof-of-concept experiments show that dynamic barcoding of the trans-cleavage activity in droplets is possible allowing targeting of single CpG methylation in the droplets. By the use of multiple crRNAs, each targeting a different part of the target sequence, multiple Cas12a proteins could contribute to the fluorescence.

The feasibility of the current assay in dynamic barcoding of the trans-cleavage activity in droplets is demonstrated for the cancer marker panel GHSR/MAL (Hentschel et al. Clin Epigenetics. 2022 Feb 5; 14(1): 19).

The GHSR/MAL target sequences and the homologous crRNA sequences (Alt-R® A.s.

Cas12a crRNA) used were according to Table 2.

It is shown that the total concentration of MAL and GHSR sequences ant the % methylation can be determined in the urine of bladder cancer patients and healthy controls even up to sub fM concentrations. Accordingly, cancer patients and healthy controls can be distinguished with appropriate sensitivity and specificity.

Similar results are obtained with other Cas effector protein with a collateral trans- cleavage activity. It is expected that the assay has an even higher sensitivity/specificity and/or or lower LOD, when using different Cas effector proteins, such as different Cas12a variants. It is known that the trans-cleavage activity varies between the different Cas12a variants, e.g. bCas12a, AsCas12a V3, or AsCas12a Ultra may be particularly effective.

It is found that with the use of multiple crRNAs, each targeting a different part of the target sequence, multiple Cas12a proteins can contribute to the fluorescence.

It is found that the evaporation of droplets (e.g. by heating) and thereby effecting droplet shrinkage can be exploited as an extra signal-amplification step. This allows fluorescence intensity of the shrunken droplets to be linked to the concentration of the target sequence present in the original volume.

It is shown that to extract the trans-cleavage information from the fluorescence output per droplet over time, the area under the curve of the droplet intensity (corrected for the background fluorescence) can be taken to extrapolate the trans-cleavage information and better understand other factors influencing the increase in fluorescence over time.

The evaporation rates can be homogenized by trapping of the droplets. In this way, droplets move less, and the increase in intensity and the decrease in diameter can be followed quickly. There are known methods in the art, such as the trapping system presented by Labanieh et al. (Micromachines (Basel). 2015 Oct;6(10):1469-1482). The evaporation (and shrinkage ratio) can be exploited the trans- cleavage rate from the increase in fluorescence can be extracted using the in-house developed Matlab script.

It is found that MSRE’s can be added prior or after droplet formation. It appears generally more beneficial to add the MSREs prior to droplet formation. At the typical concentration range to be used, the chance of multiple cleaved fragments being presented in the same droplet is putatively low (for concentrations below 100 fM, assuming 20 pm droplets < 1%). Therefore, cleaved non-methylated DNA can result in the trans-cleavage of one Cas12a protein only, inducing a slower increase in fluorescence over time, compared to droplets containing methylated, intact DNAs where multiple Cas12a proteins can bind simultaneously, all contributing to the increase in fluorescence over time.