CN114945680A - CRISPR-based diagnostics using microfluidic compartmentalized rapid multiplexing - Google Patents

Abstract

The present invention provides a method for detecting the presence of a plurality of different target nucleic acids, comprising the steps of: a portion of the nucleic acid-comprising composition is partitioned into each of a plurality of microfluidic compartments, a Cas endonuclease reaction solution is introduced into each compartment, and a change in signal in the compartment is detected.

Description

CRISPR-based diagnostics using microfluidic compartmentalization rapid multiplexing

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No.62/961,122, filed on 14/1/2020, which is incorporated herein by reference in its entirety.

Technical Field

A CRISPR-based assay for detecting up to thousands of nucleic acid targets of interest from clinical or non-clinical samples using a microfluidic device.

Background

CRISPR/Cas technology is revolutionizing the field of functional genomics as it allows for precise gene editing. Recently, researchers have found that Cas proteins cleave nucleic acids at will after binding to a target, which is referred to as Cas "side-cut" activity. Cas proteins with side-cut cleavage properties can be used to develop rapid and sensitive CRISPR-based diagnostic assays to detect nucleic acids from pathogens or other nucleic acid targets. For example, Gootenberg et al, 2017, "Nucleic acid detection with CRISPR-Cas13a/C2C2," Science 356: 438-. Cas14 also shows side-cutting activity to cut ssDNA upon binding to its ssDNA or dsDNA target. See Harrington et al,2018, "Programmed DNA determination by minor CRISPR-Cas14 enzymes," Science 362: 839-. Two groups independently found the side-cut ssDNA cleavage activity of Cas12a/Cpf1 (Li et al,2018, "CRISPR-Cas 12 a-associated nucleic acid detection," Cell Discov 4, 20; and Chen et al,2018, "CRISPR-Cas 12a target binding unknown-amplified DNase activity," Science 2018: 436-439).

Cas-bypass activity based detection assays have been developed, including Cas13 based assays ("SHERLOCK"; see, e.g., Goodenberg et al,2018, "Multiplexed and portable nuclear acid detection with Cas13, Cas12a, and Csm 6" Science 360: 439. 444, and Cas12a based detection ("DETETR"; see, e Chen et al,2018, supra.) also see, Li et al, 2019, "CRISPR/Systems TowarNews New-Generation" Trends in Biotechnology 37:7:730-743, and Sashital,2018, "genetic Medium detection in the CRISPR-era Cas," genome.10: 32. all references cited herein are incorporated by reference in their entirety for all purposes.

Disclosure of Invention

The present invention discloses a method for the detection of nucleic acid targets of interest, such as nucleic acids from pathogenic microorganisms, variant gene sequences and other target DNA or RNA.

Drawings

FIG. 1 illustrates an embodiment of the present invention. FIG. 1 illustrates the extraction of nucleic acids (e.g., the isolation of DNA and/or RNA) from a sample. In this illustration, assays are performed to determine whether nucleic acids encoding one or more of target 1(T1), target 2(T2), target 3(T3), and target 4(T4) are present in the sample. The figure identifies genomic dna (gdna), circulating fetal dna (cfdna), and cDNA as non-limiting examples. FIG. 2 illustrates amplification of various targets. In this illustration, targets 1 and 3 are amplified, targets 2 and 4 are not amplified (e.g., are not present in the sample), and other target and/or non-target amplicons are generated. Legend 3 illustrates compartmentalization by equally dividing the amplified copies into aliquots (e.g., droplets, microwells, partitions) and introducing target-specific guide RNAs, CAS proteins, and other reagents and reporters. Legend 4 illustrates that in compartments containing grnas specific for target 1, a signal is generated by activation of sidecut activity, indicating the presence of target 1 in the sample. Panel 4 illustrates that no signal was produced in the compartment containing the gRNA specific for target 2, indicating that target 2 is not present in the sample. The signals from the multiple compartments may be detected sequentially or simultaneously. Exemplary signals are optical signals and pH signals.

Detailed Description

1. Overview

For purposes of illustration and not limitation, FIG. 1 depicts one form of the present invention, which includes the following steps:

step 1: optionally extracting nucleic acid from a sample suspected of containing the nucleic acid target of interest.

Step 2: optionally amplifying the nucleic acid, including the target of interest if present in the sample.

And step 3: nucleic acids from the sample or amplification products (amplicons) from step 2, including copies of the nucleic acid target of interest, are dispensed into a micro-compartment (e.g., a microwell, a droplet, or a microfluidic chamber) using microfluidic techniques (partitioning of microfluidic channels, electrowetting droplets, droplet encapsulation in channels, etc.).

And 4, step 4: the nucleic acid substrates (nucleic acids and/or amplicons) in each of the microcompartments are exposed to reaction conditions (e.g., in combination with reagents including a Cas protein, a guide nucleic acid (gRNA), and a reporter system (e.g., a reporter oligonucleotide)). If the nucleic acid target sequence is present in the compartment, the Cas-gRNA complex will bind, resulting in activation of the Cas protein sidecut activity and activation of the reporter system (e.g., cleavage of the reporter oligonucleotide) in the compartment.

And 5: activation of the reporter system in the compartment (e.g., cleavage of the reporter oligonucleotide) results in the generation or change of a detectable signal. The signal is detected.

2. Detailed analysis

This section provides additional details and non-limiting examples.

2.1 step 1: extraction of nuclei from one or more samples suspected of containing one or more nucleic acid targets of interest And (4) acid.

The sample can be any material that contains nucleic acids (e.g., DNA or RNA) and is suspected of containing one or more target nucleic acid sequences. Suitable samples include clinical samples (e.g., blood, urine, cerebrospinal fluid, etc.), agricultural samples, environmental samples, food samples, and the like.

The nucleic acid in the sample may be DNA and/or RNA, including, for example, nucleic acids from human or non-human animals, nucleic acids from plants or fungi, nucleic acids from prokaryotes or prokaryotic populations, and mixtures thereof. The target nucleic acid to be detected may be genomic DNA, mitochondrial DNA, mRNA, rRNA or cDNA. In one approach, the nucleic acid is from a microorganism or population of microorganisms (e.g., microbiome, such as gut microbiome). In some cases, the nucleic acid is from a pathogen. In one approach, the sample comprises nucleic acids from multiple sources, such as pooled nucleic acids from multiple patients, a maternal sample comprising both fetal and maternal nucleic acids, a patient sample comprising circulating tumor DNA, or a sample comprising nucleic acids from eukaryotes (e.g., humans) and prokaryotes (e.g., microbiome bacteria or pathogenic bacteria).

The sample may be analyzed without purifying or enriching the nucleic acid. Alternatively, the nucleic acid may be purified (partially or substantially separated from non-nucleic acid components of the sample) prior to amplification. Methods for nucleic acid purification are well known in the art. In another approach, the target nucleic acid can be enriched prior to the amplification step. For example, hybridization capture (or "hybridization enrichment") methods can be used to isolate specific sequences of interest (target nucleic acids) or defined sequence classes (e.g., exon DNA). See, for example, Gaudin, M.and Christelle, D.2018, "Hybrid Capture-Based New Generation Sequencing and Its Application to Human Infections Diseases," Frontiers in Microbiology 9: 2924.

The nucleic acid from the sample may be manipulated, modified or transformed prior to, simultaneously with or after amplification. For example, RNA from a sample can be used to prepare cDNA. As another example, the adapter can be incorporated into a nucleic acid (e.g., cDNA, RNA, or an amplicon of interest). In one approach, T1-T4 may be cDNA made from ribosomal RNA from a pathogenic bacterium and include sequences that specifically recognize the pathogen. In one approach, T1-T4 may be DNA sequences encoding drug resistance markers carried by certain bacteria. The concentration of sample nucleic acid or amplicon may be adjusted (e.g., by dilution) prior to compartmentalization or other steps.

2.2 step 2: amplification of nucleic acid targets from samples

In some approaches, the nucleic acid target sequence may be amplified prior to detection. Alternatively, the assay may be performed without amplification. In this manner, the sample, typically in a buffer, or the isolated but unamplified nucleic acid may be partitioned into the micro-compartments. For convenience, the amplified and unamplified target nucleic acids can be referred to as "substrate target nucleic acids".

Many nucleic acid amplification methods are known in the art and can be used in the present invention. Amplification is typically performed prior to the compartmentalization step discussed below. When amplification is performed, the amplification may be targeted amplification or non-targeted amplification. "Targeted amplification" refers to the preferential amplification of one or more specific target sequences. For example, PCR or rt-PCR (reverse transcription PCR) can be performed using primer combinations designed to amplify nucleic acids with specific sequences present. Methods for targeted amplification include PCR, rt-PCR, RPA or rt-RPA (recombinase polymerase amplification) or NASBA (nucleic acid sequence-based amplification) using defined primers. For example, multiple primer pairs can be used to amplify (e.g., using PCR) multiple target sequences from a sample. See, for example, U.S. patent nos. 7,666,598; deiman et al, 2002, "Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA)". mol.Biotech.20: 163- "180. In one approach, multiplex amplification (e.g., multiplex PCR) is performed in a vessel and an amplification reaction mixture containing amplicon products is dispensed into compartments.

In some embodiments, the amplification is non-targeted amplification, such as Whole Genome Amplification (WGA). In some cases, non-targeted amplification is performed after the reverse transcription step. Examples are DOP-PCR, MALBAC, MDA, NASBA and LIANTI. See, e.g., Telenius et al, 1992, "Generation oligonucleotide-printed PCR," Generation amplification of target DNA by a single generation primer, "Genomics 13, 718-; dean et al, 2002, "Comprehensive human genome amplification using multiple display amplification" PNAS 99: 5261-; zong et al, 2012, "Genome-wide detection of Single-nucleotide and copy-number variations of a Single human cell," Science 338: 1622-.

The amplification step may be performed in a tube (e.g., an Eppendorf tube or a PCR strip), on a microfluidic chip, a droplet, or any other suitable compartmentalization platform. In the case of a microfluidic chip, the amplification step may be integrated with the compartmentalization step (step 3) into a single chip. Also, in a droplet platform, amplification can be performed in one microfluidic droplet; the contents are then diluted (e.g., with buffer) and divided into different droplets, each with the same number and type of templates in it.

Amplification can produce either RNA amplicons or DNA amplicons. Different amplifications can be combined with different detection methods, depending in part on whether the amplicon is DNA or RNA.

2.3 step 3: dispensing amplicons or target nucleic acids into compartments

Following amplification, the amplification product, including the copy of the nucleic acid target of interest, is partitioned or divided into aliquots or microchambers. Exemplary microchambers include, but are not limited to, microwells, microchambers, and microdroplets. Microfluidic devices and methods for such compartmentalization are known in the art, including but not limited to:

automatic pipetting into microwells;

use of self-filling Microwell arrays (see, e.g., Kang et al, 2010, "Cell definition in Patterned Nanolititer drops in a Microwell Array by Wiping," J Biomed Mater Res 93: 547-

Dispensing into microchambers using microfluidic channels (see, e.g., U.S. patent nos. 7,476,363 and 8,591,834 to Fluidigm corp.);

dispersed into droplets (see, e.g., US 20190331585 a1 by Becton Dickinson and co.);

combining droplets with different CRISPR-Cas/gRNA combinations with droplets comprising a single target amplicon

Combining;

electrowetting droplets (see, e.g., US8716015B2 by Advanced Liquid Logic inc.);

droplet encapsulation in channels (see, e.g., US9477233B2 at the university of chicago).

It should be understood that the above described partitioning or bisecting techniques are for illustration purposes, but the present invention is not limited to a particular partitioning method.

Preferably, the samples are equally divided in the sense that each compartment contains approximately the same volume of amplicon solution. It is desirable that each compartment contain about the same number and type of nucleic acid substrates, so that the primary variable between compartments is the presence of grnas. The volume of each compartment can range from picoliters to microliters (e.g., 100 picoliters to 500 microliters, such as 100 to 1,000 picoliters, 1 to 100 nanoliters, 100 nanoliters to 10 microliters, or 10 microliters to 500 microliters). The number of compartments is usually more than 10 and may be between 10 and 10 ⁶ In the range of, for example, 10 to 10,000, 10 to 100, 100 to 10,000, or 1,000 to 10,000).

2.4 step 4: substrate nucleic acid and reagent combination

The substrate (amplicon or unamplified nucleic acid) in each compartment can be combined with reagents to perform the assay. That is, an assay comprising a nucleic acid (including a template, if present) and an assay reagent is provided in a microchamber. Assay reagents include (1) Cas protein, (2) guide rna (grna), (3) reporter systems (e.g., reporter oligonucleotides), and (4) buffers, cofactors, and other ancillary reagents.

The process used to combine reagents with target nucleic acids will vary depending on the microfluidic platform. Exemplary platforms and methods are described above in section 2.3. For example, in a microwell-based platform, reagents and substrate nucleic acids can be combined by automatically pipetting a single reagent and reagent mixture into each well.

The agents may be added in any order or combination, as long as premature activation of Cas-bypass activity is avoided. Multiple different grnas are typically mixed with an aliquot of Cas protein to allow assembly of Cas-gRNA Ribonucleoprotein (RNP) complexes. In certain instances, Cas and gRNA are mixed to assemble the RNP complex prior to addition of reporter system components. In this case, the assembled RNPs can be added to individual sample compartments with the same number and type of templates. Conditions under which Cas, gRNA, activating template sequence, and reporter system are all present (e.g., in a compartment or aliquot) can be referred to as "complete assay".

If the compartment contains a target nucleic acid sequence recognized by the Cas protein/gRNA complex in that compartment, the Cas-gRNA complex will bind to the target and will activate Cas protein sidecut activity. By providing a reporter system (e.g., reporter oligonucleotide) in each compartment, activation of the Cas protein side-cleavage activity in the compartment can be detected. In one mode, the reporter oligonucleotide in the compartment is cleaved and cleavage is detected, indicating that the polynucleotide comprising the target sequence is (or was) present in the compartment.

It will be appreciated that in some multiplexing embodiments, multiple target sequences are determined by using multiple different grnas. By altering the complementary sequence (typically 17-25bp) in the gRNA, different nucleic acid targets or different regions of interest can be assayed in different compartments. In certain platforms, it is desirable to associate each compartment (e.g., well or droplet) with a gRNA contained in the compartment. In one approach, the grnas are addressable in the sense that the gDNA content of a compartment can be determined based on the location of the compartment. In this way, the signal detected from any particular compartment can correspond to the gRNA in that compartment, allowing the user to interpret the signal from a particular compartment as an indication that compartment contains the target sequence corresponding to the gRNA sequence. In one approach, there is a specific gRNA in each compartment at a known (addressable) location (e.g., a microwell in an array), so signal changes from any location can be correlated with gRNA content. The signal change can be recorded from the spatial information (e.g., by taking a fluorescent image of the microwells in the array). Alternatively, the signal may be recorded sequentially through the various compartments (e.g., droplets) of the detector. In the case of a droplet platform, droplets containing predetermined known grnas can be generated using appropriately designed microfluidic circuitry and procedures. In this manner, the signal from any droplet can be correlated to the known gRNA content of the droplet. Negative controls without amplified template or without gRNA can be included.

2.4.2Cas protein

In certain embodiments, the amplicon is DNA and the Cas protein has a side-cleavage activity that recognizes the DNA (i.e., the side-cleavage activity is a DNase activity). In some embodiments, the Cas protein is Cas12a or Cas14 or other DNA-recognizing Cas protein.

DNA amplicon and DNA recognition Cas protein

Amplification methods that generate DNA amplicons are useful for Cas proteins with DNA cleavage side-cleavage activity (e.g., Cas12a or Cas 14). Examples of amplification methods to produce DNA amplicons include DOP-PCR, MALBAC, and MDA. See Telenius et al, 1992, "Generation oligonucleotide-primer PCR" general amplification of target DNA by a single generation primer, "Genomics 13, 718-725; dean et al, 2002, "Comprehensive human genome amplification using multiple display amplification" PNAS 99: 5261-; zong et al, 2012, "Genome-wide detection of single-nucleotide and copy-number variations of a single human cell". Science 338: 1622-.

RNA amplicon and RNA recognition Cas protein

Amplification methods that generate RNA amplicons can be used for Cas proteins with RNA digestion side-cut activity (e.g., Cas 13). Amplification may include reverse transcription and/or transcription steps. One suitable amplification method is linear amplification by transposon insertion (LIANTI), which produces ssRNA. See Chen et al, 2017, "Single-cell walls-genes analysis by Linear Amplification via Transposon Insertion (LIANTI)" Science 356: 189-. Other RNA amplification methods include NASBA (nucleic acid sequence based amplification) and LAMP (Loop-mediated isothermal amplification).

Cas proteins in general

Any suitable Cas protein with sidecut activity can be used in the practice of the assays described herein in combination with a suitable gRNA. Table 1 provides examples of targets recognized by Cas gRNA complexes and exemplary substrates for side-cutting nuclease activity. Using standard assays, skilled practitioners are able to recognize Cas proteins with side-cutting activity. See, for example, Chen et al, Li et al, Gootenberg et al, and Harrington et al, all supra. Assay conditions such as optimal temperature, substrate, pH, etc. are known for various Cas proteins and/or are readily determined by skilled practitioners. In some cases, the Cas protein is from the Cas12 family (including Cas12a and engineered forms or variants of naturally occurring Cas12 proteins). In certain instances, the Cas protein is from the Cas13 family (including Cas13a/C2C2 and engineered forms or variants of the naturally occurring Cas13 protein). In certain instances, the Cas protein is from the Cas14 family (including engineered forms or variants of naturally occurring Cas14 proteins).

TABLE 1

Li et al,2018, supra; chen et al,2018, supra.

Gootenberg et al,2018, supra.

Harrington et al,2018, supra.

2.4.3 guide RNA (gRNA) and CRISPR multiplexing

To allow CAS protein-gRNA complexes to bind to DNA, grnas comprising sequences complementary to the target sequence can be used. Typically, the complementary sequence is followed by an adjacent protospacer (protospacer) or "PAM" sequence on the target strand. The CAS protein-gRNA complex may comprise crRNA alone (which comprises a guide RNA that binds to a specific sequence in the target sequence) or both the crRNA and tracrRNA. Alternatively, the complex may comprise Cas and a single guide rna (sgrna), wherein both crRNA and tracrRNA are fused in a single molecule. For convenience, all sgrnas, crrnas, and crRNA/tracrrnas may be referred to as "grnas" in this disclosure.

Similarly, to allow CAS protein-gRNA complexes to bind RNA, grnas comprising sequences complementary to the target sequence can be used.

Various methods for gRNA design are known in the art and can be used in the present invention.

Multiplexing can be achieved in the present invention by using multiplexing sets of grnas in the same compartment. grnas can be designed to detect multiple different target sequences. CRISPR-based multiplexing has been described (see, e.g., Quan et al,2018, "FLASH: A next-generation CRISPR diagnostic for multiplexed detection of interactive resistance Sequences," bioRxiv 426338; and Gu et al, 2016 "deletion of Absolute Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-absolute Sequences in sequencing protocols and molecular counting applications," Genome Biology 17: 1-13).

2.5, step 5: reporter systems and assays for sidecut Activity (cleavage)

As described above, if a nucleic acid target sequence is present in the compartment, the Cas-gRNA complex will bind, resulting in activation of Cas protein side-cleavage activity. This activation (and, by inference, the presence of the target sequence in the compartment) can be detected by a reporter system that produces a detectable signal when a bystander activity is present. Various systems may be used. Two non-limiting examples are provided below. In some approaches, a dual reporter system is used, in which Cas13a cleaves the poly (a) reporter, the product of which activates Csm6 to cleave the second reporter. See Goodenberg et al,2018 Science 360: 439-.

Optical probe

In one approach, a quenched optical probe is used as a reporter. Optical probes are widely used in the art. Typically, the probe comprises a nucleic acid moiety (the other moiety cleavable by CRISPR) conjugated to a fluorescent dye (e.g., FAM) and a quenching dye (e.g., BHQ 1). Cleavage (hydrolysis) of the probe by the Cas endonuclease results in dye separation and a detectable increase in fluorescence.

Detecting pH or other chemical changes

In one approach, Cas endonuclease activity causes a chemical change that can be detected by an electrical sensor. For example, in one approach, the presence of CAS bypass activity may produce a pH change that can be detected by a CMOS sensor. In one method, detecting a pH change involves two enzymatic reactions. For example, in a first step, Cas (e.g., Cas13) binds to the target RNA and cleaves the RNA molecule in the compartment, producing RNA2',3' -cyclic phosphates. In the next step, RNA2',3' -cyclic phosphodiesterase is producing hydrogen ions (H) ⁺ ) Converting the RNA2',3' -cyclic phosphate to RNA2' -phosphate. [ H ] ⁺ ]The increase in (d) can be detected using a pH sensor, such as an Ion Sensitive Field Effect Transistor (ISFET), available from Winsense co.

Compartment and gRNA correlation

The signal detection/recording order may be sequential or parallel. For sequential imaging or CMOS detection, the droplets must pass through the detection area in time and be associated with the gRNA, or the camera must scan the microwells sequentially. For parallel detection, the camera must cover a larger area than the negative control to accommodate enough droplets or microwells, or a single droplet/microwell has a single sensor (e.g., CMOS, CCD) for signal change detection. The signals from the multiple compartments may be detected sequentially or simultaneously.

***

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. In the event of a conflict between the present application and a reference provided herein, the present application shall control.

Claims (16)

1. An assay for determining whether a sample comprises at least one of a plurality of different nucleic acid target sequences, comprising:

(a) obtaining a composition comprising polynucleotides derived from the sample;

(b) dispensing at least a portion of the composition into each of a plurality of microfluidic compartments, wherein each compartment contains an aliquot of the composition;

(c) introducing reagents for performing a nucleic acid assay into each compartment, wherein the reagents comprise:

a CRISPR-associated (Cas) protein having target-activated side-cut cleavage activity,

a reporter system comprising a nucleic acid substrate for said target-activated side-cut cleavage activity; and

a guide RNA complementary to at least one target sequence of the plurality of different nucleic acid target sequences;

wherein the guide RNA associates with the Cas protein to form an RNA, a Cas complex, and

wherein at least two compartments have different guide RNAs complementary to different target sequences;

(d) maintaining the compartments in (c) under conditions that result in: the RNA in each compartment the Cas protein complex recognizes a target sequence of the plurality of different target sequences, if present, resulting in activation of the target-activated side-cleavage activity, and cleavage of the nucleic acid substrate in the compartment;

(e) detecting activation of activated side-cutting cleavage activity of said target in said compartment.

2. The assay according to claim 1, wherein the sample is a clinical sample from a patient.

3. The assay according to claim 1, wherein the polynucleotides in the composition in step (a) are amplification products of nucleic acids from a sample.

4. The assay according to claim 1, wherein at least one target sequence is from a microorganism.

5. The assay according to claim 4, wherein the microorganism is pathogenic.

6. The assay of claim 1, wherein in step (c), the Cas protein and the reporter system are combined prior to the introducing.

7. The assay of claim 6, wherein in step (c), the Cas protein, reporter system, and the aliquot of the composition are combined prior to the introducing.

8. An assay according to claim 1, wherein in step (c) the guide RNA is combined with a mixture of the aliquots comprising the Cas protein, a reporter system and the composition.

9. An assay according to claim 1, wherein the guide RNA is pre-deposited into a well or compartment.

10. The assay according to claim 1, wherein the guide RNA is a sgRNA.

11. The assay according to claim 1, wherein the guide RNA comprises crRNA and tracrRNA.

12. The assay of claim 1, wherein the Cas protein is Cas12a, Cas13, or Cas 14.

13. The assay according to claim 1, wherein the substrate for target-activated side-cut cleavage activity is RNA.

14. The assay according to claim 1, wherein the substrate for target-activated side-cut cleavage activity is DNA.

15. The assay of any one of the preceding claims, wherein the compartment is a microfluidic compartment, a microfluidic chamber or a microwell.

16. The assay according to claim 15, wherein the number of compartments is from 10 to 10 ⁶ 。

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