US20230279375A1

US20230279375A1 - Signal boost cascade assay

Info

Publication number: US20230279375A1
Application number: US18/078,031
Authority: US
Inventors: Anurup GANGULI; Ashish Pandey; Ariana Mostafa; Jacob Berger
Original assignee: Vedabio Inc
Current assignee: Vedabio Inc
Priority date: 2021-12-13
Filing date: 2022-12-08
Publication date: 2023-09-07
Also published as: US20230193273A1; US20240218364A1; US20240035028A1; US12129468B2; WO2023114090A3; US20250109399A1; US11884921B2; WO2023114090A2

Abstract

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

Description

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 63/289,112, filed 13 Dec. 2021; U.S. Ser. No. 63/359,183, filed 7 Jul. 2022; U.S. Ser. No. 63/395,394, filed 5 Aug. 2022; and U.S. Ser. No. 63/397,785, filed 12 Aug. 2022.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Submitted herewith is an electronically filed sequence listing via EFS-Web a Sequence Listing XML, entitled “LS004US1_seqlist_20221207”, created 7 Dec. 2022, which is 1,227,000 bytes in size. The sequence listing is part of the specification of this specification and is incorporated by reference in its entirety.

PETITION UNDER 37 CFR 1.84(a)(2)

This patent application contains at least one drawing executed in color. The color drawings are necessary as the only practical medium by which aspects of the claimed subject matter may be accurately conveyed. The claimed invention relates to variant proteins that alter the active site thereof and the color drawings are necessary to easily discern the structural difference between variants. As the color drawings are being filed electronically via EFS-Web, only one set of the drawings is required.

FIELD OF THE INVENTION

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide a signal boost upon detection of target nucleic acids of interest in less than one minute and at ambient temperatures down to 16° C. or less.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Rapid and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the present of diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment including identification of biothreats. Classic PCR and nucleic acid-guided nuclease or CRISPR (clustered regularly interspaced short palindromic repeats) detection methods rely on pre-amplification of target nucleic acids of interest to enhance detection sensitivity. However, amplification increases time to detection and may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results. Improved technologies that allow very rapid and accurate detection of nucleic acids are therefore needed for timely diagnosis and treatment of disease, to identify toxins in consumables and the environment, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
The present disclosure provides compositions of matter and assay methods to detect target nucleic acids of interest. The “nucleic acid-guided nuclease cascade assays” or “signal boost cascade assays” or “cascade assays” described herein comprise two different ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep one of the ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the other ribonucleoprotein complex. The present nucleic acid-guided nuclease cascade assay can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in less than one minute and in some embodiments virtually instantaneously without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex DNA amplification, such as primer-dimerization. Further, the cascade assay prevents “leakiness” that can lead to non-specific signal generation resulting in false positives by preventing unwinding of the blocked nucleic acid molecules or blocked primer molecules (double-stranded molecules); thus, the cascade assay is quantitative in addition to being rapid. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1, the cascade assay components are the same in each assay no matter what target nucleic acid(s) of interest is being detected; moreover, the gRNA in the RNP1 is easily reprogrammed using traditional guide design methods.
The present disclosure is related first, to the instantaneous cascade assay, and second, to three modalities for preventing any “leakiness” in the cascade assay leading to false positives. The three modalities enhance the cascade assay and are in addition to using blocked nucleic acid molecules or blocked primer molecules in the cascade assay.
A first embodiment provides a method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. or more comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at an equal or higher molar concentration than the RNP2s in the reaction mixture, wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, and wherein the reaction mixture comprises at least one of a variant nuclease, the concentration ratio of the blocked nucleic acid molecules at a higher molar concentration than the molar concentration of RNP2s in the reaction mixture, and/or the blocked nucleic acid molecules comprise at least one bulky modification; contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1, wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, and wherein the at least one unblocked nucleic acid molecule binds to RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample in one minute or less.
An additional embodiment provides a method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. or more comprising the steps of: providing a reaction mixture comprising: first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to the nucleic acid target of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on a synthesized activating molecule, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the second gRNA; a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the plurality of blocked primer molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, wherein the blocked primer molecules each optionally comprise at least one bulky modification, and wherein the reaction mixture comprises at least one of a variant nuclease, a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or the blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides; contacting the reaction mixture with the sample under conditions that allow nucleic acid targets of interest in the sample to bind to RNP1, wherein: upon binding of the nucleic acid targets of interest to the RNP1, the RNP1 becomes active trans-cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase; the at least one unblocked primer molecule binds to one of the template molecules and is extended by the polymerase and nucleotides to form at least one synthesized activating molecule having a sequence complementary to the second gRNA; and the at least one synthesized activating molecule binds to the second gRNA, and RNP2 becomes active cleaving at least one further blocked primer molecule and at least one reporter moiety in a cascade; allowing the cascade to continue; and detecting the unblocked primer molecules, thereby detecting the target nucleic acid of interest in the sample in one minute or less.
Aspects of the embodiments of the methods for identifying a target nucleic acid of interest in a sample in one minute or less can be substituted for any assay for identifying target nucleic acids; for example, for detecting human pathogens; animal pathogens; disease biomarkers; pathogens in laboratories, food processing facilities, hospitals, and in the environment, including bioterrorism applications (see the exemplary organisms listed in Tables 1, 2, 3, 5 and 6 and the exemplary human biomarkers listed in Table 4). Suitable samples for testing include any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal (including humans), or microbe.
There is also provided in an embodiment a method of detecting a target nucleic acid molecule in a sample in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) contacting the target nucleic acid molecule with the reaction mixture under conditions that, relative to a control reaction, reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, wherein: (i) upon binding of the target nucleic acid molecule, the RNP1 becomes active wherein the first nucleic acid-guided nuclease cleaves at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and (ii) at least one unblocked nucleic acid molecule binds to the second gRNA, and the RNP2 becomes active wherein the second nucleic acid-guided nuclease cleaves at least one further blocked nucleic acid molecule; and (c) detecting the cleavage products of step (b), thereby detecting the target nucleic acid molecule in the sample.
There is also provided a second embodiment comprising a method of increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the second ribonucleoprotein complex (RNP2) in a cascade reaction comprising: (a) a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) the RNP2 comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, and (b) the target nucleic acid molecule comprising a sequence complementary to the first gRNA; and the method comprising the step of initiating the cascade reaction by contacting (a) and (b) under conditions that reduce the probability of R-loop formation between the blocked nucleic acid molecules and the second gRNA, thereby reducing increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the RNP2 relative to a control reaction.
There is also provided in a third embodiment a method of increasing the signal-to-noise ratio in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) initiating the cascade reaction by contacting the target nucleic acid molecule with the reaction mixture under conditions that reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, thereby increasing the signal-to-noise ratio in the cascade reaction relative to a control reaction, wherein: (i) upon binding of the target nucleic acid molecule, the RNP1 becomes active cleaving at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and (ii) the least one unblocked nucleic acid molecule binds to the second gRNA, and the RNP2 becomes active cleaving at least one further blocked nucleic acid molecule; and (c) detecting the cleavage products of the cascade reaction in step (b); and (d) determining the signal-to-noise ratio of the cascade reactions in step (b).
A fourth embodiment provides a method of increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of a second ribonucleoprotein complex (RNP2) in a cascade reaction comprising the steps of: (a) providing a reaction mixture comprising: a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA) comprising a sequence complementary to a target nucleic acid molecule; the RNP2 comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second guide RNA, (b) initiating the cascade reaction by contacting the target nucleic acid molecule with the reaction mixture under conditions that reduce the probability of R-loop formation between the second gRNA and the plurality of blocked nucleic acid molecules, thereby increasing the efficiency, reducing the background, increasing the signal-to-noise ratio, reducing cis-cleavage of blocked nucleic acid molecules and preventing unwinding of the RNP2 in the cascade reaction relative to a control reaction.
In some aspects of these embodiments, the conditions that reduce R-loop formation comprise one or more of the steps of: 1) providing a molar concentration of blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; 2) engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less.
Another embodiment provides a method for preventing unwinding of blocked nucleic acid molecules in the presence of an RNP in a cascade reaction comprising the steps of: providing blocked nucleic acid molecules; providing ribonucleoprotein complexes comprising a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule resulting from trans-cleavage of the blocked nucleic acid molecules; and providing a molar concentration of the blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less thereby preventing unwinding of the blocked nucleic acid molecules in the cascade reaction.
In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules are blocked primer molecules.
In a further embodiment, there is provided a method for preventing unwinding of blocked nucleic acid molecules or blocked primer molecules in the presence of an RNP comprising the steps of: providing blocked nucleic acid molecules or blocked primer molecules; providing ribonucleoprotein complexes comprising a nucleic acid-guided nuclease that exhibits both cis- and trans-cleavage activity upon activation and a gRNA that recognizes an unblocked nucleic acid molecule or an unblocked primer molecule resulting from trans-cleavage of the blocked nucleic acid molecule or blocked primer molecule; and providing a molar concentration of blocked nucleic acid molecules that exceeds the molar concentration of ribonucleoprotein complexes; engineering the nucleic acid-guided nuclease used in the ribonucleoprotein complex to result in a variant nucleic acid-guided nuclease such that single stranded DNA is cleaved times faster than double stranded DNA is cleaved; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications of a size of about 1 nm or less.
Other embodiments provide a method for detecting target nucleic acid molecules in a sample in less than one minute without amplifying the target nucleic acid molecules; and instantaneously detecting target nucleic acid molecules in a sample without amplifying the target nucleic acid molecules.
In some aspects of the methods, the reaction mixture is provided at 16° C., and in some aspects, the reaction mixture is provided at 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C. or higher.
Other embodiments provide reaction mixtures for identifying a target nucleic acid of interest in a sample in one minute or less comprising: first ribonucleoprotein (RNP1) complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; and a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, and wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the reaction mixture comprises at least one of a variant nuclease, a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification.
Also provided is a reaction mixture for identifying a target nucleic acid of interest in a sample in one minute or less comprising: first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to the nucleic acid target of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on synthesized activating molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the second gRNA; a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked primer molecules optionally each comprise at least one bulky modification and wherein the plurality of blocked primer molecules and the RNP2s optionally are at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the reaction mixture comprises at least one of a variant nuclease, at a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.
Further provided is a composition of matter comprising: ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guided nuclease and a gRNA that is not complementary to the target nucleic acid of interest; wherein the nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; and a plurality of the blocked nucleic acid molecules comprising a sequence corresponding to the gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked nucleic acid molecules each comprise at least one bulky modification, wherein the blocked nucleic acid molecules optionally each comprise at least one bulky modification, and wherein the plurality of blocked nucleic acid molecules and the RNP2s optionally are at a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and wherein the composition comprises at least one of a variant nuclease, a concentration ratio where the blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.
Additionally provided is a composition of matter comprising: ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guided nuclease and a gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity; a plurality of template molecules comprising sequence homology to the gRNA; and a plurality of the blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecules cannot be extended by a polymerase, and wherein the blocked primer molecules comprise: a first region recognized by the RNP2; one or more second regions not complementary to the first region forming at least one loop; and one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein the blocked primer molecules optionally each comprise at least one bulky modification, and wherein the plurality of blocked primer molecules and the RNPs optionally are at a concentration where the blocked nucleic acid molecules are at a molar concentration equal to or greater than the molar concentration of the RNPs in the reaction mixture, and wherein the composition comprises at least one of a variant nuclease, a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, and/or blocked nucleic acid molecules comprising at least one bulky modification; and a polymerase and a plurality of nucleotides.
In some aspects of these embodiments, the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample. In some aspects, the reporter moieties are not coupled to the blocked primer molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected; yet in other aspects, the reporter moieties are coupled to the blocked primer molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected.
In some aspects of all embodiments comprising bulky modifications, the bulky modifications are about 1 nm in size, and in some aspects, the bulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the bulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the blocked nucleic acid molecules include bulky modifications and wherein there are two bulky modifications with one bulky modification located on the 5′ end of the blocked nucleic acid molecule and one bulky modification located on the 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ ends comprising the two bulky modifications are less than 11 nm from one another. In other aspects, the bulky modification is on a 5′ end of blocked nucleic acid molecules and may be selected from the group of 5′ Fam (6-fluorescein amidite); Black Hole Quencher-1-5′; biotin TEG (15 atom triethylene glycol spacer); biotin-5′; and cholesterol TEG (15 atom triethylene glycol spacer). In other aspects, the bulky modification is on a 3′ end of the blocked nucleic acid molecules and may be selected from the group of Black Hole Quencher-1-3′; biotin-3′; and TAMRA-3′ (carboxytetramethylrhodamine). In some aspects, a bulky modification is between two internal nucleic acid residues of the blocked nucleic acid molecules and may be selected from the group of Cy3 internal and Cy5, and in some aspects, the bulky modification is an internal nucleotide base modification and may be selected from the group of biotin deoxythymidine dT; disthiobiotin NHS; and fluorescein dT.
In some aspects of these embodiments, the blocked nucleic acid molecules or blocked primer molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:

(a) A-(B-L)J-C-M-T-D (Formula I);
- wherein A is 0-15 nucleotides in length;
- B is 4-12 nucleotides in length;
- L is 3-25 nucleotides in length;
- J is an integer between 1 and 10;
- C is 4-15 nucleotides in length;
- M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands;
- T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and
- D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A;
(b) D-T-T′-C-(L-B)J-A (Formula II);
- wherein D is 0-10 nucleotides in length;
- T-T′ is 17-135 nucleotides in length;
- T′ is 1-10 nucleotides in length and does not hybridize with T;
- C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T;
- L is 3-25 nucleotides in length and does not hybridize with T;
- B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
- J is an integer between 1 and 10;
- A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
(c) T-D-M-A-(B-L)J-C (Formula III);
- wherein T is 17-135 nucleotides in length;
- D is 0-10 nucleotides in length;
- M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)J-C are separate nucleic acid strands;
- A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D;
- B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T;
- L is 3-25 nucleotides in length;
- J is an integer between 1 and 10; and
- C is 4-15 nucleotides in length; or
(d) T-D-M-A-Lp-C (Formula IV);
- wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
- D is 0-15 nucleotides in length;
- M is 1-25 nucleotides in length;
- A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
- L is 3-25 nucleotides in length;
- p is 0 or 1;
- C is 4-15 nucleotides in length and comprises a sequence complementary to T.

In some aspects, (a) T of Formula I comprises at least 80% sequence complementarity to B and C; (b) D of Formula I comprises at least 80% sequence complementarity to A; (c) C of Formula II comprises at least 80% sequence complementarity to T; (d) B of Formula II comprises at least 80% sequence complementarity to T; (e) A of Formula II comprises at least 80% sequence complementarity to D; (f) A of Formula III comprises at least 80% sequence complementarity to D; (g) B of Formular III comprises at least 80% sequence complementarity to T; (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or (i) C of Formula IV comprises at least 80% sequence complementarity to T.
In some aspects, the variant nucleic acid-guided nuclease is a Type V variant nucleic acid-guided nuclease. In some aspects, the one or both of the RNP1 and the RNP2 comprise a nucleic acid-guided nuclease selected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b.
In some aspects of the embodiments that comprise a variant nucleic acid-guided nuclease, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules wherein the mutation is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. In some embodiments, there are at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs and in other aspects, there are at least three mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs. In some aspects, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, wherein the at least one mutation is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15.
In some aspects, the variant nucleic acid-guided nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, wherein single stranded DNA is cleaved 1.2 to 2.5 times faster than double stranded DNA is cleaved, at least three to four times faster than double stranded DNA is cleaved, and in some aspects, single stranded DNA is cleaved at least five times faster than double stranded DNA is cleaved. In aspects, the variant nucleic acid-guided nuclease exhibits cis- and trans-cleavage activity.
In some aspects, the variant nucleic acid-guided nuclease comprises at least two mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and in some aspects, the variant nuclease comprises at least three mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules.
In any of the embodiments comprising a concentration ratio where blocked nucleic acid molecules are at a higher molar concentration than the RNP2s in the reaction mixture, certain aspects provide that the concentration of the blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 1.5 blocked nucleic acid molecules to 1 RNP2 in the reaction mixture, and in some aspects, the concentration of the blocked nucleic acid molecules and the RNP2s are at a concentration ratio of at least 2 blocked nucleic acid molecules to 1 RNP2 in the reaction mixture or at least 3 blocked nucleic acid molecules to 1 RNP2, or at least 3.5 blocked nucleic acid molecules to 1 RNP2, or at least 4 blocked nucleic acid molecules to 1 RNP2, or at least 4.5 blocked nucleic acid molecules to 1 RNP2, or at least 5 blocked nucleic acid molecules to 1 RNP2, or at least 5.5 blocked nucleic acid molecules to 1 RNP2, or at least 6 blocked nucleic acid molecules to 1 RNP2, or at least 6.5 blocked nucleic acid molecules to 1 RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 8 blocked nucleic acid molecules to 1 RNP2, or at least 8.5 blocked nucleic acid molecules to 1 RNP2, or at least 9 blocked nucleic acid molecules to 1 RNP2, or at least 9.5 blocked nucleic acid molecules to 1 RNP2, or at least 10 blocked nucleic acid molecules to 1 RNP2.
In further embodiments there is provided a variant Cas12a nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant Cas12a nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules and wherein the variant Cas12a nuclease exhibits both cis- and trans-cleavage activity. In some aspects, wherein the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or the at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules is selected from mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15 including and equivalent amino acid residues in Cas12a orthologs to these SEQ ID Nos: 1-15.
In some aspects, the variant Cas12a nuclease that has been engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved comprises any one of SEQ ID NOs: 16-600.
Alternatively, an embodiment provides a single-strand-specific Cas12a nucleic acid-guided nucleases comprising an LbCas12a (i.e., SEQ ID NO: 1) with an acetylated K595 (K595K^Ac) residue; an AsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^Ac) residue; a CtCas12a (i.e., SEQ ID NO: 3) with an acetylated R591 (R591R^Ac) residue; an EeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^Ac) residues; an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635 (K635K^Ac) residue; an FnCas12a (i.e., SEQ ID NO: 6) with an acetylated K671 (K671K^Ac) residue; an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylated N671 (N671K^Ac) residue; an FbCas12a (i.e., SEQ ID NO: 8) with an acetylated K678 (K678K^Ac) residue; an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylated K601 (K601K^Ac) residue; an MbCas12a (i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^Ac) residue; a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^Ac) residue; a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732 (K732K^Ac) residue; an AaCas12a (i.e., SEQ ID NO: 13) with an acetylated K607 (K607K^Ac) residue; a BoCas12a (i.e., SEQ ID NO: 14) with an acetylated K653 (K653K^Ac) residue; or an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylated K577 (K577K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Cas12a ortholog acetylated at the amino acid of the ortholog equivalent to K595 of SEQ ID NO:1.
These aspects and other features and advantages of the invention are described below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A is an overview of a prior art quantitative PCR (“qPCR”) assay where target nucleic acids of interest from a sample are amplified before detection.

FIG. 1B is an overview of the general principles underlying the nucleic acid-guided nuclease cascade assay described in detail herein where target nucleic acids of interest from a sample do not need to be amplified before detection.

FIG. 1C is an illustration of the unwinding issue that is mitigated by the modalities described herein.

FIG. 2A is a diagram showing the sequence of steps in an exemplary cascade assay utilizing blocked nucleic acid molecules.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule and a method for unblocking the blocked nucleic acid molecules of the disclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid molecule with a design able to block R-loop formation with an RNP complex, thereby blocking activation of the trans-nuclease activity of an RNP complex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acid molecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5 is a simplified block diagram of an exemplary method for designing, synthesizing and screening variant nucleic acid-guided nucleases.

FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacterium Cas12a).

FIG. 6B shows the result of example mutations on the LbCas12a protein structure prediction using Rosetta and SWISS modeling of LbCas12a and indicating the PAM regions.

FIG. 7 is a simplified diagram of acetylating the K595 amino acid in the wildtype sequence of LbCas12a (K595K^Ac).

FIG. 8A is an illustration of a blocked nucleic acid molecule with bulky modifications, cleavage thereof, and steric hindrance at the PAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by 5′ and 3′ modifications to a blocked nucleic acid molecule.

FIG. 8B illustrates five exemplary variations of blocked nucleic acid molecules with bulky modifications.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, and internal positions in blocked nucleic acid molecules.

FIG. 9 is an illustration of a lateral flow assay that can be used to detect the cleavage and separation of a signal from a reporter moiety.

FIG. 10A depicts Molecule U29 and describes the properties thereof, where MU29 was used to generate the data shown in FIGS. 10B-10H.

FIG. 11A shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation G532A in the wildtype sequence.

FIG. 11B shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K538A in the wildtype sequence.

FIG. 11C shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation Y542A in the wildtype sequence.

FIG. 11D shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K595A in the wildtype sequence.

FIG. 11E shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations G532A, K538A, Y5442A and K595A in the wildtype sequence.

FIG. 11F shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K595D in the wildtype sequence.

FIG. 11G shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutation K595E in the wildtype sequence.

FIG. 11H shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations K538A, Y542A and K595D in the wildtype sequence.

FIG. 11I shows the result of protein structure prediction using Rosetta and SWISS modeling of LbCas12a comprising the mutations K538A, Y542A and K595E in the wildtype sequence.

FIGS. 12A-12G are a series of graphs showing the time for detection of dsDNA and ssDNA both with and without PAM sequences for wildtype LbaCas12a and engineered variants of LbaCas12a.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

DEFINITIONS

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.
All of the functionalities described in connection with one embodiment of the compositions and/or methods described herein are intended to be applicable to the additional embodiments of the compositions and/or methods except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. Conventional methods are used for the procedures described herein, such as those provided in the art, and demonstrated in the Examples and various general references. Unless otherwise stated, nucleic acid sequences described herein are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA, as RNA, or a combination of DNA and RNA (e.g., a chimeric nucleic acid).
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.
The term “and/or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without another. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
As used herein, the term “about,” as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
As used herein, the terms “binding affinity” or “dissociation constant” or “K_d” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high K_d(which in the context of the present disclosure refers to blocked nucleic acid molecules or blocked primer molecules binding to RNP2) indicates the presence of more unbound molecules, and a low K_d(which in the context of the present disclosure refers to unblocked nucleic acid molecules or unblocked primer molecules binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_dvalues are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_dvalues are in the range of 100 nM-100 μM (10 mM) and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_dvalues.
As used herein, the terms “binding domain” or “binding site” refer to a region on a protein, DNA, or RNA, to which specific molecules and/or ions (ligands) may form a covalent or non-covalent bond. By way of example, a polynucleotide sequence present on a nucleic acid molecule (e.g., a primer binding domain) may serve as a binding domain for a different nucleic acid molecule (e.g., an unblocked primer nucleic acid molecule). Characteristics of binding sites are chemical specificity, a measure of the types of ligands that will bond, and affinity, which is a measure of the strength of the chemical bond.
As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second RNP complex to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to the second RNP complex (RNP2) to activate trans-cleavage of additional blocked nucleic acid molecules. A “blocked nucleic acid molecule” may be a “blocked primer molecule” in some embodiments of the cascade assay.
The terms “Cas RNA-guided nucleic acid-guided nuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated protein that is an RNA-guided nucleic acid-guided nuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP) complex.
As used herein, the terms “cis-cleavage”, “cis-nucleic acid-guided nuclease activity”, “cis-mediated nucleic acid-guided nuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence-specific cleavage of a target nucleic acid of interest, including an unblocked nucleic acid molecule or synthesized activating molecule, by a nucleic acid-guided nuclease in an RNP complex. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event.
The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-ATCGAT-5′ is 100% complementary to a region of the nucleotide sequence 5′-GCTAGCTAG-3′.
As used herein, the term “contacting” refers to placement of two moieties in direct physical association, including in solid or liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in samples or in vivo by administering an agent to a subject.
The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains comprises glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains comprises serine and threonine; a group of amino acids having amide containing side chains comprises asparagine and glutamine; a group of amino acids having aromatic side chains comprises phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains comprises lysine, arginine, and histidine; a group of amino acids having acidic side chains comprises glutamate and aspartate; and a group of amino acids having sulfur containing side chains comprises cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.
A “control” is a reference standard of a known value or range of values.
The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. The crRNA region of the gRNA is a customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein (RNP) complex containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid. Target nucleic acids of interest may include a protospacer adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region on the target nucleic acid of interest, including on an unblocked nucleic acid molecule or synthesized activating molecule. A gRNA may contain a spacer sequence including a plurality of bases complementary to a protospacer sequence in the target nucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its corresponding target nucleic acid of interest. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. A guide RNA may be from about 20 nucleotides to about 300 nucleotides long. Guide RNAs may be produced synthetically or generated from a DNA template.
“Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, a nucleic acid molecule (for example, a blocked nucleic acid molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and/or internucleoside linkages. In another embodiment, a modified protein (e.g., a modified or variant nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.
The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410 (1990)).
As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and hybridizes to it. The hybridized target nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease. In the cascade assays described herein, a first ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, cas12a or cas14a for a DNA target nucleic acid, or cas13a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification includes a second guide RNA specific to an unblocked nucleic acid or synthesized activating molecule, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease.
As used herein, the terms “protein” and “polypeptide” are used interchangeably. Proteins may or may not be made up entirely of amino acids.
As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample” may also refer to specimens or aliquots from food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water (from both natural and treatment sites), air, or sewer samples; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.
The terms “target DNA sequence”, “target sequence”, “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by a gRNA sequence in vitro or in vivo. The “target strand” of a target nucleic acid of interest is the strand of the double-stranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or RNA polynucleotides. A target nucleic acid of interest may be located in the nucleus or cytoplasm of a cell such as, for example, within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast, or it can be exogenous to a host cell, such as a eukaryotic cell or a prokaryotic cell. The target nucleic acid of interest may be present in a sample, such as a biological or environmental sample, and it can be a viral nucleic acid molecule, a bacterial nucleic acid molecule, a fungal nucleic acid molecule, or a polynucleotide of another organism, such as a coding or a non-coding sequence, and it may include single-stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The target nucleic acid of interest may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids can be detected by the disclosed method.
As used herein, the terms “trans-cleavage”, “trans-nucleic acid-guided nuclease activity”, “trans-mediated nucleic acid-guided nuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity” and variations thereof refer to indiscriminate, non-sequence-specific cleavage of a target nucleic acid molecule by a nucleic acid-guided nuclease (such as by a Cas12, Cas13, and Cas14) which is triggered by binding of N nucleotides of a target nucleic acid molecule to a gRNA and/or by cis- (sequence-specific) cleavage of a target nucleic acid molecule. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation by a single turn-over cis-cleavage event.
Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR/Cas effector nucleases such as, but not limited to, engineered Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), CasY (Cas12d), Cas 13a nucleases or naturally-occurring proteins, such as a Cas12a isolated from, for example, Francisella tularensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID: 15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.
The term “variant” in the context of the present disclosure refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in one or more amino acid residues (e.g., substitutions, additions, and/or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications—including chemical modifications—to one or more amino acids that do not involve amino acid substitutions, additions or deletions.
As used herein, the terms “variant engineered nucleic acid-guided nuclease” or “variant nucleic acid-guided nuclease” refer to nucleic acid-guided nucleases have been engineered to mutate the PAM interacting domains in the LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas 12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guided nucleases such that double-stranded DNA (dsDNA) substrates bind to the variant nucleic acid-guided nuclease and are cleaved by the variant nucleic acid-guided nuclease at a slower rate than single-stranded DNA (ssDNA) substrates.
A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter and methods for cascade assays that detect nucleic acids. The cascade assays allow for massive multiplexing, and provide high accuracy, low cost, minimum workflow and results in less than one minute or, in some embodiments, virtually instantaneously, even at ambient temperatures of about 16-20° C. or less up to 48° C. The cascade assays described herein comprise first and second ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep the second ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex. The methods comprise the steps of providing cascade assay components, contacting the cascade assay components with a sample, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample.
Early and accurate identification of, e.g., infectious agents, microbe contamination, variant nucleic acid sequences that indicate the presence of diseases such as cancer or contamination by heterologous sources is important in order to select correct treatment; identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. Nucleic acid-guided nucleases, such as Type V nucleic acid-guided nucleases, can be utilized for the detection of target nucleic acids of interest associated with diseases, food contamination and environmental threats. However, currently available nucleic acid detection such as quantitative PCR (also known as real time PCR or qPCR) or CRISPR-based detection assays such as SHERLOCK™ and DETECTR™ rely on DNA amplification, which requires time and may lead to changes to the relative proportion of nucleic acids, particularly in multiplexed nucleic acid assays. The lack of rapidity for these detection assays is due to the fact that there is a significant lag phase early in the amplification process where fluorescence above background cannot be detected. With qPCR, for example, there is a lag until the cycle threshold or Ct value, which is the number of amplification cycles required for the fluorescent signal to exceed the background level of fluorescence, is achieved and can be quantified.
The present disclosure describes a signal boost cascade assay and improvements thereto that can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in less than one minute and in some embodiments virtually instantaneously without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. As described in detail below, the cascade assays utilize signal boost mechanisms comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes), blocked nucleic acid molecules or blocked primer molecules, reporter moieties, and, in some embodiments, polymerases and template molecules. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1 (i.e., gRNA1), the cascade assay components are essentially identical no matter what target nucleic acid(s) of interest are being detected, and gRNA1 is easily programmable.
The improvements to the signal amplification or signal boost cascade assay described herein result from preventing undesired unwinding of the blocked nucleic acid molecules in the reaction mix by the second ribonucleoprotein complex (RNP2) before the blocked nucleic acid molecules are unblocked via trans-cleavage, leading to increased efficiency, reduced background, and increased signal-to-noise ratio in the cascade assay. Minimizing undesired unwinding serves two purposes. First, preventing undesired unwinding that happens not as a result of unblocking due to trans-cleavage subsequent to cis-cleavage of the target nucleic acid of interest or trans-cleavage of unblocked nucleic acid molecules—but due to other factors—leads to a “leaky” cascade assay system, which in turn leads to non-specific signal generation.
Second, preventing undesired unwinding limits non-specific interactions between the nucleic acid-guided nucleases (here, in the RNP2s) and blocked nucleic acid molecules such that only blocked nucleic acid molecules that become unblocked due to trans-cleavage activity react with the nucleic acid-guided nucleases. This “fidelity” in the cascade assay leads primarily to desired interactions and limits “wasteful” interactions where the nucleic acid-guided nucleases are essentially acting on blocked nucleic acid molecules rather than unblocked nucleic acid molecules. That is, the nucleic acid-guided nucleases are focused on desired interactions which then leads to immediate signal amplification or boost in the cascade assay.
The present disclosure provides three modalities to minimize leakiness leading to minimal false positives or higher background signal. The present disclosure demonstrates that undesired unwinding of the blocked nucleic acid molecules can be lessened substantially by 1) increasing the molar ratio of the concentration of blocked nucleic acid molecules (equivalent to a target nucleic acid molecule for the RNP2) to be equal to or greater than the molar concentration of RNP2 (e.g., the nucleic acid-guided nuclease in RNP2); 2) engineering the nucleic acid-guided nuclease used in RNP2 so as to increase the time it takes the nucleic acid-guided nuclease to recognize double-strand DNA at least two-fold and preferably three-fold or more; and/or 3) engineering the blocked nucleic acid molecules to include bulky modifications (that is, molecules with a size of about 1 nm or less).
The first modality for minimizing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules) is to adjust the relative concentrations of the blocked nucleic acid molecules (or blocked primer molecules) and RNP2s such that the molar concentration of the blocked nucleic acid molecules (or blocked primer molecules) is equal to or greater than the molar concentration of RNP2s. Before the present disclosure, the common wisdom in performing CRISPR detection assays was to use a vast excess of nucleic acid-guided nuclease (e.g., RNP complex) to target.
In most detection assays, the quantity of the target nucleic acid of interest is not known (e.g., the detection assay is performed on a sample with an unknown concentration of target); however, in experiments conducted to determine the level of detection of two CRISPR detection assays known in the art, DETECTR™ and SHERLOCK™, the nucleic acid nuclease was present at ng/μL concentrations and the target of interest was present at very low copy numbers or at femtomolar to attamolar concentration. Thus, the present methods and reagent mixtures not only adjust the relative concentrations of the blocked nucleic acid molecules (or blocked primer molecules) and RNP2s such that the molar concentration of the blocked nucleic acid molecules (or blocked primer molecules) is equal to or greater than the molar concentration of RNP2s, but the molar concentration of RNP2s may still exceed the molar concentration of the blocked nucleic acid molecules by a lesser amount, such as where the molar concentration of RNP2s exceeds the molar concentration of blocked nucleic acid molecules (or blocked target molecules) by 100,000×, 50,000×, 25,000×, 10,000×, 5,000×, 1000×, 500×, 100×, 50×, or 10× or less.
For example, Sun, et al. ran side-by-side comparisons of the DETECTR™ and SHERLOCK™ detection assays, using a concentration of 100 ng/μL LbCas12a in the DETECTR™ assay and a concentration of 20 ng/μL LwCas13a in the SHERLOCK™ assay, where the concentration of the target nucleic acid molecules ranged from 0 copies/μL, 0.1 copies/μL, 0.2 copies/μL, 1.0 copy/μL, 2.0 copies/μL, 5.0 copies/μL, 10.0 copies/μL, and so on up to 200.0 copies/μL. (Sun, et al., J. of Translational Medicine, 12:74 (2021).) In addition, Broughton, et al., ran the DETECTR™ assay using a concentration range of 2.5 copies/μL, to 1250 copies/μL, target nucleic acid molecules to 40 nM LbCas12 (see, Broughton, et al., Nat. Biotech., 38:870-74 (2020)); and Lee, et al., ran the SHERLOCK™ assay using a concentration range of 10 fM to 50 aM target nucleic acid molecules to 150 nM Cas12 (see Lee, et al., PNAS, 117(41):25722-31 (2020). Thus, the ratio of nucleic acid-guided nuclease to blocked nucleic acid molecule (e.g., target for RNP2) described herein is very different from ratios practiced in the art and this ratio has been determined to limit undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules).
In a second modality, variant nucleic acid-guided nucleases have been engineered to mutate the domains in the variants that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules in, e.g., Type V nucleic acid-guided nucleases such as the LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), and other related nucleic acid-guided nucleases (e.g., homologs and orthologs of these nucleic acid-guided nucleases) also limit unwinding. These variant nucleic acid-guided nucleases have been engineered such that double-stranded DNA (dsDNA) substrates bind to and activate to the variant nucleic acid-guided nucleases slowly, but single-stranded DNA (ssDNA) substrates continue to bind and activate the variant nucleic acid-guided nuclease at a high rate. Thus, the variant nucleic acid-guided nucleases effect a “lock” on the RNP complex (here, the RNP2) vis-à-vis double-strand DNA. Locking RNP2 in this way lessens the likelihood of undesired unwinding of the blocked nucleic acid molecules as described in detail herein (see FIG. 1C and the accompanying discussion). Modifying the nucleic acid-guided nucleases to not recognize dsDNA or to recognize dsDNA is contrary to what is desired in other CRISPR-based diagnostic/detection assays.
Finally, another modality for minimizing undesired unwinding of the blocked nucleic acid molecules is to use “bulky modifications” at the 5′ and/or 3′ ends of the blocked nucleic acid molecules and/or at internal nucleic acid bases of the blocked nucleic acid molecules. Doing so creates steric hindrance at the domains of the nucleic acid-guided nuclease in RNP2 that interact with the PAM region or that interact with surrounding sequences on the blocked nucleic acid molecules, disrupting, e.g., PAM recognition in the target strand and preventing displacement of the non-target strand. Using bulky modifications is yet another path to locking RNP2 to double-strand DNA molecules thereby lessening the likelihood of undesired unwinding of the blocked nucleic acid molecules as described in detail herein (again, see FIG. 1C and the accompanying discussion). “Bulky modifications” include molecules with a size of about 1 nm or less.
FIG. 1A provides a simplified diagram demonstrating a prior art method for quantifying target nucleic acids of interest in a sample; namely, the quantitative polymerase chain reaction or qPCR, which to date may be considered the gold standard for quantitative detection assays. The difference between PCR and qPCR is that PCR is a qualitative technique that indicates the presence or absence of a target nucleic acid of interest in a sample, where qPCR allows for quantification of target nucleic acids of interest in a sample. qPCR involves selective amplification and quantitative detection of specific regions of DNA or cDNA (i.e., the target nucleic acid of interest) using oligonucleotide primers that flank the specific region(s) in the target nucleic acid(s) of interest. The primers are used to amplify the specific regions using a polymerase. Like PCR, repeated cycling of the amplification process leads to an exponential increase in the number of copies of the region(s) of interest; however, unlike traditional PCR, the increase is tracked using an intercalating dye or, as shown in FIG. 1A, a sequence-specific probe (e.g., a “Taq-man probe”) the fluorescence of which is detected in real time. RT-qPCR differs from qPCR in that a reverse transcriptase is used to first copy RNA molecules to produce cDNA before the qPCR process commences.
FIG. 1A is an overview of a qPCR assay where target nucleic acids of interest from a sample are amplified before detection. FIG. 1A shows the qPCR method 10, comprising a double-stranded DNA template 12 and a sequence specific Taq-man probe 14 comprising a region complementary to the target nucleic acid of interest 20, a quencher 16, a quenched fluorophore 18 where 22 denotes quenching between the quencher 16 and quenched fluorophore 18. Upon denaturation, the two strands of the double-stranded DNA template 12 separate into complementary single strands 26 and 28. In the next step, primers 24 and 24′ anneal to complementary single strands 26 and 28, as does the sequence-specific Taq-man probe 14 via the region complementary 20 to the complementary strand 26 of the target nucleic acid of interest. Initially the Taq-man probe is annealed to complementary strand 26 of the target region of interest intact; however, primers 24 and 24′ are extended by polymerase 30 but the Taq-man probe is not, due to the absence of a 3′ hydroxy group. Instead, the exonuclease activity of the polymerase “chews up” the Taq-man probe, thereby separating the quencher 16 from the quenched fluorophore 18 resulting in an unquenched or excited-state fluorophore 34. The fluorescence quenching ensures that fluorescence occurs only when target nucleic acids of interest are present and being copied, where the fluorescent signal is proportional to the number of single-strand target nucleic acids being amplified.
As noted above, the downside to the prior art, currently available detection assays such as qPCR, as well as CRISPR-based reaction assays such as SHERLOCK™ and DETECTR™ is that these assays rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid testing, e.g., in the field. That is, where the present cascade assay works at ambient temperatures, including room temperatures and below, assays that require amplification of the target nucleic acids of interest do not work well at lower temperatures—even those assays utilizing isothermal amplification—due to non-specific binding of the primers and low polymerase activity. Further, primer design is far more challenging. As for the lack of rapidity of detection assays that require amplification of the target nucleic acids of interest, a significant lag phase occurs early in the amplification process where fluorescence above background cannot be detected, particularly in samples with very low copy numbers of the target nucleic acid of interest. And, again, amplification, particularly multiplex amplification, may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results.
FIG. 1B provides a simplified diagram demonstrating a method (100) of a cascade assay. The cascade assay is initiated when the target nucleic acid of interest (104) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides an RNP complex to the target nucleic acid of interest and hybridizes to it. Typically, preassembled RNP complexes are employed in the reaction mix—as opposed to separate nucleic acid-guided nucleases and gRNAs—to facilitate rapid (and in the present cascade assays, virtually instantaneous) detection of the target nucleic acid(s) of interest.
“Activation” of RNP1 refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) by binding of the target nucleic acid-guided nuclease to the gRNA of RNP1, initiating cis-cleavage where the target nucleic acid of interest is cleaved by the nucleic acid-guided nuclease. This binding and/or cis-cleavage activity then initiates trans-cleavage activity (i.e., multi-turnover activity) of the nucleic acid-guided nuclease, where trans-cleavage is indiscriminate, leading to non-sequence-specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP1 (102). This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) in various ways, which are described in detail below. Each newly activated RNP2 (110) activates more RNP2 (108→110), which in turn cleave reporter moieties (112). The reporter moieties (112) may be a synthetic molecule linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart (or about 10-11 nm apart) or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties also are described in greater detail below.
As more RNP2s are activated (108→110), more trans-cleavage activity is activated and more reporter moieties are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (104) to RNP1 (102) initiates what becomes a cascade of signal production (120), which increases exponentially; hence, the terms “signal amplification” or “signal boost.” The cascade assay thus comprises a single turnover event that triggers a multi-turnover event that then triggers another multi-turnover event in a “cascade.” As described below in relation to FIG. 4 , the reporter moieties (112) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease cascade assay, or the reporter moieties may be covalently or non-covalently linked to the blocked nucleic acid molecules or synthesized activating molecules (i.e., the target molecules for the RNP2).
As described in detail below, the present description presents three modalities for minimizing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules), which possess regions of double-strand DNA, where such unwinding can lead to non-specific signal generation and false positives. The modalities are 1) altering the ratio of the nucleic acid-guided nuclease in RNP2 to the blocked nucleic acid molecules in contravention to the common wisdom for CRISPR detection/diagnostic assays; 2) engineering the nucleic acid-guided nuclease used in RNP2 so that recognition of double-stranded DNA occurs more slowly than for single-strand DNA, in contravention to nucleic acid-guided nucleases that are used in other CRISPR-based detection assays; and 3) modifying the 5′ and/or 3′ ends and/or various internal nucleic acid bases of the blocked nucleic acid molecules. One, two or all three of these modalities may be employed in a given assay.
FIG. 1C is an illustration of the effects of unwinding. FIG. 1C shows at left a double-strand blocked nucleic acid molecule comprising a target strand and a non-target strand, where the non-target strand comprises regions (shown as loops) unhybridized to the target strand. Proceeding right at top, cleavage of the loops in the non-target strand by trans-cleavage initiated by RNP1 or RNP2 destabilizes the double-strand blocked nucleic acid molecule; that is, the now short regions of the non-target strand that are hybridized to the target strand become destabilized and dehybridize. As these short regions dehybridize, the target strand is released and can bind to gRNA2 in RNP2, triggering cis-cleavage of the target strand followed by trans-cleavage of additional blocked nucleic acid molecules. This process is the signal boost assay working as designed.
The pathway at the bottom of FIG. 1C illustrates the effect of undesired unwinding; that is, unwinding due not to trans-cleavage as designed but by other unwinding due to recognition of the blocked nucleic acid molecule by gRNA2 and the nucleic acid-guided nuclease in RNP2. As seen in the alternative pathway at bottom of FIG. 1C, R-loop formation between RNP2 and the blocked nucleic acid molecule (or blocked primer molecule) can still occur due to unwinding of the blocked nucleic acid molecule after gRNA2 identifies the PAM. Indeed, this unwinding can occur even in the absence of a PAM. It is an inherent characteristic of the biology of nucleic acid-guided nucleases.
Various components of the cascade assay, descriptions of how the cascade assays work, and the modalities used to minimize undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules) are described in detail below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques (e.g., RT-PCR). The target nucleic acids of interest are identified in a sample, such as a biological sample from a subject (including non-human animals or plants), items of manufacture, or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep, and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrial sites (including food processing sites) and products, plants and grains, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.
In some embodiments, the target nucleic acid of interest is from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus) that affects mammals, including humans. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from bacteria, such as Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum, Mycoplasma genitalium, and/or Gardnerella vaginalis. Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and/or human parvovirus B19 (B19V). Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a fungus, such as Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and/or Cryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such as Trichomonas vaginalis. See, e.g., Table 1 for an exemplary list of human pathogens, Table 2 for an exemplary list of human sexually transmissible diseases.

TABLE 1

Human Pathogens

		NCBI
		Taxonomy	NCBI Sequence ID
Name	Category	ID	Number

Acinetobacter baumannii	Bacteria	470	GCF_008632635.1
Acinetobacter calcoaceticus	Bacteria	471	GCF_002055515.1
Acinetobacter	Bacteria	909768	Not applicable
calcoaceticus-baumannii
complex
Anaplasma	Bacteria	948	GCF_000439775.1
phagocytophilum
Bacillus anthracis	Bacteria	1392	GCF_000008445.1
Bacteroides fragilis	Bacteria	817	GCF_016889925.1
Bartonella henselae	Bacteria	38323	GCF_000612965.1
Bordetella parapertussis	Bacteria	519	GCF_004008295.1
Bordetella pertussis	Bacteria	520	GCF_004008975.1
Borrelia mayonii	Bacteria	1674146	GCF_001936295.1
Borrelia miyamotoi	Bacteria	47466	GCF_003431845.1
Brucella abortus	Bacteria	235	GCF_000054005.1
Brucella melitensis	Bacteria	29459	GCF_000007125.1
Brucella suis	Bacteria	29461	GCF_000007505.1
Burkholderia mallei	Bacteria	13373	GCF_002346025.1
Burkholderia pseudomallei	Bacteria	28450	GCF_000756125.1
Campylobacter jejuni	Bacteria	197	GCF_000009085.1
Chlamydia pneumoniae	Bacteria	83558	GCF_000007205.1
Chlamydia psittaci	Bacteria	83554	GCF_000204255.1
Chlamydia Tracomatis	Bacteria	813	GCF_000008725.1
Clostridium botulinum	Bacteria	1491	GCF_000063585.1
Clostridium perfringens	Bacteria	1502	GCF_020138775.1
Coxiella burnetii	Bacteria	777	GCF_000007765.2
Ehrlichia chaffeesis	Bacteria	945	GCF_000632965.1
Ehrlichia ewingii	Bacteria	947	Not available
Ehrlichia ruminantium	Bacteria	779	GCF_013460375.1
Enterobacter cloacae	Bacteria	550	GCF_000770155.1
Enterobacter cloacae	Bacteria	354276	Not applicable
complex
Enterococcus faecalis	Bacteria	1351	GCF_000393015.1
Enterococcus faecium	Bacteria	1352	GCF_009734005.1
Escherichia coli	Bacteria	562	GCF_000008865.2
Francisella tularensis	Bacteria	263	GCF_000156415.1
Gardnerella vaginalis	Bacteria	2702	GCF_002861965.1
Haemophilus influenzae	Bacteria	727	GCF_000931575.1
Klebsiella aerogenes	Bacteria	548	GCF_007632255.1
Klebsiella oxytoca	Bacteria	571	GCF_003812925.1
Klebsiella pneumoniae	Bacteria	573	GCF_000240185.1
Legionella pneumophila	Bacteria	446	GCF_001753085.1
Leptospira interrogans	Bacteria	173	GCF_002073495.2
Leptospira kirschneri	Bacteria	29507	GCF_000243695.2
Leptospira wolffii	Bacteria	409998	GCF_004770635.1
Listeria monocytogenes	Bacteria	1639	GCF_000196035.1
Moraxella catarrhalis	Bacteria	480	GCF_002080125.1
Mycobacterium tuberculosis	Bacteria	1773	GCF_000195955.2
Mycoplasma genitalium	Bacteria	2097	GCF_000027325.1
Mycoplasma pneumoniae	Bacteria	2104	GCF_900660465.1
Neisseria gonorrhoeae	Bacteria	485	GCF_013030075.1
Neisseria meningitidis	Bacteria	487	GCF_008330805.1
Proteus hauseri	Bacteria	183417	GCF_004116975.1
Proteus mirabilis	Bacteria	584	GCF_000069965.1
Proteus penneri	Bacteria	102862	GCF_022369495.1
Proteus vulgaris	Bacteria	585	GCF_000754995.1
Pseudomonas aeruginosa	Bacteria	287	GCF_000006765.1
Rickettsia parkeri	Bacteria	35792	GCF_005549115.1
			GCA_018610945.1
			GCF_000965075.1
			GCF_000965085.1
			GCF_000284195.1
			GCF_000965145.1
Rickettsia prowazekii	Bacteria	782	GCF_000277165.1
Rickettsia rickettsii	Bacteria	783	GCF_000017445.4
Salmonella bongori	Bacteria	54736	GCF_000439255.1
Salmonella enterica	Bacteria	28901	GCF_000006945.2
Salmonella enterica	Bacteria	28901	GCF_000006945.2
Serratia marcescens	Bacteria	615	GCF_003516165.1
Shigella boydii	Bacteria	621	GCF_001905915.1
Shigella dysenteriae	Bacteria	622	GCF_001932995.2
Shigella flexneri	Bacteria	623	GCF_000006925.2
Shigella sonnei	Bacteria	624	GCF_013374815.1
Staphylococcus auerus	Bacteria	1280	GCF_000013425.1
Staphylococcus enterotoxin	Bacteria	1280	U93688.2
B
Staphylococcus epidermidis	Bacteria	1282	GCF_006094375.1
Staphylococcus lugdunensis	Bacteria	28035	GCF_001558775.1
Stenotrophomonas	Bacteria	40324	GCF_900475405.1
maltophilia
Streptococcus agalactiae	Bacteria	1311	GCF_001552035.1
Streptococcus pneumoniae	Bacteria	1313	GCF_002076835.1
Streptococcus pyogenes	Bacteria	1314	GCF_900475035.1
Treponema pallidum	Bacteria	160	GCF_000246755.1
Ureaplasma urealyticum	Bacteria	2130	GCF_000021265.1
Vibrio parahaemolyticus	Bacteria	670	GCF_000196095.1
Vibrio vulnificus	Bacteria	672	GCF_002204915.1
Yersinia enterocolitica	Bacteria	630	GCF_001160345.1
Yersinia pestis	Bacteria	632	GCF_000222975.1
Candida albicans	Fungus	5476	GCF_000182965.3
Candida auris	Fungus	498019	GCF_002775015.1
Candida glabrata	Fungus	5478	GCF_000002545.3
Candida parapsilosis	Fungus	5480	GCF_000182765.1
Candida tropicalis	Fungus	5482	GCF_000006335.3
Coccidioides immitis	Fungus	5501	GCF_000149335.2
Coccidioides posadasii	Fungus	199306	GCF_000151335.2
Cokeromyces recurvatus	Fungus	90255	GCA_000697235.1
Cryptococcus gattii	Fungus	37769	GCF_000185945.1
Cryptococcus neoformans	Fungus	5207	GCF_000091045.1
Cunninghamella	Fungus	90251	GCA_000697215.1
bertholletiae
Encephalitozoon cuniculi	Fungus	6035	GCF_000091225.1
Encephalitozoon hellem	Fungus	27973	GCF_000277815.2
Encephalitozoon intestinalis	Fungus	58839	GCF_000146465.1
Enterocystozoon bieneusi	Fungus	31281	GCF_000209485.1
Mortierella wolfii	Fungus	90253	GCA_016098105.1
Pichia kudriavzevii	Fungus	4909	GCF_003054445.1
Saksenaea vasiformis	Fungus	90258	GCA_000697055.1
Syncephalastrum	Fungus	13706	GCA_002105135.1
racemosum
Trichomonas vaginalis	Fungus	5722	GCF_000002825.2
Ricinus communis	Plant	3988	GCF_019578655.1
Acanthamoeba castellanii	Protozoa	5755	GCF_000313135.1
Babesia divergens	Protozoa	32595	GCA_001077455.2
Babesia microti	Protozoa	5868	GCF_000691945.2
Balamuthia mandrillaris	Protozoa	66527	GCA_001185145.1
Cryptosporidium parvum	Protozoa	5807	GCF_000165345.1
Cyclospora cayatanensis	Protozoa	88456	GCF_002999335.1
Entamoeba histolytica	Protozoa	5759	GCF_000208925.1
Giardia lamblia	Protozoa	5741	GCF_000002435.2
Naegleria fowleri	Protozoa	5763	GCF_008403515.1
Toxoplasma gondii	Protozoa	5811	GCF_000006565.2
Alkhumra hemorrhagic	Virus	172148	JF416961.1
fever virus
Argentinian	Virus	2169991	GCF_000856545.1
mammarenavirus
Betacoronavirus 1	Virus	694003	GCF_000862505.1
			GCF_003972325.1
Black Creek Canal	Virus	1980460	GCF_002817355.1
orthohantavirus
California encephalitis	Virus	1933264	GCF_003972565.1
orthobunyavirus
Chapare mammarenavirus	Virus	499556	GCF_000879235.1
Chikungunya virus	Virus	37124	GCF_000854045.1
Crimean-Congo	Virus	1980519	GCF_000854165.1
hemorrhagic fever
orthnairovirus
Dabie bandavirus	Virus	2748958	GCF_000897355.1
			GCF_003087855.1
Deer tick virus	Virus	58535	MZ148230 to
			MZ148271
Dengue virus 1	Virus	11053	GCF_000862125.1
Dengue virus 2	Virus	11060	GCF_000871845.1
Dengue virus 3	Virus	11069	GCF_000866625.1
Dengue virus 4	Virus	11070	GCF_000865065.1
Eastern equine encephalitis	Virus	11021	GCF_000862705.1
virus
Enterovirus A	Virus	138948	GCF_002816655.1
			GCF_000861905.1
			GCF_001684625.1
Enterovirus B	Virus	138949	GCF_002816685.1
			GCF_000861325.1
Enterovirus C	Virus	138950	GCF_000861165.1
Enterovirus D	Virus	138951	GCF_000861205.1
			GCF_002816725.1
Guanarito mammarenavirus	Virus	45219	GCF_000853765.1
Heartland bandavirus	Virus	2747342	GCF_000922255.1
Hendra henipavirus	Virus	63330	GCF_000852685.1
Hepacivirus C	Virus	11103	GCF_002820805.1
			GCF_000861845.1
			GCF_000871165.1
			GCF_000874285.1
			GCF_001712785.1
hepatitis A virus	Virus	208726	K02990.1
			M14707.1
			M20273.1
			X75215.1
			AB020564.1
hepatitis B virus	Virus	10407	GCF_000861825.2
hepatitis C virus	Virus	11103	GCF_002820805.1
			GCF_000861845.1
			GCF_000871165.1
			GCF_000874285.1
			GCF_000874265.1
			GCF_001712785.1
Hepatovirus A	Virus	12092	GCF_000860505.1
Human adenovirus A	Virus	129875	GCF_000846805.1
Human adenovirus B	Virus	108098	GCF_000857885.1
Human adenovirus C	Virus	129951	GCF_000858645.1
Human adenovirus D	Virus	130310	GCF_000885675.1
Human adenovirus E	Virus	130308	GCF_000897015.1
Human adenovirus F	Virus	130309	GCF_000846685.1
Human adenovirus G	Virus	536079	GCF_000847325.1
Human alphaherpesvirus 1	Virus	10298	GCF_000859985.2
Human alphaherpesvirus 2	Virus	10310	GCF_000858385.2
human betaherpesvirus 6A	Virus	32603	GCF_000845685.2
human betaherpesvirus 6B	Virus	32604	GCF_000846365.1
Human coronavirus 229E	Virus	11137	GCF_001500975.1
			GCF_000853505.1
Human coronavirus HKU1	Virus	290028	GCF_000858765.1
Human coronavirus NL63	Virus	277944	GCF_000853865.1
Human coronavirus OC43	Virus	31631	GCF_003972325.1
Human gammaherpesvirus	Virus	37296	GCF_000838265.1
8
Human immunodeficiency	Virus	11676	GCF_000864765.1
virus 1
Human immunodeficiency	Virus	11709	GCF_000856385.1
virus 2
human metapneumovirus	Virus	162145	GCF_002815375.1
human papillomavirus	Virus		GCF_001274345.1
Human polyomavirus 1	Virus	1891762	GCF_000837865.1
Human polyomavirus 2	Virus	1891763	GCF_000863805.1
human rhinovirus A	Virus	147711	GCF_000862245.1
			GCF_002816835.1
human rhinovirus B	Virus	147712	GCF_000861265.1
			GCF_002816855.1
human rhinovirus C	Virus	463676	GCF_002816885.1
			GCF_000872325.1
Influenza A virus	Virus	11320	GCF_001343785.1
			GCF_000851145.1
			GCF_000866645.1
Influenza B virus	Virus	11520	GCF_000820495.2
Influenza C virus	Virus	11552	GCF_000856665.10
Influenza D virus	Virus	1511084	GCF_002867775.1
Japanese encephalitis virus	Virus	11072	GCF_000862145.1
Kyasanur Forest disease	Virus	33743	GCF_002820625.1
virus
La Crosse orthobunyavirus	Virus	2560547	GCF_000850965.1
Lassa virus	Virus	11620	GCF_000851705.1
Lujo mammarenavirus	Virus	649188	GCF_000885555.1
Lyssavirus australis	Virus	90961	GCF_000850325.1
Marburg virus	Virus		NC_001608.3
Measles morbillivirus	Virus	11234	GCF_000854845.1
Middle East respiratory	Virus	1335626	GCF_002816195.1
syndrome-related			GCF_000901155.1
coronavirus
Monongahela hantavirus	Virus	2259728	MH539865
			MH539866
			MH539867
New York hantavirus	Virus	44755	U36803.1
			U36802.1
			U36801.1
			U09488.1
Nipah henipavirus	Virus	121791	GCF_000863625.1
Norwalk virus	Virus	11983	GCF_000864005.1
			GCF_008703965.1
			GCF_008703985.1
			GCF_008704025.1
			GCF_010478905.1
			GCF_000868425.1
Omsk hemorrhagic fever	Virus	12542	GCF_000855505.1
virus
parainfluenza virus 1	Virus	12730	GCF_000848705.1
			NC_003461
parainfluenza virus 2	Virus		X57559.1
			AF533010
			AF533011
			AF533012
parainfluenza virus 3	Virus	11216	GCA_006298365.1
			GCA_000850205.1
parainfluenza virus 4	Virus	2560526	NC_021928.1
Paslahepevirus balayani	Virus	1678141	GCF_000861105.1
Poliovirus	Virus	138950	GCF_000861165.1
Primate erythroparvovirus 1	Virus	1511900	GCF_000839645.1
Rabies lyssavirus	Virus	11292	GCF_000859625.1
respiratory syncytial virus	Virus	12814	GCF_000856445.1
Rift Valley virus	Virus	11588	HE687302
			HE687307
Saint Louis encephalitis	Virus	11080	GCF_000866785.1
virus
			GCF_000849945.1
			GCF_000855765.1
Sapporo virus	Virus	95342	GCF_000854265.1
			GCF_001008475.1
			GCF_000853825.1
SARS-related coronavirus	Virus	694009	GCF_000864885.1
			GCF_009858895.2
Severe acute respiratory	Virus	2901879	NC_004718.3
syndrome coronavirus 1
Severe acute respiratory	Virus	2697049	NC_045512.2
syndrome coronavirus 2
Sin Nombre virus	Virus	1980491	GCF_000854765.1
Tick-borne encephalitis	Virus	11084	GCF_000863125.1
virus
Variola major	Virus	12870	not available
Variola minor	Virus	53258	not available
Variola virus	Virus	10255	GCF_000859885.1
Venezuelan equine	Virus	11036	GCF_000862105.1
encephalitis virus
West Nile virus	Virus	11082	GCF_000861085.1
			GCF_000875385.1
Western equine encephalitis	Virus	11039	GCF_000850885.1
virus
Yellow fever virus	Virus	11089	GCF_000857725.1
Zaire ebolavirus	Virus	186538	GCF_000848505.1
Zika virus	Virus	64320	GCF_000882815.3
			GCF_002366285.1

TABLE 2

Human STD pathogens

		NCBI
		Taxonomy	NCBI Sequence
Name	Category	ID	ID Number

Pthirus pubis	Animal	121228	MT721740.1
Sarcoptes scabiei	Animal	52283	GCA_020844145.1
Chlamydia trachomatis	Bacteria	813	GCF_000008725.1
Gardnerella vaginalis	Bacteria	2702	GCF_002861965.1
Haemophilus ducreyi	Bacteria	730	GCF_001647695.1
Mycoplasma genitalium	Bacteria	2097	GCF_000027325.1
Neisseria gonorrhoeae	Bacteria	485	GCF_013030075.1
Treponema pallidum	Bacteria	160	GCF_000246755.1
Trichomonas vaginalis	Protozoa	5722	GCF_000002825.2
Hepacivirus C	Virus	11103	GCF_002820805.1
Hepatitis B virus	Virus	10407	GCF_000861825.2
Hepatitis delta virus	Virus	12475	GCF_000856565.1
Hepatovirus A	Virus	12092	GCF_000860505.1
Human alphaherpesvirus 1	Virus	10298	GCF_000859985.2
Human immunodeficiency	Virus	11676	GCF_000864765.1
virus 1
Human immunodeficiency	Virus	11709	GCF_000856385.1
virus 2
Human papillomavirus	Virus	10566	GCF_001274345.1

Additionally, the target nucleic acid of interest may originate in an organism such as a bacterium, virus, fungus or other pest that infects livestock or agricultural crops. Such organisms include avian influenza viruses, mycoplasma and other bovine mastitis pathogens, Clostridium perfringens, Campylobacter sp., Salmonella sp., Pospirivoidae, Avsunvirodiae, Panteoea stewartii, Mycoplasma genitalium, Sprioplasma sp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora, Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens, Spiroplasma citri, Phytophthora infestans, Endothia parasitica, Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis, Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytis cincerea, Plasmopara viticola, or Botryotinis fuckleina. See, e.g., Table 3 for an exemplary list of non-human animal pathogens.

TABLE 3

Animal Pathogens

		NCBI
		Taxonomy	NCBI Sequence
Name	Category	ID	ID Number

Acarapis woodi	Animal	478375	GCA_023170135.1
Aethina tumida	Animal	116153	GCF_001937115.1
Chorioptes bovis	Animal	420257
Chrysomya bezziana	Animal	69364
Cochliomyia hominivorax	Animal	115425	GCA_004302925.1
Echinococcus granulosus	Animal	6210	GCF_000524195.1
Echinococcus	Animal	6211	GCA_000469725.3
multilocularis
Gyrodactylus salaris	Animal	37629	GCA_000715275.1
Psoroptes ovis	Animal	83912	GCA_002943765.1
Sarcoptes scabiei	Animal	52283	GCA_020844145.1
Taenia solium	Animal	6204	GCA_001870725.1
Trichinella britovi	Animal	45882	GCA_001447585.1
Trichinella nativa	Animal	6335	GCA_001447565.1
Trichinella nelsoni	Animal	6336	GCA_001447455.1
Trichinella papuae	Animal	268474	GCA_001447755.1
Trichinella pseudospiralis	Animal	6337	GCA_001447645.1
Trichinella spiralis	Animal	6334	GCF_000181795.1
Trichinella zimbabwensis	Animal	268475	GCA_001447665.1
Tropilaelaps clareae	Animal	208209
Tropilaelaps koenigerum	Animal	208208
Tropilaelaps mercedesae	Animal	418985	GCA_002081605.1
Tropilaelaps thaii	Animal	418986
Varroa destructor	Animal	109461	GCF_002443255.1
Varroa jacobsoni	Animal	62625	GCF_002532875.1
Varroa rindereri	Animal	109259
Varroa underwoodi	Animal	109260
Anaplasma centrale	Bacteria	769	GCF_000024505.1
Anaplasma marginale	Bacteria	770	GCF_000020305.1
Bacillus anthracis	Bacteria	1392	GCF_000008445.1
Brucella abortus	Bacteria	235	GCF_000054005.1
Brucella melitensis	Bacteria	29459	GCF_000007125.1
Brucella ovis	Bacteria	236	GCF_000016845.1
Brucella suis	Bacteria	29461	GCF_000007505.1
Burkholderia mallei	Bacteria	13373	GCF_002346025.1
Burkholderia pseudomallei	Bacteria	28450	GCF_000756125.1
Campylobacter fetus	Bacteria	196	GCF_000015085.1
Candidatus Xenohaliotis	Bacteria	84677
californiensis
Candidatus Hepatobacter	Bacteria	1274402	GCF_000742475.1
penaei
Chlamydia abortus	Bacteria	83555	GCF_900416725.2
Chlamydia psittaci	Bacteria	83554	GCF_000204255.1
Corynebacterium	Bacteria	1719	GCF_001865765.1
pseudotuberculosis
Coxiella burnetii	Bacteria	777	GCF_000007765.2
Ehrlichia ruminantium	Bacteria	779	GCF_013460375.1
Francisella tularensis	Bacteria	263	GCF_000156415.1
Melissococcus plutonius	Bacteria	33970	GCF_003966875.1
Mycobacterium avium	Bacteria	1764	GCF_000696715.1
Mycobacterium	Bacteria	1773	GCF_000195955.2
tuberculosis
Mycoplasma capricolum	Bacteria	2095	GCF_000012765.1
Mycoplasma gallisepticum	Bacteria	2096	GCF_000286675.1
Mycoplasma mycoides	Bacteria	2102	GCF_000023685.1
Mycoplasma putrefaciens	Bacteria	2123	GCF_900476175.1
Mycoplasmopsis agalactiae	Bacteria	2110	GCF_009150585.1
Mycoplasmopsis synoviae	Bacteria	2109	GCF_013393745.1
Paenibacillus larvae	Bacteria	1464	GCF_002951935.1
Pasteurella multocida	Bacteria	747	GCF_000006825.1
Salmonella enterica	Bacteria	28901	GCF_000006945.2
Streptococcus equi	Bacteria	1336	GCF_015689455.1
Taylorella equigenitalis	Bacteria	29575	GCF_002288025.1
Vibrio parahaemolyticus	Bacteria	670	GCF_000196095.1
Batrachochy trium	Fungi	109871	GCF_000203795.1
dendrobatidis
Batrachochy trium	Fungi	1357716	GCA_021556675.1
salamandrivorans
Aphanomyces astaci	Oomycota	112090	GCF_000520075.1
Aphanomyces invadans	Oomycota	157072	GCF_000520115.1
Babesia bigemina	Protozoa	5866	GCF_000981445.1
Babesia bovis	Protozoa	5865	GCA_000165395.2
Babesia caballi	Protozoa	5871
Bonamia exitiosa	Protozoa	362532
Bonamia ostreae	Protozoa	126728
Leishmania amazonensis	Protozoa	5659	GCA_005317125.1
Leishmania braziliensis	Protozoa	5660	GCF_000002845.2
Leishmania donovani	Protozoa	5661	GCF_000227135.1
Leishmania infantum	Protozoa	5671	GCF_000002875.2
Leishmania major	Protozoa	5664	GCF_000002725.2
Leishmania mexicana	Protozoa	5665	GCF_000234665.1
Leishmania tropica	Protozoa	5666	GCA_014139745.1
Marteilia refringens	Protozoa	107386
Perkinsus marinus	Protozoa	31276	GCF_000006405.1
Perkinsus olseni	Protozoa	32597	GCA_013115135.1
Theileria annulata	Protozoa	5874	GCF_000003225.4
Theileria equi	Protozoa	5872	GCF_000342415.1
Theileria parva	Protozoa	5875	GCF_000165365.1
Tritrichomonas foetus	Protozoa	1144522	GCA_001839685.1
Trypanosoma brucei	Protozoa	5691	GCF_000002445.2
Trypanosoma congolense	Protozoa	5692	GCA_002287245.1
Trypanosoma equiperdum	Protozoa	5694	GCA_001457755.2
Trypanosoma evansi	Protozoa	5697	GCA_917563935.1
Trypanosoma vivax	Protozoa	5699	GCA_021307395.1
African horse	Virus	40050	GCF_000856125.1
sickness virus
African swine fever virus	Virus	10497	GCF_000858485.1
Akabane orthobunyavirus	Virus	1933178	GCF_000871205.1
Alcelaphine	Virus	35252	GCF_000838825.1
gammaherpesvirus 1
Alphaarterivirus equid	Virus	2499620	GCF_000860865.1
Alphacoronavirus 1	Virus	693997	GCF_000856025.1
Ambystoma tigrinum virus	Virus	265294	GCF_000841005.1
Avian coronavirus	Virus	694014	GCF_012271565.1
Avian influenza virus	Virus	11309
Avian metapneumovirus	Virus	38525	GCF_002989735.1
Avian orthoavulavirus 1	Virus	2560319	GCF_002834085.1
Avihepatovirus A	Virus	691956	GCF_000869945.1
Betaarterivirus suid 1	Virus	2499680	GCF_003971765.1
Bluetongue virus	Virus	40051	GCF_000854445.3
Bovine alphaherpesvirus 1	Virus	10320	GCF_008777455.1
Bovine leukemia virus	Virus	11901	GCF_000853665.1
Camelpox virus	Virus	28873	GCF_000839105.1
Caprine arthritis	Virus	11660	GCF_000857525.1
encephalitis virus
Crimean-Congo	Virus	1980519	GCF_000854165.1
hemorrhagic fever
orthonairovirus
Cyprinid herpesvirus 3	Virus	180230	GCF_000871465.1
Decapod iridescent virus 1	Virus	2560405	GCF_00478 8555.1
Decapod	Virus	1513224	GCF_000844705.1
penstyldensovirus 1
Deformed wing virus	Virus	198112	GCF_000852585.1
Eastern equine	Virus	11021	GCF_000862705.1
encephalitis virus
Epizootic haematopoietic	Virus	100217	GCF_001448375.1
necrosis virus
Epizootic hemorrhagic	Virus	40054	GCF_000885335.1
disease virus
Equid alphaherpesvirus 1	Virus	10326	GCF_000844025.1
Equid alphaherpesvirus 4	Virus	10331	GCF_000846345.1
Equine infectious	Virus	11665	GCF_000847605.1
anemia virus
Foot-and-mouth disease	Virus	12110	GCF_002816555.1
virus
Frog virus 3	Virus	10493	GCF_002826565.1
Gallid alphaherpesvirus 1	Virus	10386	GCF_000847005.1
Goatpox virus	Virus	186805	GCF_000840165.1
Haliotid herpesvirus 1	Virus	1513231	GCF_000900375.1
Hendra henipavirus	Virus	63330	GCF_000852685.1
Infectious bursal	Virus	10995	GCF_000855485.1
disease virus
Infectious spleen	Virus	180170	GCF_000848865.1
and kidney necrosis virus
Influenza A virus	Virus	11320	GCF_000851145.1
Isavirus salaris	Virus	55987	GCF_000854145.2
Japanese encephalitis virus	Virus	11072	GCF_000862145.1
Lumpy skin disease virus	Virus	59509	GCF_000839805.1
Lyssavirus rabies	Virus	11292	GCF_000859625.1
Macrobrachium	Virus	222557	GCA_000856985.1
rosenbergii nodavirus
Middle East respiratory	Virus	1335626	GCF_002816195.1
syndrome-related
coronavirus
Myxoma virus	Virus	10273	GCF_000843685.1
Nairobi sheep	Virus	1980526	GCF_002117695.1
disease orthonairovirus
Nipah henipavirus	Virus	121791	GCF_000863625.1
Norwegian salmonid	Virus	344701
alphavirus
Novirhabdovirus piscine	Virus	1980916	GCF_000856505.1
Novirhabdovirus salmonid	Virus	1980917	GCF_000850065.1
Penaeid shrimp infectious	Virus	282786	GCA_000866305.1
myonecrosis virus
Peste des petits ruminants	Virus	2593991	GCF_000866445.1
virus
Pestivirus C	Virus	2170082	GCF_000864685.1
			GCF_003034095.1
Pestivirus A	Virus	2170080	GCF_000861245.1
Rabbit hemorrhagic	Virus	11976	GCF_000861285.1
disease virus
Rift Valley fever	Virus	1933187	GCF_000847345.1
phlebovirus
Rinderpest morbillivirus	Virus	11241	GCF_000856645.1
Severe acute	Virus	694009	GCF_000864885.1
respiratory syndrome-
related coronavirus
Sheeppox virus	Virus	10266	GCF_000840205.1
Slow bee paralysis virus	Virus	458132	GCF_0008 87395.1
Sprivirus cyprinus	Virus	696863	GCF_000850305.1
Suid alphaherpesvirus 1	Virus	10345	GCF_000843 825.1
Swine vesicular	Virus	12075
disease virus
Taura syndrome virus	Virus	142102	GCF_000849385.1
Tilapinevirus tilapiae	Virus	2034996	GCF_001630085.1
Venezuelan equine	Virus	11036	GCF_000862105.1
encephalitis virus
Vesiculovirus Indiana	Virus	1972577	GCF_000850045.1
Visna-maedi virus	Virus	2169971	GCF_000849025.1
West Nile Virus	Virus	11082	GCF_000861085.1
Western equine	Virus	11039	GCF_OOO85O885.1
encephalitis virus
White spot syndrome virus	Virus	342409	GCF_000848085.2
Yellow head virus	Virus	96029	GCF_003972805.1

In some embodiments, other target nucleic acids of interest may be for non-infectious conditions, e.g., to be used for genotyping, including non-invasive prenatal diagnosis of, e.g, trisomies, other chromosomal abnormalities, and known genetic diseases such as Tay Sachs disease and sickle cell anemia. Other target nucleic acids of interest and samples are described herein, such as human biomarkers for cancer. An exemplary list of human biomarkers is in Table 4. Target nucleic acids of interest may include engineered biologics, including cells such as CAR-T cells, or target nucleic acids of interest from very small or rare samples, where only small volumes are available for testing.

TABLE 4

Human Biomarkers

			NCBI	NCBI
			Taxonomy	Gene
Biomarker	Disease	Sample	ID	ID

Aβ42, amyloid beta-	Alzheimer disease	CSF	9606	351
protein
prion protein	Alzheimer disease,	CSF	9606	5621
	prion disease
Vitamin D binding	multiple sclerosis	CSF	9606	2638
protein	progression
CXCL13	multiple sclerosis	CSF	9606	10563
alpha-synuclein	parkinsonian disorders	CSF	9606	6622
tau protein	parkinsonian disorders	CSF	9606	4137
Apo II	parkinsonian disorders	CSF	9606	336
ceruloplasmin	parkinsonian disorders	CSF	9606	1356
peroxisome	parkinsonian disorders	CSF	9606	5467
proliferation-
activated PD receptor
parkin	neurogenerative	CSF	9606	5071
	disorders
PTEN induced	neurogenerative	CSF	9606	65018
putative kinase I	disorders
DJ-1 (PARK7)	neurogenerative	CSF	9606	11315
	disorders
leucine-rich repeat	neurogenerative	CSF	9606	120892
kinase	disorders
secretogranin II	bipolar disorder	CSF	9606	7857
neurofilament light	axonal degeneration	CSF	9606	4747
chain
IL-12B, CXDL13,	Intrathecal	CSF	9606	3593, 10563,
IL-8	inflammation			3576
ACE2	cardiovascular disease	blood	9606	59272
alpha-amylase	cardiovascular disease	saliva	9606	276
alpha-feto protein	pregnancy	blood	9606	174
albumin	urine	diabetes	9606	213
albumin, urea	albuminuria	urine	9606	213
neutrophil gelatinase-	acute kidney injury	urine	9606	3934
associated lipocalin
(NGAL)
IL-18	acute kidney injury	urine	9606	3606
liver fatty acid	acute kidney injury	urine	9606	2168
binding protein
Dkk-3	prostate cancer	semen	9606	27122
autoantibody to	early diagnosis	blood	9606
CD25	esophageal squamous
	cell carcinoma
hTERT	lung cancer	blood	9606	7015
CAI 25 (MUC16)	lung cancer	blood	9606	94025
VEGF	lung cancer	blood	9606	7422
IL-2	lung cancer	blood	9606	3558
osteopontin	lung cancer	blood	9606	6696
BRAF, CCNI, EGRF,	lung cancer	saliva	9606	673, 16007,
FGF19, FRS2,				1956, 9965,
GREB1, and LZTS1				10818, 9687,
				11178
human epididymis	ovarian cancer	blood	9606	10406
protein 4
CA125	ovarian cancer	saliva	9606	94025
EMP1	nasopharyngeal	saliva	9606	13730
	carcinoma
IL-8	oral cancer	saliva	9606	3576
carcinoembryonic	oral or salivary	saliva	9606	1048
antigen	malignant tumors
thioredoxin	Spinalcellular carcinoma	saliva	9606	7295
AIP (aryl	Acute intermittent	blood	9606	9049
hydrocarbon receptor	porphyria, somatotroph
interacting protein)	adenoma, prolactin-
	producing pituitary
	gland adenoma
ALK receptor	Neuroblastoma	blood	9606	238
tyrosine kinase	susceptibility, large cell
	lymphoma
BAP1 (BRCA1	BAP1-related tumor	blood	9606	8314
associated protein 1)	predisposition,
	melanoma susceptibility
BLM	Bloom syndrome	blood	9606	641
BRCA1	Breast-ovarian cancer	blood	9606	672
	susceptibility, familial
	breast cancer
BRCA2	Breast-ovarian cancer	blood	9606	675
	susceptibility, familial
	breast cancer, glioma
	susceptibility
CASR (calcium	Epilepsy susceptibility	blood	9606	846
sensing receptor)
CDC73	Hyperparathyroidism 2	blood	9606	79577
	with jaw tumors
CEBPA	Acute myloid leukemia	blood	9606	1050
EPCAM	Colorectal cancer	blood	9606	4072
FH	hypercholesterolemia	blood	9606	2271
GATA2	Acute myeloid leukemia	blood	9606	2642
MITF	Melanoma susceptibility	blood	9606	4286
MSH2	Lynch syndrome	blood	9606	4436
MSH3	Endometrial carcinoma	blood	9606	4437
MSH6	Endometrial carcinoma,	blood	9606	2956
	colorectal cancer
NF1	Neurofibromatosis,	blood	9606	4763
	juvenile
	myelomonocytic
	leukemia
PDGRA	Eosinophilic leukemia,	blood	9606	5156
	recurrent inflammatory
	gastrointestinal fibroids
PHOX2B	Neuroblastoma	blood	9606	8929
	susceptibility
POTI	Melanoma	blood	9606	25913
	susceptibility, glioma
	susceptibility

The target nucleic acids of interest may be taken from environmental samples. A list of exemplary biosafety pathogens is in Table 5, and an exemplary list of known viruses is in Table 6.

TABLE 5

Exemplary Laboratory Biosafety Parasites and Pathogens

		NCBI
		Taxonomy
Name	Category	ID

Acarapis woodi	Animal	478375
Aethina tumida	Animal	116153
Alaria americana	Animal	2282137
Amblyomma	Animal	6943
americanum
Amblyomma maculatum	Animal	34609
Amphimerus	Animal
pseudofelineus
Ancylostoma braziliense	Animal	369059
Ancylostoma caninum	Animal	29170
Ancylostoma duodenale	Animal	51022
Anisakis pegreffii	Animal	303229
Anisakis simplex	Animal	6269
Baylisascaris columnaris	Animal	575210
Baylisascaris melis	Animal
Baylisascaris procyonis	Animal	6259
Bunostomum	Animal	577651
phlebotomum
Ceratonova shasta	Animal	60662
Chrysomya bezziana	Animal	69364
Cochliomyia	Animal	115425
hominivorax
Dicrocoelium	Animal	57078
dendriticum
Diphyllobothrium	Animal	28845
dendriticum
Diphyllobothrium latum	Animal	60516
Echinococcus granulosa	Animal
Echinococcus multilocularis	Animal	6211
Echinococcus oligarthrus	Animal	6212
Echinococcus shiquicus	Animal	260967
Echinococcus vogeli	Animal	6213
Echinostoma cinetorchis	Animal	1873862
Echinostoma hortense	Animal	48216
Echinostoma liei	Animal	48214
Echinostoma revolutum	Animal	48217
Fasciola hepatica	Animal	6192
Fascioloides magna	Animal	394415
Gyrodactylus salaris	Animal	37629
Ixodes pacificus	Animal	29930
Ixodes ricinus	Animal	34613
Ixodes scapularis	Animal	6945
Metagonimus yokogawai	Animal	84529
Metorchis conjunctus	Animal
Myxobolus cerebralis	Animal	59783
Nanophyetuss almincola	Animal	240278
Necator americanus	Animal	51031
Oestrus ovis	Animal	123737
Opisthorchis felineus	Animal	147828
Opisthorchis viverrini	Animal	6198
Parafilaria bovicola	Animal	2282233
Paragonimus kellicotti	Animal	100269
Paragonimus miyazakii.	Animal	59628
Paragonimus	Animal	34504
westermani
Psoroptes ovis	Animal	83912
Rhipicephalus annulatus	Animal	34611
Rhipicephalus sanguineus	Animal	34632
Sarcoptes scabiei	Animal	52283
Taenia multiceps	Animal	94034
Taenia saginata	Animal	6206
Taenia solium	Animal	6204
Toxocara canis	Animal	6265
Toxocara cati	Animal	6266
Trichinella spiralis	Animal	6334
Trichuris suis	Animal	68888
Trichuris trichiura	Animal	36087
Trichuris vulpis	Animal	219738
Tropilaelaps clareae	Animal	208209
Tropilaelaps mercedesae	Animal	418985
Uncinaria stenocephala	Animal	125367
Varroa destructor	Animal	109461
Actinobacillus	Bacteria	715
pleuropneumoniae
Aeromonas hydrophila	Bacteria	644
Aeromonas salmonicida	Bacteria	645
Aliarcobacter butzleri	Bacteria	28197
Aliarcobacter	Bacteria	28198
cryaerophilus
Aliarcobacter skirrowii	Bacteria	28200
Anaplasma centrale	Bacteria	769
Anaplasma marginale	Bacteria	770
Anaplasma	Bacteria	948
phagocytophilum
Bacillus anthracis	Bacteria	1392
Bacillus cereus	Bacteria	1396
Bartonella henselae	Bacteria	38323
Bibersteinia trehalosi	Bacteria	47735
Borrelia burgdorferi	Bacteria	139
Brucella abortus	Bacteria	235
Brucella canis	Bacteria	36855
Brucella melitensis	Bacteria	29459
Brucella ovis	Bacteria	236
Brucella suis	Bacteria	29461
Burkholderia mallei	Bacteria	13373
Burkholderia	Bacteria	28450
pseudomallei
Campylobacter coli	Bacteria	195
Campylobacter fetus fetus	Bacteria	32019
Campylobacter fetus	Bacteria	32020
venerealis
Campylobacter jejuni	Bacteria	197
Chlamydia caviae	Bacteria	83557
Chlamydia felis	Bacteria	83556
Chlamydia muridarum	Bacteria	83560
Chlamydia pecorum	Bacteria	85991
Chlamydia pneumoniae	Bacteria	83558
Chlamydia psittaci	Bacteria	83554
Chlamydia suis	Bacteria	83559
Chlamydia trachomatis	Bacteria	813
Chlamydophilus abortus	Bacteria
Clostridium botulinum	Bacteria	1491
Clostridium difficile	Bacteria	1496
Clostridium perfringens	Bacteria
Types A, B, C, and D
Coxiella burnetii	Bacteria	777
Cronobacter sakazakii	Bacteria	28141
Ehrlichia canis	Bacteria	944
Ehrlichia chaffeensis	Bacteria	945
Ehrlichia ewingii	Bacteria	947
Ehrlichia ondiri	Bacteria
Ehrlichia ruminantium	Bacteria	779
Escherichia coli	Bacteria	562
Klebsiella aerogenes	Bacteria	548
Klebsiella granulomatis	Bacteria	39824
Klebsiella grimontii	Bacteria	2058152
Klebsiella huaxiensis	Bacteria	2153354
Klebsiella kielensis	Bacteria	2042302
Klebsiella michiganensis	Bacteria	1134687
Klebsiella milletis	Bacteria	223378
Klebsiella oxytoca	Bacteria	571
Klebsiella pneumoniae	Bacteria	573
Klebsiella quasipneumoniae	Bacteria	1463165
Klebsiella quasivariicola	Bacteria	2026240
Klebsiella senegalensis	Bacteria	223379
Klebsiella steroids	Bacteria	1641362
Klebsiella variicola	Bacteria	244366
Proteus mirabilis	Bacteria	584
Pseudomonas abietaniphila	Bacteria	89065
Pseudomonas acephalitica	Bacteria	407029
Pseudomonas acidophila	Bacteria	1912599
Pseudomonas adelgestsugas	Bacteria	1302376
Pseudomonas aeruginosa	Bacteria	287
Pseudomonas aestus	Bacteria	1387231
Pseudomonas agarici	Bacteria	46677
Pseudomonas akappageensis	Bacteria
Pseudomonas alcaligenes	Bacteria	43263
Pseudomonas alcaliphila	Bacteria	101564
Pseudomonas alginovora	Bacteria	37638
Pseudomonas alkanolytica	Bacteria
Pseudomonas	Bacteria	237609
alkylphenolica
Pseudomonas allii	Bacteria	2740531
Pseudomonas alliivorans	Bacteria	2810613
Pseudomonas	Bacteria	2774460
allokribbensis
Pseudomonas alloputida	Bacteria	1940621
Pseudomonas alvandae	Bacteria	2842348
Pseudomonas amygdali	Bacteria	47877
Pseudomonas	Bacteria	32043
amyloderamosa
Pseudomonas anatoliensis	Bacteria	2710589
Pseudomonas andersonii	Bacteria	147728
Pseudomonas	Bacteria	53406
anguilliseptica
Pseudomonas antarctica	Bacteria	219572
Pseudomonas	Bacteria	485870
anuradhapurensis
Pseudomonas	Bacteria	2710591
arcuscaelestis
Pseudomonas	Bacteria	289370
argentinensis
Pseudomonas	Bacteria	702115
arsenicoxydans
Pseudomonas	Bacteria	2842349
asgharzadehiana
Pseudomonas asiatica	Bacteria	2219225
Pseudomonas asplenii	Bacteria	53407
Pseudomonas asturiensis	Bacteria	1190415
Pseudomonas asuensis	Bacteria	1825787
Pseudomonas atacamensis	Bacteria	2565368
Pseudomonas atagonensis	Bacteria	2609964
Pseudomonas aurantiaca	Bacteria	86192
Pseudomonas aureofaciens	Bacteria	587851
Pseudomonas avellanae	Bacteria	46257
Pseudomonas	Bacteria	1869229
aylmerensis
Pseudomonas azadiae	Bacteria	2843612
Pseudomonas	Bacteria
azerbaij anoccidentalis
Pseudomonas	Bacteria
azerbaij anorientalis
Pseudomonas azotifigens	Bacteria	291995
Pseudomonas	Bacteria	47878
azotoformans
Pseudomonas baetica	Bacteria	674054
Pseudomonas balearica	Bacteria	74829
Pseudomonas baltica	Bacteria	2762576
Pseudomonas	Bacteria	2843610
bananamidigenes
Pseudomonas bathycetes	Bacteria
Pseudomonas batumici	Bacteria	226910
Pseudomonas	Bacteria	556533
benzenivorans
Pseudomonas bijieensis	Bacteria	2681983
Pseudomonas	Bacteria	254015
blatchfordae
Pseudomonas bohemica	Bacteria	2044872
Pseudomonas borbori	Bacteria	289003
Pseudomonas borealis	Bacteria	84586
Pseudomonas botevensis	Bacteria	2842352
Pseudomonas	Bacteria	930166
brassicacearum
Pseudomonas	Bacteria	2708063
brassicae
Pseudomonas brenneri	Bacteria	129817
Pseudomonas bubulae	Bacteria	2316085
Pseudomonas campi	Bacteria	2731681
Pseudomonas canadensis	Bacteria	915099
Pseudomonas	Bacteria	2859001
canavaninivorans
Pseudomonas cannabina	Bacteria	86840
Pseudomonas capeferrum	Bacteria	1495066
Pseudomonas capsici	Bacteria	2810614
Pseudomonas	Bacteria	46678
caricapapayae
Pseudomonas carnis	Bacteria	2487355
Pseudomonas caspiana	Bacteria	1451454
Pseudomonas cavernae	Bacteria	2320867
Pseudomonas	Bacteria	2320866
cavernicola
Pseudomonas cedrina	Bacteria	651740
Pseudomonas cellulosa	Bacteria	155077
Pseudomonas cerasi	Bacteria	1583341
Pseudomonas chaetocerotis	Bacteria
Pseudomonas chengduensis	Bacteria	489632
Pseudomonas	Bacteria	203192
chloritidismutans
Pseudomonas chlororaphis	Bacteria	587753
Pseudomonas cichorii	Bacteria	36746
Pseudomonas citronellolis	Bacteria	53408
Pseudomonas clemancea	Bacteria	416340
Pseudomonas coenobios	Bacteria
Pseudomonas	Bacteria	1605838
coleopterorum
Pseudomonas composti	Bacteria	658457
Pseudomonas congelans	Bacteria	200452
Pseudomonas	Bacteria	53409
coronafaciens
Pseudomonas corrugata	Bacteria	47879
Pseudomonas costantinii	Bacteria	168469
Pseudomonas	Bacteria	157783
cremoricolorata
Pseudomonas cremoris	Bacteria	2724178
Pseudomonas crudilactis	Bacteria	2697028
Pseudomonas	Bacteria	543360
cuatrocienegasensis
Pseudomonas cyclaminis	Bacteria	2781239
Pseudomonas daroniae	Bacteria	2487519
Pseudomonas	Bacteria	882211
deceptionensis
Pseudomonas defluvii	Bacteria	1876757
Pseudomonas delhiensis	Bacteria	366289
Pseudomonas denitrificans	Bacteria	43306
Pseudomonas	Bacteria
diazotrophicus
Pseudomonas	Bacteria	135830
diterpeniphila
Pseudomonas donghuensis	Bacteria	1163398
Pseudomonas dryadis	Bacteria	2487520
Pseudomonas duriflava	Bacteria	459528
Pseudomonas edaphica	Bacteria	2006980
Pseudomonas ekonensis	Bacteria	2842353
Pseudomonas elodea	Bacteria	179878
Pseudomonas endophytica	Bacteria	1563157
Pseudomonas entomophila	Bacteria	312306
Pseudomonas eucalypticola	Bacteria	2599595
Pseudomonas excibis	Bacteria
Pseudomonas	Bacteria	359110
extremaustralis
Pseudomonas	Bacteria	169669
extremorientalis
Pseudomonas fakonensis	Bacteria	2842355
Pseudomonas farris	Bacteria	2841207
Pseudomonas farsensis	Bacteria	2745492
Pseudomonas ficuserectae	Bacteria	53410
Pseudomonas fildesensis	Bacteria	1674920
Pseudomonas flavescens	Bacteria	29435
Pseudomonas flexibilis	Bacteria	706570
Pseudomonas floridensis	Bacteria	1958950
Pseudomonas fluorescens	Bacteria	294
Pseudomonas fluvialis	Bacteria	1793966
Pseudomonas foliumensis	Bacteria	2762593
Pseudomonas fragi	Bacteria	296
Pseudomonas	Bacteria	104087
frederiksbergensis
Pseudomonas fulgida	Bacteria	200453
Pseudomonas fulva	Bacteria	47880
Pseudomonas furukawaii	Bacteria	1149133
Pseudomonas fuscovaginae	Bacteria	50340
Pseudomonas gelidicola	Bacteria	1653853
Pseudomonas gessardii	Bacteria	78544
Pseudomonas gingeri	Bacteria	117681
Pseudomonas glareae	Bacteria	1577705
Pseudomonas glycinae	Bacteria	1785145
Pseudomonas gozinkensis	Bacteria	2774461
Pseudomonas graminis	Bacteria	158627
Pseudomonas granadensis	Bacteria	1421430
Pseudomonas	Bacteria	1628277
gregormendelii
Pseudomonas grimontii	Bacteria	129847
Pseudomonas	Bacteria	1245526
guangdongensis
Pseudomonas	Bacteria	1288410
guariconensis
Pseudomonas guezennei	Bacteria	310348
Pseudomonas guguanensis	Bacteria	1198456
Pseudomona sguineae	Bacteria	425504
Pseudomonas guryensis	Bacteria	2759165
Pseudomonas haemolytica	Bacteria	2600065
Pseudomonas	Bacteria	53411
halodenitrificans
Pseudomonas halodurans	Bacteria	28258
Pseudomonas	Bacteria
halosaccharolytica
Pseudomonas	Bacteria
halosensibilis
Pseudomonas hamedanensis	Bacteria	2745504
Pseudomonas helianthi	Bacteria	251654
Pseudomonas helleri	Bacteria	1608996
Pseudomonas	Bacteria	1471381
helmanticensis
Pseudomonas huaxiensis	Bacteria	2213017
Pseudomonas hunanensis	Bacteria	1247546
Pseudomonas hutmensis	Bacteria	2707027
Pseudomonas	Bacteria	297
hydrogenothermophila
Pseudomonas	Bacteria	39439
hydrogenovora
Pseudomonas hydrolytica	Bacteria	2493633
Pseudomonas indica	Bacteria	137658
Pseudomonas indoloxydans	Bacteria	404407
Pseudomonas inefficax	Bacteria	2078786
Pseudomonas iranensis	Bacteria	2745503
Pseudomonas iridis	Bacteria	2710587
Pseudomonas izuensis	Bacteria	2684212
Pseudomonas japonica	Bacteria	256466
Pseudomonas jessenii	Bacteria	77298
Pseudomonas jinanensis	Bacteria
Pseudomonas jinjuensis	Bacteria	198616
Pseudomonas juntendi	Bacteria	2666183
Pseudomonas	Bacteria	2293832
kairouanensis
Pseudomonas karstica	Bacteria	1055468
Pseudomonas	Bacteria	2745482
kermanshahensis
Streptococcus uberis	Bacteria	1349
Besnoitia besnoiti	Chromista	94643
Bonamia exitiosa	Chromista	362532
Bonamia ostreae	Chromista	126728
Amniculicola longissima	Fungus	2566060
Arthroderma amazonicum	Fungus	1592210
Aschersonia hypocreoidea	Fungus	370936
Aspergillago clavatoflava	Fungus	41064
Aspergillus acidohumus	Fungus	1904037
Aspergillus acidus	Fungus	1069201
Aspergillus aculeatinus	Fungus	487661
Aspergillus aculeatus	Fungus	5053
Aspergillus aeneus	Fungus	41754
Aspergillus affinis	Fungus	1070780
Aspergillus alabamensis	Fungus	657433
Aspergillus alliaceus	Fungus	209559
Aspergillus amazonicus	Fungus	710228
Aspergillus ambiguus	Fungus	176160
Aspergillus amoenus	Fungus	1220191
Aspergillus	Fungus	296546
amyloliquefaciens
Aspergillus amylovorus	Fungus	176161
Aspergillus angustatus	Fungus	2783700
Aspergillus anomalus	Fungus	454240
Aspergillus anthodesmis	Fungus	37233
Aspergillus apicalis	Fungus	478867
Aspergillus	Fungus	1140386
appendiculatus
Aspergillus arachidicola	Fungus	656916
Aspergillus ardalensis	Fungus	1458899
Aspergillus arvii	Fungus	368784
Aspergillus	Fungus	1695225
askiburgiensis
Aspergillus asperescens	Fungus	176163
Aspergillus assulatus	Fungus	1245746
Aspergillus astellatus	Fungus	1810904
Aspergillus	Fungus	41725
aurantiobrunneus
Aspergillus	Fungus	2663348
aurantiopurpureus
Aspergillus aureolatus	Fungus	41755
Aspergillus aureoterreus	Fungus	41288
Aspergillus aureus	Fungus	309747
Aspergillus auricomus	Fungus	138274
Aspergillus austr aliensis	Fungus	1250384
Aspergillus austroafricanus	Fungus	1220192
Aspergillus avenaceus	Fungus	36643
Aspergillus awamori	Fungus	105351
Aspergillus baarnensis	Fungus	2070749
Aspergillus baeticus	Fungus	1194636
Aspergillus bahamensis	Fungus	522521
Aspergillus bertholletiae	Fungus	1226010
Aspergillus biplanus	Fungus	176164
Aspergillus bisporus	Fungus	41753
Aspergillus bombycis	Fungus	109264
Aspergillus botswanensis	Fungus	1810893
Candida albicans	Fungus	5476
Candida glabrata	Fungus	5478
Candida krusei	Fungus	4909
Candida parapsilosis	Fungus	5480
Candida tropicalis	Fungus	5482
Cryptococcus gattii	Fungus	37769
Cryptococcus neoformans	Fungus	5207
Epidermophyton	Fungus	34391
floccosum
Epidermophyton	Fungus	74042
stockdaleae
Fusarium acaciae	Fungus
Fusarium acaciae-mearnsii	Fungus	282272
Fusarium acicola	Fungus
Fusarium acremoniopsis	Fungus
Fusarium acridiorum	Fungus
Fusarium acutatum	Fungus	78861
Fusarium aderholdii	Fungus
Fusarium adesmiae	Fungus
Fusarium aduncisporum	Fungus
Fusarium aecidii-	Fungus
tussilaginis
Fusarium aeruginosum	Fungus
Fusarium aethiopicum	Fungus	569394
Fusarium affine	Fungus
Fusarium agaricorum	Fungus
Fusarium ailanthinum	Fungus
Fusarium alabamense	Fungus
Fusarium albedinis	Fungus
Fusarium albertii	Fungus
Fusarium	Fungus
albidoviolaceum
Fusarium albiziae	Fungus
Fusarium albocarneum	Fungus
Fusarium album	Fungus
Fusarium aleurinum	Fungus
Fusarium aleyrodis	Fungus
Fusarium alkanophilum	Fungus
Fusarium allescheri	Fungus
Fusarium allescherianum	Fungus
Fusarium allii-sativi	Fungus
Trichophyton simii	Fungus	63406
Trichophyton	Fungus	69891
soudanense
Trichophyton tonsurans	Fungus	34387
Trichophyton verrucosum	Fungus	63417
Trichophyton violaceum	Fungus	34388
Ochroma pyramidale	Plant	66662
Babesia bigemina	Protozoa	5866
Babesia bovis	Protozoa	5865
Babesia divergens	Protozoa	32595
Babesia jakimovi	Protozoa
Babesia major	Protozoa	127461
Babesia occultans	Protozoa	536930
Babesia ovata	Protozoa	189622
Cryptosporidium parvum	Protozoa	5807
Eimeria acervulina	Protozoa	5801
Eimeria brunetti	Protozoa	51314
Eimeria maxima	Protozoa	5804
Eimeria meleagridis	Protozoa	1431345
Eimeria necatrix	Protozoa	51315
Eimeria tenella	Protozoa	5802
Entamoeba	Protozoa	5759
histolytica
Giardia duodenalis	Protozoa	5741
Giardia lambia	Protozoa
Histomonas meleagridis	Protozoa	135588
Ichthyobodo necator	Protozoa	155203
Ichthyophthirius	Protozoa	5932
multifiliis
Isospora burrowsi	Protozoa
Isospora canis	Protozoa	1662860
Isospora felis	Protozoa	482539
Isospora neorivolta	Protozoa
Isospora ohioensis	Protozoa	279926
Leishmania braziliensis	Protozoa	5660
Leishmania chagasi	Protozoa	44271
Leishmania infantum	Protozoa	5671
Marteilia refringens	Protozoa	107386
Mikrocytos mackini	Protozoa	195010
Perkinsus marinus	Protozoa	31276
Perkinsus olensi	Protozoa
Sarcocystis cruzi	Protozoa	5817
Sarcocystis hirsuta	Protozoa	61649
Sarcocystis hominis	Protozoa	61650
Theileria annulata	Protozoa	5874
Theileria buffei	Protozoa
Theileria lestoquardi	Protozoa	77054
Theileria luwenshuni	Protozoa	540482
Theileria mutans	Protozoa	27991
Theileria orientalis	Protozoa	68886
Theileria parva	Protozoa	5875
Theileria sergenti	Protozoa	5877
Theileria uilenbergi	Protozoa	507731
Toxoplasma gondii	Protozoa	5811
Trichomonas fetus	Protozoa
Trichomonas gallinae	Protozoa	56777
Trichomonas stableri	Protozoa	1440121
Trypanosoma brucei	Protozoa	5691
Trypanosoma congolense	Protozoa	5692
Trypanosoma cruzi	Protozoa	5693
Abras virus	Virus	2303487
Absettarov virus	Virus
Abu Hammad virus	Virus	248058
Abu Mina virus	Virus	248059
Acado virus	Virus
Acara virus	Virus	2748201
Achiote virus	Virus	2036702
Adana virus	Virus	1611877
Adelaide River virus	Virus	31612
Adria virus	Virus
Aedes aegypti densovirus	Virus	186156
Aedes albopictus	Virus	35338
densovirus
Aedes flavivirus	Virus	390845
Aedes galloisi flavivirus	Virus	1046551
Aedes pseudoscutellaris	Virus
densovirus
Aedes pseudoscutellaris	Virus	341721
reovirus
Aedes vexans	Virus	7163
African horse sickness	Virus	40050
virus
African swine fever virus	Virus	10497
Aguacate virus	Virus	1006583
Aino virus	Virus	11582
Akabane virus	Virus	70566
Alajuela virus	Virus	1552846
Alcelaphine	Virus	35252
gammaherpesvirus 1
Alenquer virus	Virus	629726
Aleutian Mink Disease	Virus
Alfuy virus	Virus	44017
Alkhumra hemorrhagic	Virus	172148
fever virus
Allpahuayo	Virus	144752
mammarenavirus
Almeirim virus	Virus
Almendravirus arboretum	Virus	1972683
Almendravirus cootbay	Virus	1972685
Almpiwar virus	Virus	318843
Alocasia macrorrhizos	Virus	4456
Altamira virus	Virus
Amapari virus	Virus
Ambe virus	Virus	1926500
Amga virus	Virus	1511732
Amur/Soochong virus	Virus
Anadyr virus	Virus	1642852
Anajatuba virus	Virus	379964
Ananindeua virus	Virus	1927813
Andasibe virus	Virus
Andes orthohantavirus	Virus	1980456
Anhanga virus	Virus	904722
Anhembi virus	Virus	273355
Anopheles A virus	Virus	35307
Anopheles B virus	Virus	35308
Anopheles flavivirus	Virus	2053814
Anopheles gambiae	Virus	487311
densovirus
Antequera virus	Virus	2748239
Apoi virus	Virus	64280
Araguari virus	Virus	352236
Aransas Bay virus	Virus	1428582
Araraquara virus	Virus	139032
Bluetongue virus	Virus	40051
Bobaya virus	Virus	2818228
Bobia virus	Virus
Boraceia virus	Virus
Borna disease virus	Virus	12455
Botambi virus	Virus
Boteke virus	Virus	864698
Bouboui virus	Virus	64295
Bourbon virus	Virus	1618189
Bovine ephemeral fever	Virus	11303
virus
Bovine Herpes Virus 1	Virus
Bovine leukemia virus	Virus	11901
Bovine orthopneumovirus	Virus	11246
Bovine viral	Virus	11099
diarrhea virus 1
Bowe virus	Virus	1400425
Bozo virus	Virus	273349
Cumuto virus	Virus	1457166
Cupixi mammarenavirus	Virus	208899
Curionopolis virus	Virus	490110
Cyprinid herpesvirus 3	Virus	180230
Czech Aedes vexans	Virus
flavivirus virus
D’Aguilar virus	Virus
Dabakala virus	Virus
Dabieshan virus	Virus	1167310
Dak Nong virus	Virus	1238455
Dakar bat virus	Virus	64282
Dandenong virus	Virus	483046
Dashli virus	Virus	1764087
Deer tick virus	Virus	58535
Dengue virus	Virus	12637
Dengue virus 1 virus	Virus
Cumuto virus	Virus	1457166
Cupixi mammarenavirus	Virus	208899
Curionopolis virus	Virus	490110
Lymphocytic	Virus	11623
choriomeningitis
mammarenavirus
Lyssavirus aravan	Virus	211977
Lyssavirus australis	Virus	90961
Lyssavirus lagos	Virus	38766
Lyssavirus spp.	Virus	11286
Lyssavirus bokeloh	Virus	1072176
Lyssavirus caucasicus	Virus	249584
Lyssavirus duvenhage	Virus	38767
Lyssavirus irkut	Virus	249583
Lyssavirus khujand	Virus	237716
Lyssavirus mokola	Virus	12538
Lyssavirus rabies	Virus	11292
Lyssavirus shimoni	Virus	746543
Marisma mosquito virus	Virus	1105173
Marituba virus	Virus	292278
Marondera virus	Virus	108092
Marrakai virus	Virus	108088
Massila virus	Virus
Matariya virus	Virus	1272948
Matruh virus	Virus	1678229
Matucare virus	Virus	908873
Mayaro virus	Virus	59301
Mboke virus	Virus	273342
Mburo virus	Virus	2035534
Meaban virus	Virus	35279
Medjerda Valley virus	Virus	1775957
Melao virus	Virus	35515
Meno virus	Virus
Mercadeo virus	Virus	1708574
Semliki Forest virus	Virus	11033
Sena Madureira virus	Virus	1272957
Seoul virus	Virus	1980490
Sepik virus	Virus	44026
Serra Do Navio virus	Virus	45768
Serra Norte virus	Virus	1000649
Severe fever with	Virus	1003835
thrombocytopenia
syndrome virus
Shamonda virus	Virus	159150
Shark River virus	Virus	2303490
Shiant Island virus	Virus
Shokwe virus	Virus	273359
Shuni virus	Virus	159148
Silverwater virus	Virus	1564099
Simbu orthobunyavirus	Virus	35306
Sin Nombre virus	Virus	1980491
Sindbis virus	Virus	11034
Sixgun City virus	Virus
Skinner Tank virus	Virus	481886
Snowshoe hare virus	Virus	11580
Sokoluk virus	Virus	64317
Soldado virus	Virus	426791
Solwezi virus	Virus
Somone virus	Virus
Sororoca virus	Virus	273354
Souris virus	Virus	2010246
South Bay virus	Virus	1526514
South River virus	Virus	45769
Spanish Culex flavivirus	Virus
virus
Spanish Ochlerotatus	Virus
flavivirus virus
Spondweni virus	Virus	64318
Sprivirus cyprinus	Virus	696863
Sripur virus	Virus	1620897
St. Abbs Head virus	Virus
St. Croix River virus	Virus
St. Louis encephalitis	Virus	11080
virus
Stanfield virus	Virus
Stratford virus	Virus	44027

TABLE 6

Exemplary list of viruses

		NCBI
		Taxonomy
	Name	ID

	Aalivirus A	2169685
	Aarhusvirus	2732762
	dagda
	Aarhusvirus	2732763
	katbat
	Aarhusvirus	2732764
	luksen
	Aarhusvirus	2732765
	mysterion
	Abaca bunchy	438782
	top virus
	Abatino	2734574
	macacapox
	virus
	Abbeymikolon-	2734213
	virus
	abbeymikolon
	Abouovirus	1984774
	abouo
	Abouovirus	1984775
	davies
	Abutilon	1926117
	golden mosaic
	virus
	Abutilon	932071
	mosaic
	Bolivia virus
	Abutilon	1046572
	mosaic Brazil
	virus
	Abutilon	10815
	mosaic virus
	Abutilon	169102
	yellows virus
	Acadevirus	2733576
	PM116
	Acadevirus	2733577
	Pm5460
	Acadevirus	2733574
	PM85
	Acadevirus	2733575
	PM93
	Acadianvirus	1982901
	acadian
	Acadianvirus	1982902
	baee
	Acadianvirus	1982903
	reprobate
	Acanthamoeba	212035
	polyphaga
	mimivirus
	Acanthocystis	322019
	turfacea
	chlorella virus 1
	Acara	2170053
	orthobunyavirus
	Achimota	2560259
	pararubulavirus 1
	Achimota	2560260
	pararubulavirus 2
	Achromobacter	2169962
	virus Axp3
	Acidianus	437444
	bottle-shaped
	virus
	Acidianus	300186
	filamentous
	virus 2
	Acidianus	346881
	filamentous
	virus 3
	Acidianus	346882
	filamentous
	virus 6
	Acidianus	346883
	filamentous
	virus 7
	Acidianus	346884
	filamentous
	virus 8
	Acidianus	512792
	filamentous
	virus 9
	Acidianus	309181
	rod-shaped
	virus 1
	Acidianus	693629
	spindle-
	shaped virus 1
	Acidianus	315953
	two-tailed
	virus
	Acinetobacter	279006
	virus 133
	Acintetobacter
	virus B2
	Acintetobacter
	virus B5
	Acionnavirus	2734078
	monteraybay
	Acipenserid	2871198
	herpesvirus 2
	Aconitum	101764
	latent virus
	Acrobasis
	zelleri
	entomopoxvirus
	Actinidia seed	2560282
	borne latent
	virus
	Actinidia	2024724
	virus 1
	Actinidia	1112769
	virus A
	Actinidia	1112770
	virus B
	Actinidia	1331744
	virus X
	Acute bee	92444
	paralysis virus
	Adana	2734433
	phlebovirus
	Adeno-	1511891
	associated
	dependoparvo
	virus A
	Adeno-	1511892
	associated
	dependoparvo
	virus B
	Adoxophyes	1993630
	honmai
	entomopoxvirus
	Adoxophyes	224399
	honmai
	nucleopolyhedro-
	virus
	Adoxophyes	170617
	orana
	granulovirus
	Aedes aegypti
	entomopoxvirus
	Aedes aegypti
	Mosqcopia
	virus
	Aedes	341721
	pseudoscutellaris
	reovirus
	Aegirvirus	2733888
	SCBP42
	Aeonium	1962503
	ringspot virus
	Aeromonas
	virus 43
	Aeropyrum	1157339
	coil-shaped
	virus
	Aeropyrum	700542
	pernix
	bacilliform
	virus 1
	Aeropyrum	1032474
	pernix ovoid
	virus 1
	Aerosvirus	2733365
	AS7
	Aerosvirus	2733364
	av25AhydR2PP
	Aerosvirus	2733366
	ZPAH7
	Affertcholeram-	141904
	virus
	CTXphi
	African	2560285
	cassava
	mosaic
	Burkina Faso
	virus
	African	10817
	cassava
	mosaic virus
	African	2056161
	eggplant
	mosaic virus
	African horse	40050
	sickness virus
	African oil	185218
	palm ringspot
	virus
	African swine	10497
	fever virus
	Agaricus	2734345
	bisporus
	alphaendorna-
	virus 1
	Agaricus
	bisporus virus 4
	Agatevirus	1910935
	agate
	Agatevirus	1910936
	bobb
	Agatevirus	1910937
	Bp8pC
	Ageratum	1260769
	enation
	alphasatellite
	Ageratum	188333
	enation virus
	Ageratum	1386090
	latent virus
	Ageratum leaf	912035
	curl Buea
	betasatellite
	Ageratum leaf	635076
	curl
	Cameroon
	betasatellite
	Ageratum leaf	2182585
	curl Sichuan
	virus
	Ageratum leaf	333293
	curl virus
	Ageratum	169687
	yellow leaf
	curl
	betasatellite
	Ageratum	187850
	yellow vein
	alphasatellite
	Ageratum	185750
	yellow vein
	betasatellite
	Ageratum	1454227
	yellow vein
	China
	alphasatellite
	Ageratum	437063
	yellow vein
	Hualian virus
	Ageratum	1407058
	yellow vein
	India
	alphasatellite
	Ageratum	2010316
	yellow vein
	India
	betasatellite
	Ageratum	915293
	yellow vein
	Singapore
	alphasatellite
	Ageratum	2010317
	yellow vein
	Sri Lanka
	betasatellite
	Ageratum	222079
	yellow vein
	Sri Lanka
	virus
	Ageratum	44560
	yellow vein
	virus
	Aghbyvirus	2733367
	ISAO8
	Aglaonema	1512278
	bacilliform
	virus
	Agricanvirus	1984777
	deimos
	Agricanvirus	2560433
	desertfox
	Agricanvirus	1984778
	Ea3570
	Agricanvirus	1984779
	ray
	Agricanvirus	1984780
	simmy50
	Agricanvirus	1984781
	specialG
	Agropyron	41763
	mosaic virus
	Agrotis	208013
	ipsilon
	multiple
	nucleopolyhed
	rovirus
	Agrotis	10464
	segetum
	granulovirus
	Agrotis	1962501
	segetum
	nucleopolyhed
	rovirus A
	Agrotis	1580580
	segetum
	nucleopolyhed
	rovirus B
	Agtrevirus	1987994
	AG3
	Agtrevirus	2169690
	SKML39
	Aguacate	2734434
	phlebovirus
	Ahlum
	waterborne
	virus
	Ahphunavirus	2733368
	Ahp1
	Ahphunavirus	2733369
	CF7
	Ahtivirus	2734079
	sagseatwo
	Aichivirus A	72149
	Aichivirus B	194965
	Aichivirus C	1298633
	Aichivirus D	1897731
	Aichivirus E	1986958
	Aichivirus F	1986959
	Ailurivirus A	2560287
	Aino	2560289
	orthobunyavirus
	Air potato	2560290
	ampelovirus 1
	Akabane	1933178
	orthobunyavirus
	Akhmeta virus	2200830
	Alajuela	1933181
	orthobunyavirus
	Alasvirus	2501934
	muscae
	Alcelaphine	35252
	gammaherpes
	virus 1
	Alcelaphine	138184
	gammaherpes
	virus 2
	Alcube	2734435
	phlebovirus
	Alcyoneusvirus	2560541
	K641
	Alcyoneusvirus	2560545
	RaK2
	Alefpapilloma	2169692
	virus 1
	Alenquer	2734436
	phlebovirus
	Alexandravirus	2734080
	AD1
	Alexandravirus	2734081
	alexandra
	Alfalfa
	betanucleorha
	bdovirus
	Alfalfa cryptic
	virus 1
	Alfalfa	1770265
	enamovirus 1
	Alfalfa leaf	1306546
	curl virus
	Alfalfa mosaic	12321
	virus
	Alfalfa virus S	1985968
	Algerian	515575
	watermelon
	mosaic virus
	Allamanda	452758
	leaf curl virus
	Allamanda	1317107
	leaf mottle
	distortion
	virus
	Alligatorweed
	stunting virus
	Allium cepa	2058778
	amalgavirus 1
	Allium cepa	2058779
	amalgavirus 2
	Allium virus	317027
	X
	Allpahuayo	144752
	mammarenavius
	Almendravirus	1972686
	almendras
	Almendravirus	1972683
	arboretum
	Almendravirus	1972684
	balsa
	Almendravirus	1972687
	chico
	Almendravirus	1972685
	cootbay
	Almendravirus	2734366
	menghai
	Bat associated	1987731
	cyclovirus 6
	Bat associated	1987732
	cyclovirus 7
	Bat associated	1987733
	cyclovirus 8
	Bat associated	1987734
	cyclovirus 9
	Bat	1913643
	coronavirus
	CDPHE15
	Bat	1244203
	coronavirus
	HKU10
	Bat Hp-	2501961
	betacoronavirus
	Zhejiang2013
	Bat	1146877
	mastadenovirus A
	Bat	1146874
	mastadenovirus B
	Bat	2015370
	mastadenovirus C
	Bat	2015372
	mastadenovirus D
	Bat	2015374
	mastadenovirus E
	Bat	2015375
	mastadenovirus F
	Bat	2015376
	mastadenovirus G
	Bat
	mastadenovirus H
	Bat
	mastadenovirus I
	Bat
	mastadenovirus J
	Batai	2560341
	orthobunyavirus
	Batama	1933177
	orthobunyavirus
	Batfish	2560342
	actinovirus
	Bavaria virus	2560343
	Baxtervirus	2169730
	baxterfox
	Baxtervirus	2169731
	yeezy
	Baylorvirus	2734055
	bv1127AP1
	Baylorvirus	376820
	PHL101
	Bayou	1980459
	orthohantavirus
	Bcepfunavirus	417280
	bcepF1
	Bcepmuvirus	264729
	bcepMu
	Bcepmuvirus	431894
	E255
	Bdellomicrovirus	1986027
	MH2K
	Bdellovibrio
	virus MAC1
	Beak and	77856
	feather disease
	virus
	Bean calico	31602
	mosaic virus
	Bean chlorosis	1227354
	virus
	Bean common	43240
	mosaic
	necrosis virus
	Bean common	12196
	mosaic virus
	Bean dwarf	10838
	mosaic virus
	Bean golden	10839
	mosaic virus
	Bean golden	220340
	yellow mosaic
	virus
	Bean leaf	2004460
	crumple virus
	Bean leafroll	12041
	virus
	Bean mild
	mosaic virus
	Bean necrotic	2560344
	mosaic
	orthotospovirus
	Bean pod	12260
	mottle virus
	Bean rugose	128790
	mosaic virus
	Bean white	2169732
	chlorosis
	mosaic virus
	Bean yellow	267970
	disorder virus
	Bean yellow	714310
	mosaic
	Mexico virus
	Bean yellow	12197
	mosaic virus
	Bear Canyon	192848
	mammarenavirus
	Beauveria	1740646
	bassiana
	polymycovirus 1
	Beauveria	1685109
	bassiana
	victorivirus 1
	Bebaru virus	59305
	Beecentumtre	10778
	virus B103
	Beet black	196375
	scorch virus
	Beet chlorosis	131082
	virus
	Beet cryptic	509923
	virus 1
	Beet cryptic	912029
	virus 2
	Beet cryptic	29257
	virus 3
	Beet curly top	391228
	Iran virus
	Beet curly top	10840
	virus
	Beet mild	156690
	yellowing
	virus
	Beet mosaic	114921
	virus
	Beet necrotic	31721
	yellow vein
	virus
	Beet	72750
	pseudoyellows
	virus
	Beet ringspot	191547
	virus
	Beet soil-	76343
	borne mosaic
	virus
	Beet soil-	46436
	borne virus
	Beet virus Q	71972
	Beet western	12042
	yellows virus
	Beet yellow	35290
	stunt virus
	Beet yellows	12161
	virus
	Beetle mivirus
	Beetrevirus	2560656
	B3
	Beetrevirus	2560663
	JBD67
	Beetrevirus	2560664
	JD18
	Beetrevirus	2560675
	PM105
	Beihai
	picobirnavirus
	Beilong	2560345
	jeilongvirus
	Bell pepper	354328
	alphaendorna-
	virus
	Bell pepper	368735
	mottle virus
	Belladonna	12149
	mottle virus
	Bellamyvirus	2734095
	bellamy
	Bellavista	2560346
	orthobunyavirus
	Bellflower	1720595
	vein chlorosis
	virus
	Bellflower	1982660
	veinal mottle
	virus
	Beluga whale	694015
	coronavirus
	SW1
	Bendigovirus	2560495
	GMA6
	Benedictvirus	1071502
	cuco
	Benedictvirus	1993876
	tiger
	Benevides	2170054
	orthobunyavirus
	Bequatrovirus	1984785
	avesobmore
	Bequatrovirus	1918005
	B4
	Bequatrovirus	1918006
	bigbertha
	Bequatrovirus	1918007
	riley
	Bequatrovirus	1918008
	spock
	Bequatrovirus	1918009
	troll
	Berhavirus	2509379
	beihaiense
	Berhavirus	2509380
	radialis
	Berhavirus	2509381
	sipunculi
	Berisnavirus 1	2734518
	Cacao yellow	12150
	mosaic virus
	Cacao yellow	2169726
	vein banding
	virus
	Cache Valley	2560364
	orthobunyavirus
	Cachoeira	2560365
	Porteira
	orthobunyavirus
	Cacipacore	64305
	virus
	Cactus mild	229030
	mottle virus
	Cactus virus 2
	Cactus virus X	112227
	Cadicivirus A	1330068
	Cadicivirus B	2560366
	Caenorhabditis
	elegans Cer1
	virus
	Caenorhabditis
	elegans
	Cer13 virus
	Caeruleovirus	1985175
	Bc431
	Caeruleovirus	1985176
	Bcp1
	Caeruleovirus	1985177
	BCP82
	Caeruleovirus	1985178
	BM15
	Caeruleovirus	1985179
	deepblue
	Caeruleovirus	1985180
	JBP901
	Cafeteria	1513235
	roenbergensis
	virus
	Cafeteriavirus-	1932923
	dependent
	mavirus
	Caimito	2734421
	pacuvirus
	Cajanus cajan
	Panzee virus
	Caladenia	1198147
	virus A
	Calanthe mild	73840
	mosaic virus
	Cali	2169993
	mammarenavirus
	Calibrachoa	204928
	mottle virus
	California	1933264
	encephalitis
	orthobunyavirus
	California	2170175
	reptarenavirus
	Caligid
	hexartovirus
	Caligrhavirus	2560367
	caligus
	Caligrhavirus	2560551
	lepeophtheirus
	Caligrhavirus	2560736
	salmonlouse
	Calla lily	2560368
	chlorotic spot
	orthotospovirus
	Calla lily	243560
	latent virus
	Callistephus	1886606
	mottle virus
	Callitrichine	106331
	gammaherpes
	virus
3
	Calopogonium
	yellow vein
	virus
	Camel	2169876
	associated
	drosmacovirus 1
	Camel	2169877
	associated
	drosmacovirus 2
	Camel	2170105
	associated
	porprismaco-
	virus 1
	Camel	2170106
	associated
	porprismaco-
	virus 2
	Camel	2170107
	associated
	porprismaco-
	virus 3
	Camel	2170108
	associated
	porprismaco-
	virus 4
	Camelpox	28873
	virus
	Campana	2734442
	phlebovirus
	Campoletis
	aprilis
	ichnovirus
	Campoletis
	flavicincta
	ichnovirus
	Camptochiron
	omus tentans
	entomopoxvirus
	Campylobacter	1006972
	virus IBB35
	Camvirus	1982882
	amela
	Camvirus	1982883
	CAM
	Canary	142661
	circovirus
	Canarypox	44088
	virus
	Candida
	albicans Tca2
	virus
	Candida
	albicans Tca5
	virus
	Candiru	1933182
	phlebovirus
	Canid	170325
	alphaherpesvirus 1
	Canine	1985425
	associated
	gemygorvirus 1
	Canine	1194757
	circovirus
	Canine	10537
	mastadenovirus A
	Canine	11232
	morbillivirus
	Canna yellow	2560371
	mottle
	associated
	virus
	Canna yellow	419782
	mottle virus
	Canna yellow	433462
	streak virus
	Cannabis	1115692
	cryptic virus
	Cano	1980463
	Delgadito
	orthohantavirus
	Canoevirus	2734056
	canoe
	Cao Bang	1980464
	orthohantavirus
	Caper latent	1031708
	virus
	Capim	1933265
	orthobunyavirus
	Capistrivirus	2011077
	KSF1
	Capraria	2049955
	yellow spot
	virus
	Caprine	39944
	alphaherpesvirus 1
	Caprine	11660
	arthritis
	encephalitis
	virus
	Caprine	135102
	gammaherpes
	virus
2
	Caprine	2560372
	respirovirus 3
	Capsicum	2560373
	chlorosis
	orthotospovirus
	Capsicum	2734586
	India
	alphasatellite
	Captovirus	235266
	AFV1
	Capuchin	2163996
	monkey
	hepatitis B
	virus
	Caraparu	1933290
	orthobunyavirus
	Carbovirus	2136037
	queenslandense
	Dyonupapillo	1513250
	mavirus 1
	Dyoomega-	1918731
	papillomavirus 1
	Dyoomikron-	1513251
	papillomavirus 1
	Dyophipapilloma-	1920493
	virus 1
	Dyopipapilloma-	1513252
	virus 1
	Dyopsipapilloma-	1920498
	virus 1
	Dyorhopapilloma-	1513253
	virus 1
	Dyosigmapapilloma-	1513254
	virus 1
	Dyotau-	1932910
	papillomavirus 1
	Dyotheta-	1235662
	papillomavirus 1
	Dyoupsilon-	1932912
	papillomavirus 1
	Dyoxipapilloma-	1513255
	virus 1
	Dyoxipapilloma-	2169881
	virus 2
	Dyozeta-	1177766
	papillomavirus 1
	Eapunavirus	2733615
	Eap1
	East African	223262
	cassava
	mosaic
	Cameroon
	virus
	East African	393599
	cassava
	mosaic Kenya
	virus
	East African	223264
	cassava
	mosaic
	Malawi virus
	East African	62079
	cassava
	mosaic virus
	East African	223275
	cassava
	mosaic
	Zanzibar virus
	East Asian	2734556
	Passiflora
	distortion
	virus
	East Asian	341167
	Passiflora
	virus
	Eastern	2170195
	chimpanzee
	simian foamy
	virus
	Eastern equine	11021
	encephalitis
	virus
	Eastern	2734571
	kangaroopox
	virus
	Eastlansingvirus	2734004
	Sf12
	Echarate	2734447
	phlebovirus
	Echinochloa	42630
	hoja blanca
	tenuivirus
	Echinochloa
	ragged stunt
	virus
	Eclipta yellow	2030126
	vein
	alphasatellite
	Eclipta yellow	875324
	vein virus
	Eclunavirus	2560414
	EcL1
	Ectocarpus	2083183
	fasciculatus
	virus a
	Ectocarpus	37665
	siliculosus
	virus
1
	Ectocarpus
	siliculosus
	virus a
	Ectromelia	12643
	virus
	Ectropis	59376
	obliqua
	nucleopolyhedro-
	virus
	Ectropis	1225732
	obliqua virus
	Edenvirus	2734230
	eden
	Edge Hill	64296
	virus
	Efquatrovirus	2560415
	AL2
	Efquatrovirus	2560416
	AL3
	Efquatrovirus	2560417
	AUEF3
	Efquatrovirus	2560424
	EcZZ2
	Efquatrovirus	2560420
	EF3
	Efquatrovirus	2560421
	EF4
	Efquatrovirus	2560425
	EfaCPT1
	Efquatrovirus	2560426
	IME196
	Efquatrovirus	2560427
	LY0322
	Efquatrovirus	2560428
	PMBT2
	Efquatrovirus	2560429
	SANTOR1
	Efquatrovirus	2560430
	SHEF2
	Efquatrovirus	2560431
	SHEF4
	Efquatrovirus	2560432
	SHEF5
	Eganvirus EtG	2734059
	Eganvirus	29252
	ev186
	Enterovirus A	138948
	Enterovirus B	138949
	Enterovirus C	138950
	Enterovirus D	138951
	Enterovirus E	12064
	Enterovirus F	1330520
	Enterovirus G	106966
	Enterovirus H	310907
	Enterovirus I	2040663
	Enterovirus J	1330521
	Enterovirus K	2169884
	Enterovirus L	2169885
	Entnonaginta-	2734061
	virus ENT90
	Entoleuca	2734428
	entovirus
	Enytus
	montanus
	ichnovirus
	Ephemerovirus	1972589
	adelaide
	Ephemerovirus	1972594
	berrimah
	Ephemerovirus	1972593
	febris
	Ephemerovirus	1972595
	kimberley
	Ephemerovirus	1972596
	koolpinyah
	Ephemerovirus	1972587
	kotonkan
	Ephemerovirus	1972592
	obodhiang
	Ephemerovirus	1972597
	yata
	Epichloe	382962
	festucae virus 1
	Epinotia	166056
	aporema
	granulovirus
	Epiphyas	70600
	postvittana
	nucleopolyhed
	rovirus
	Epirus cherry	544686
	virus
	Epizootic	100217
	haematopoietic
	necrosis
	virus
	Epizootic	40054
	hemorrhagic
	disease virus
	Eponavirus	2734105
	epona
	Epseptimavirus	1982565
	118970sal2
	Epseptimavirus	491003
	EPS7
	Epseptimavirus	2732021
	ev123
	Epseptimavirus	2732022
	ev329
	Epseptimavirus	2732023
	LVR16A
	Epseptimavirus	2732019
	mar003J3
	Epseptimavirus	2732024
	S113
	Epseptimavirus	2732025
	S114
	Epseptimavirus	2732026
	S116
	Epseptimavirus	2732027
	S124
	Epseptimavirus	2732028
	S126
	Epseptimavirus	2732029
	S132
	Epseptimavirus	2732030
	S133
	Epseptimavirus	2732031
	S147
	Epseptimavirus	2732020
	saus132
	Epseptimavirus	2732032
	seafire
	Epseptimavirus	2732033
	SH9
	Epseptimavirus	2732034
	STG2
	Epseptimavirus	1540099
	stitch
	Epseptimavirus	2732035
	Sw2
	Epsilonarterivirus	2501964
	hemcep
	Epsilonarterivirus	2501965
	safriver
	Epsilonarterivirus	2501966
	zamalb
	Epsilonpapilloma-	40537
	virus 1
	Epsilonpapilloma-	2169886
	virus 2
	Epsilonpolyoma-	1891754
	virus bovis
	Eptesipox	1329402
	virus
	Equid	10326
	alphaherpesvirus 1
	Equid	80341
	alphaherpesvirus 3
	Equid	10331
	alphaherpesvirus 4
	Equid	39637
	alphaherpesvirus 8
	Equid	55744
	alphaherpesvirus 9
	Equid	12657
	gammaherpes
	virus
2
	Equid	10371
	gammaherpes
	virus
5
	Equid	291612
	gammaherpes
	virus 7
	Equine	1985379
	associated
	gemycircular-
	virus 1
	Equine	201490
	encephalosis
	virus
	Equine foamy	109270
	virus
	Equine	11665
	infectious
	anemia virus
	Equine	129954
	mastadenovirus A
	Equine	129955
	mastadenovirus B
	Equine	2723956
	picobirnavirus
	Equine rhinitis	47000
	A virus
	Equine	329862
	torovirus
	Eracentumvirus	1985737
	era103
	Eracentumvirus	2733579
	S2
	Eragrostis	638358
	curvula streak
	virus
	Eragrostis	1030595
	minor streak
	virus
	Eragrostis	496807
	streak virus
	Erbovirus A	312185
	Erectites	390443
	yellow mosaic
	virus
	Eriborus
	terebrans
	ichnovirus
	Erinnyis ello	307444
	granulovirus
	Eriocheir	273810
	sinensis
	reovirus
	Ermolevavirus	2733903
	PGT2
	Ermolevavirus	2733904
	PhiKT
	Erskinevirus	2169882
	asesino
	Erskinevirus	2169883
	EaH2
	Erysimum	12152
	latent virus
	Feline	1987742
	associated
	cyclovirus 1
	Feline	11978
	calicivirus
	Feline foamy	53182
	virus
	Feline	11673
	immunodeficiency
	virus
	Feline	11768
	leukemia virus
	Feline	1170234
	morbillivirus
	Felipivirus A
	Felixounavirus	2560439
	Alf5
	Felixounavirus	1965378
	AYO145A
	Felixounavirus	2560723
	BPS15Q2
	Felsduovirus	2734062
	4LV2017
	Felsduovirus	194701
	Fels2
	Felsduovirus	2734063
	RE2010
	Felsduovirus	2734062
	4LV2017
	Felsduovirus	194701
	Fels2
	Fernvirus	1921560
	shelly
	Fernvirus	1921561
	sitara
	Festuca leaf
	streak
	cytorhabdovirus
	Fibralongavirus	2734233
	fv2638A
	Fibralongavirus	2734234
	QT1
	Fibrovirus fs1	70203
	Fibrovirus	1977140
	VGJ
	Ficleduovirus	2560473
	FCL2
	Ficleduovirus	2560474
	FCV1
	Fig badnavirus 1	1034096
	Fig cryptic	882768
	virus
	Figulus
	sublaevis
	entomopoxvirus
	Figwort	10649
	mosaic virus
	Fiji disease	77698
	virus
	Finch	400122
	circovirus
	Finkel-Biskis-	353765
	Jinkins murine
	sarcoma virus
	Finnlakevirus	2734591
	FLiP
	Fionnbharthvirus	2955891
	fionnbharth
	Fipivirus A
	Fipvunavirus	2560476
	Fpv4
	Firehammervirus	1190451
	CP21
	Firehammervirus	722417
	CP220
	Firehammervirus	722418
	CPt10
	Fischettivirus	230871
	C1
	Fishburnevirus	1983737
	brusacoram
	Flamingopox	503979
	virus
	Flammulina	568090
	velutipes
	browning
	virus
	Flaumdravirus	2560665
	KIL2
	Flaumdravirus	2560666
	KIL4
	Fletchervirus	1980966
	CP30A
	Gaiavirus gaia	1982148
	Gaillardia	1468172
	latent virus
	Gairo	1535802
	mammarenavirus
	Gajwadongvirus	2733916
	ECBP5
	Gajwadongvirus	2733917
	PP99
	Galaxyvirus	2560298
	abidatro
	Galaxyvirus	2560303
	galaxy
	Galinsoga	60714
	mosaic virus
	Gallid	10386
	alphaherpesvirus 1
	Gamaleyavirus	1920761
	Sb1
	Gambievirus	2501933
	bolahunense
	Gamboa	1933270
	orthobunyavirus
	Gammaarterivirus	2499678
	lacdeh
	Gammanucleo	2748968
	rhabdovirus
	maydis
	Gammapapilloma-	333926
	virus 1
	Gammapapilloma-	1175852
	virus 10
	Gammapapilloma-	1513256
	virus 11
	Gayfeather	578305
	mild mottle
	virus
	Gecko	2560481
	reptillovirus
	Gelderlandvirus	2560727
	melville
	Gelderlandvirus	1913658
	s16
	Gelderlandvirus	1913657
	stml198
	Gelderlandvirus	2560734
	stp4a
	Gentian	182452
	mosaic virus
	Gentian ovary	1920772
	ringspot virus
	Geotrupes
	sylvaticus
	entomopoxvirus
	Gequatrovirus	1986034
	G4
	Gequatrovirus	1910968
	ID52
	Gequatrovirus	1910969
	talmos
	Gerygone	1985381
	associated
	gemycircular-
	virus 1
	Gerygone	1985382
	associated
	gemycircular-
	virus 2
	Harrisina	115813
	brillians
	granulovirus
	Harrisonvirus	1982221
	harrison
	Harvey	11807
	murine
	sarcoma virus
	Hautre virus	1982895
	hau3
	Havel River	254711
	virus
	Hawkeyevirus	2169910
	hawkeye
	Hazara	1980522
	orthonairovirus
	Heartland	2747342
	banda virus
	Hebius
	tobanivirus 1
	Hedgehog	1965093
	coronavirus 1
	Hedwigvirus	2560502
	hedwig
	Hedyotis	1428190
	uncinella
	yellow mosaic
	virus
	Hedyotis	1428189
	yellow mosaic
	betasatellite
	Heilongjiangvirus	2734110
	Lb
	Helenium	12171
	virus S
	Helianthus	2184469
	annuus
	alphaendornavirus
	Helicobasidium	675833
	mompa
	alphaendorna-
	virus 1
	Helicobasidium	344866
	mompa
	partitivirus
	V70
	Helicobasidium	196690
	mompa
	totivirus 1-17
	Helicoverpa	489830
	armigera
	granulovirus
	Helicoverpa	51313
	armigera
	nucleopolyhedro-
	virus
	Helicoverpa	37206
	armigera stunt
	virus
	Heliothis	10290
	armigera
	entomopoxvirus
	Heliothis	113366
	virescens
	ascovirus 3a
	Heliothis zea	29250
	nudivirus
	Helleborus	592207
	mosaic virus
	Helleborus net	592206
	necrosis virus
	Helminthos-	2560520
	porium victoriae
	virus 145S
	Helminthos-	45237
	porium victoriae
	virus 190S
	Helsettvirus	2733626
	fPS53
	Helsettvirus	2733628
	fPS54ocr
	Helsettvirus	2733627
	fPS59
	Helsettvirus	2733625
	fPS9
	Helsingorvirus	1918193
	Cba121
	Helsingorvirus	1918194
	Cba171
	Jujube	2020956
	mosaic-
	associated
	virus
	Jun	2560536
	jeilongvirus
	Juncopox
	virus
	Jutiapa virus	64299
	Jwalphavirus	2169963
	jwalpha
	Kabuto	2747382
	mountain
	uukuvirus
	Kadam virus	64310
	Kadipiro virus	104580
	Kaeng Khoi	1933275
	orthobunyavirus
	Kafavirus	2733923
	SWcelC56
	Kafunavirus	1982588
	KF1
	Kagunavirus	2560464
	golestan
	Kagunavirus	1911008
	K1G
	Kagunavirus	1911010
	K1H
	Kagunavirus	1911007
	Klind1
	Kagunavirus	1911009
	Klind2
	Kagunavirus	2734197
	RP180
	Merremia	77813
	mosaic virus
	Mesta yellow	1705093
	vein mosaic
	alphasatellite
	Mesta yellow	508748
	vein mosaic
	Bahraich virus
	Metamorphoo	2734253
	virus fireman
	Metamorphoo	2734254
	virus
	metamorphoo
	Metamorphoo	2734255
	virus robsfeet
	Metrivirus	2560269
	ME3
	Mguuvirus	2733593
	JG068
	Microbacterium
	virus
	MuffinTheCat
	[2]
	Microcystis	340435
	virus Ma-
	LMM01
	Microhyla
	letovirus 1
	Micromonas	338781
	pusilia
	reovirus
	Micromonas	373996
	pusilia virus
	SP1
	Microplitis
	croceipes
	bracovirus
	Microtus	2006148
	arvalis
	polyomavirus
1
	Mukerjeevirus	2734186
	mv52B1
	Mulberry	1227557
	badnavirus 1
	Mulberry	1631303
	mosaic dwarf
	associated
	virus
	Mulberry	1527441
	mosaic leaf
	roll associated
	virus
	Mulberry
	ringspot virus
	Mulberry vein
	banding
	associated
	orthotospovirus
	Mule deerpox	304399
	virus
	Mume virus A	2137858
	Mumps	2560602
	orthorubulavirus
	Mungbean	2010322
	yellow mosaic
	betasatellite
	Mukerjeevirus	2734186
	mv52B1
	Mulberry	1227557
	badnavirus 1
	Mulberry	1631303
	mosaic dwarf
	associated
	virus
	Mycobacterium	1993864
	virus
	Tweety
	Mycobacterium	1993860
	virus Wee
	Mycobacterium	1993859
	virus
	Wildcat
	Mycoreovirus 1	311228
	Mycoreovirus 2	404237
	Mycoreovirus 3	311229
	Mylasvirus	1914020
	persius
	Mynahpox	2169711
	virus
	Myodes
	coronavirus
	2JL14
	Myodes	2006147
	glareolus
	polyomavirus
1
	Myodes	2560609
	jeilongvirus
	Myodes	2560610
	narmovirus
	Myohalovirus	1980944
	phiH
	Noxifervirus	2560671
	noxifer
	Ntaya virus	64292
	Ntepes	2734464
	phlebovirus
	Nuarterivirus
	guemel
	Nudaurelia	85652
	capensis beta
	virus
	Nudaurelia	12541
	capensis
	omega virus
	Nupapilloma-	334205
	virus 1
	Nyando	1933306
	orthobunyavirus
	Nyavirus	644609
	midwayense
	Nyavirus	644610
	nyamaniniense
	Nyavirus	1985708
	sierranevadaense
	Nyceiraevirus	2560506
	nyceirae
	Nyctalus	2501928
	velutinus
	alphacoronavirus
	SC-2013
	Nylanderia	1871153
	fulva virus 1
	Nymphadoravirus	2170041
	kita
	Nymphadoravirus	2560507
	nymphadora
	Nymphadoravirus	2170042
	zirinka
	Oat blue	56879
	dwarf virus
	Oat chlorotic	146762
	stunt virus
	Oat dwarf	497863
	virus
	Oat golden	45103
	stripe virus
	Oxbow	1980484
	orthohantavirus
	Oxyplax	2083176
	ochracea
	nucleopolyhedro-
	virus
	Paadamvirus	2733939
	RHEph01
	Pacific coast
	uukuvirus
	Pacui	2560617
	pacuvirus
	Paenibacillus
	virus Willow
	Pagavirus	2733940
	S05C849
	Pagevirus	1921185
	page
	Pagevirus	1921186
	palmer
	Pagevirus	1921187
	pascal
	Pagevirus	1921188
	pony
	Pagevirus	1921189
	pookie
	Pagoda yellow	1505530
	mosaic
	associated
	virus
	Paguronivirus 1	2508237
	Pahexavirus	1982252
	ATCC29399BC
	Pahexavirus	1982303
	pirate
	Pahexavirus	1982304
	procrass1
	Pahexavirus	1982305
	SKKY
	Pahexavirus	1982306
	solid
	Pahexavirus	1982307
	stormborn
	Pahexavirus	1982308
	wizzo
	Pahsextavirus	2733975
	pAh6C
	Pairvirus	2733941
	Lo5R7ANS
	Pakpunavirus	1921409
	CAb02
	Pahexavirus	1982303
	pirate
	Pahexavirus	1982304
	procrass1
	Pahexavirus	1982305
	SKKY
	Pea necrotic	753670
	yellow dwarf
	virus
	Pea seed-	12208
	borne mosaic
	virus
	Pea stem	199361
	necrosis virus
	Pea streak	157777
	virus
	Pea yellow	1436892
	stunt virus
	Peach	471498
	chlorotic
	mottle virus
	Peach latent	12894
	mosaic viroid
	Peach	2169999
	marafivirus D
	Peach mosaic	183585
	virus
	Peach rosette	65068
	mosaic virus
	Peanut	35593
	chlorotic
	streak virus
	Peanut clump	28355
	virus
	Peanut yellow
	mosaic virus
	Pear blister	12783
	canker viroid
	Peaton	2560627
	orthobunyavirus
	Peatvirus	2560629
	peat2
	Pecan mosaic-	1856031
	associated
	virus
	Pecentumvirus	40523
	A511
	Penicillum	2734569
	brevicompactum
	polymycovirus 1
	Pennisetum	221262
	mosaic virus
	Pepino mosaic
	virus[3]
	Pepo aphid-	1462681
	borne yellows
	virus
	Pepper chat	574040
	fruit viroid
	Pepper	2734493
	chlorotic spot
	orthotospovirus
	Phietavirus X2	320850
	Phifelvirus	1633149
	FL1
	Phikmvvirus	2733349
	15pyo
	Phlox virus S	436066
	Phnom Penh	64894
	bat virus
	Phocid	47418
	alphaherpes-
	virus 1
	Phocid	47419
	gammaherpes
	virus
2
	Phocid	2560643
	gammaherpes
	virus
3
	Phocine	11240
	morbillivirus
	Pholetesor
	ornigis
	bracovirus
	Phthorimaea	192584
	operculella
	granulovirus
	Phutvirus	2733655
	PPpW4
	Phyllosphere
	sclerotimonavirus
	Physalis	72539
	mottle virus
	Physarum
	polycephalum
	Tpl virus
	Phytophthora	310750
	alphaendorna-
	virus 1
	Picardvirus	2734264
	picard
	Pidgey	2509390
	pidchovirus
	Piedvirus	2733947
	IMEDE1
	Pienvirus	2733373
	R801
	Pifdecavirus	2733657
	IBBPF7A
	Plum bark	675077
	necrosis stem
	pitting-
	associated
	virus
	Plum pox	12211
	virus
	Plumeria	1501716
	mosaic virus
	Plutella	98383
	xylostella
	granulovirus
	Poa semilatent	12328
	virus
	Poaceae	1985392
	associated
	gemycircular-
	virus 1
	Podivirus	2733948
	S05C243
	Poecivirus A	2560644
	Pogseptimavirus	2733996
	PG07
	Pogseptimavirus	2733997
	VspSw1
	Poindextervirus	2734196
	BL10
	Poindextervirus	2748760
	rogue
	Poinsettia	305785
	latent virus
	Poinsettia	113553
	mosaic virus
	Pokeweed	1220025
	mosaic virus
	Pokrovskaiavirus	2733374
	fHeYen301
	Pokrovskaiavirus	2733375
	pv8018
	Polar bear
	mastadenovirus A
	Pollockvirus	2170215
	pollock
	Pollyceevirus	2560679
	pollyC
	Polybotosvirus	2560286
	Atuph07
	Polygonum	430606
	ringspot
	orthotospovirus
	Pomona bat	2049933
	hepatitis B
	virus
	Pongine	159603
	gammaherpes
	virus
2
	Poplar mosaic	12166
	virus
	Popoffvirus	2560283
	pv56
	Porcine	1985393
	associated
	gemycircular-
	virus 1
	Potato virus Y	12216
	Potato yellow	2230887
	blotch virus
	Potato yellow	223307
	mosaic
	Panama virus
	Potato yellow	10827
	mosaic virus
	Potato yellow	103881
	vein virus
	Pothos latent	44562
	virus
	Potosi	2560646
	orthobunyavirus
	Poushouvirus	2560396
	Poushou
	Pouzolzia	1225069
	golden mosaic
	virus
	Primate T-	194443
	lymphotropic
	virus
3
	Primolicivirus	2011081
	Pf1
	Primula	1511840
	malacoides
	virus 1
	Priunavirus	2560652
	PR1
	Privet ringspot	2169960
	virus
	Prochlorococcus
	virus
	PHM1
	Prospect Hill	1980485
	orthohantavirus
	Protapanteles
	paleacritae
	bracovirus
	Providence	213633
	virus
	Prune dwarf	33760
	virus
	Prunus latent	2560653
	virus
	Prunus	37733
	necrotic
	ringspot virus
	Przondovirus	2733672
	KN31
	Pseudomonas	462590
	virus Yua
	Pseudoplusia
	includens virus
	Pseudotevenvirus	329381
	RB16
	Pseudotevenvirus	115991
	RB43
	Psimunavirus	2734265
	psiM2
	Psipapillomavirus 1	1177762
	Psipapillomavirus 2	2170170
	Psipapillomavirus 3	2170171
	Psittacid	50294
	alphaherpesvirus 1
	Psittacine	2003673
	atadenovirus A
	Psittacine	2169709
	aviadenovirus B
	Psittacine	2734577
	aviadenovirus C
	Psittacinepox	2169712
	virus
	Pteridovirus	2734351
	filicis
	Pteridovirus	2734352
	maydis
	Pteropodid	2560693
	alphaherpesvirus 1
	Pteropox virus	1873698
	Pteropus	1985395
	associated
	gemycircularvirus 1
	Pteropus	1985404
	associated
	gemycircularvirus 10
	Ptyasnivirus 1	2734501
	Pukovnikvirus	540068
	pukovnik
	Pulverervirus	2170091
	PFR1
	Puma lentivirus	12804
	Pumpkin	2518373
	polerovirus
	Pumpkin yellow	1410062
	mosaic virus
	Punavirus P1	10678
	Punavirus RCS47	2560452
	Punavirus SJ46	2560732
	Punique	2734468
	phlebovirus
	Punta Toro	1933186
	phlebovirus
	Puumala	1980486
	orthohantavirus
	Pyrobaculum	1805492
	filamentous virus 1
	Pyrobaculum	270161
	spherical virus
	Qadamvirus	2733953
	SB28
	Qalyub	1980527
	orthonairovirus
	Qingdao virus J21	2734135
	Qingling	2560694
	orthophasmavirus
	Quail pea mosaic
	virus
	Quailpox virus	400570
	Quaranjavirus	688437
	johnstonense
	Quaranjavirus	688436
	quaranfilense
	Qubevirus durum	39803
	Qubevirus	39804
	faecium
	Quezon	2501382
	mobatvirus
	Quhwahvirus	2283289
	kaihaidragon
	Quhwahvirus	2201441
	ouhwah
	Quhwahvirus	2182400
	paschalis
	Rabbit associated	1985420
	gemykroznavirus 1
	Rabbit fibroma	10271
	virus
	Rabbit	11976
	hemorrhagic
	disease virus
	Rabovirus A	1603962
	Rabovirus B	2560695
	Rabovirus C	2560696
	Rabovirus D	2560697
	Raccoonpox	10256
	virus
	Radish leaf curl	435646
	virus
	Radish mosaic	328061
	virus
	Radish yellow	319460
	edge virus
	Rafivirus A
	Rafivirus B	2560699
	Rafivirus C
	Raleigh virus	2734266
	darolandstone
	Raleigh virus	2734267
	raleigh
	Ramie mosaic	1874886
	Yunnan virus
	Ranid	85655
	herpesvirus 1
	Ranid	389214
	herpesvirus 2
	Ranid	1987509
	herpesvirus 3
	Ranunculus leaf	341110
	distortion virus
	Ranunculus mild	341111
	mosaic virus
	Ranunculus	341112
	mosaic virus
	Raptor	691961
	siadenovirus A
	Raspberry bushy	12451
	dwarf virus
	Raspberry leaf	326941
	mottle virus
	Raspberry	12809
	ringspot virus
	Rat associated	1985405
	gemycircularvirus 1
	Rat associated	2170126
	porprismacovirus 1
	Rattail cactus	1123754
	necrosis-
	associated virus
	Rattus norvegicus	1679933
	polyomavirus 1
	Rauchvirus BPP1	194699
	Raven circovirus	345250
	Ravin virus N15	40631
	Recovirus A	2560702
	Red clover
	associated
	luteovirus
	Red clover	1323524
	cryptic virus 2
	Red clover mottle	12262
	virus
	Red clover	12267
	necrotic mosaic
	virus
	Red clover vein	590403
	mosaic virus
	Red deerpox
	virus
	Redspotted	43763
	grouper nervous
	necrosis virus
	Reginaelenavirus	2734071
	rv3LV2017
	Rehmannia	425279
	mosaic virus
	Rehmannia virus 1	2316740
	Reptilian	122203
	ferlavirus
	Reptilian	226613
	orthoreovirus
	Rerduovirus	1982376
	RER2
	Rerduovirus	1109716
	RGL3
	Restivirus RSS1	2011075
	Reston ebolavirus	186539
	Reticuloendo-	11636
	theliosis virus
	Reyvirus rey	1983751
	Rhesus macaque	2170199
	simian foamy
	virus
	Rhinolophus	2004965
	associated
	gemykibivirus 1
	Rhinolophus	2004966
	associated
	gemykibivirus 2
	Rhinolophus bat	693998
	coronavirus
	HKU2
	Rhinolophus	2501926
	ferrumequinum
	alphacoronavirus
	HuB-2013
	Rhinovirus A	147711
	Rhinovirus B	147712
	Rhinovirus C	463676
	Rhizidiomyces
	virus
	Rhizoctonia	1408133
	cerealis
	alphaendornavirus
1
	Rhizoctonia	2560704
	magoulivirus 1
	Sabo	2560716
	orthobunyavirus
	Saboya virus	64284
	Sacbrood virus	89463
	Saccharomyces	186772
	20S RNA
	narnavirus
	Saccharum streak	683179
	virus
	Saclayvirus	2734138
	Aci011
	Saclayvirus	2734139
	Aci022
	Saclayvirus	2734137
	Aci05
	Saetivirus fs2	1977306
	Saetivirus VFJ	1977307
	Saffron latent	2070152
	virus
	Saguaro cactus	52274
	virus
	Saguinine	2169901
	gammaherpesvirus 1
	Saikungvirus	2169924
	HK633
	Saikungvirus	2169925
	HK75
	Saimiri sciureus	1236410
	polyomavirus 1
	Saimiriine	10353
	alphaherpesvirus 1
	Saimiriine	1535247
	betaherpesvirus 4
	Saimiriine	10381
	gammaherpesvirus 2
	Saint Floris
	phlebovirus
	Saint Louis	11080
	encephalitis virus
	Saint Valerien
	virus
	Sakhalin	1980528
	orthonairovirus
	Sakobuvirus A	1659771
	Sal Vieja virus	64301
	Salacisavirus	2734140
	pssm2
	Salanga	2734471
	phlebovirus
	Salasvirus phi29	10756
	Salchichonvirus	298338
	LP65
	Salehabad	1933188
	phlebovirus
	Salem salemvirus	2560718
	Salivirus A	1330524
	Salmo	2749930
	aquapar amyxovirus
	Salmon gillpox	2734576
	virus
	Saphexavirus	1982380
	VD13
	Sapporo virus	95342
	Sarcochilus virus	104393
	Y
	Sashavirus sasha	2734275
	Sasquatchvirus	2734143
	Y3
	Sasvirus BFK20	2560392
	Satsuma dwarf	47416
	virus
	Sauletekiovirus	2734030
	AAS23
	Saumarez Reef	40012
	virus
	Saundersvirus	2170234
	Tp84
	Sauropus leaf	1130981
	curl virus
	Sawgrhavirus	2734397
	connecticut
	Sawgrhavirus	2734398
	longisland
	Sawgrhavirus	2734399
	minto
	Sawgrhavirus	2734400
	sawgrass
	Scale drop	1697349
	disease virus
	Scallion mosaic	157018
	virus
	Scapularis	2734431
	ixovirus
	Scapunavirus	2560792
	scapl
	Scheffersomyces	1300323
	segobiensis virus L
	Schefflera	2169729
	ringspot virus
	Schiekvirus	2560422
	EFDG1
	Schiekvirus	2734044
	EFP01
	Schiekvirus	2734045
	EfV12
	Schistocerca
	gregaria
	entomopoxvirus
	Saphexavirus	1982380
	VD13
	Sophora yellow	2169837
	stunt
	alphasatellite
5
	Sorex araneus	2734504
	coronavirus T14
	Sorex araneus	2560769
	polyomavirus 1
	Sorex coronatus	2560770
	polyomavirus 1
	Sorex minutus	2560771
	polyomavirus 1
	Sorghum	107804
	chlorotic spot
	virus
	Sorghum mosaic	32619
	virus
	Sororoca	2560772
	orthobunyavirus
	Sortsnevirus	2734190
	IME279
	Sortsnevirus	2734189
	sortsne
	Sosuga	2560773
	pararubulavirus
	Soupsvirus soups	1982563
	Soupsvirus	2560510
	strosahl
	Soupsvirus wait	2560513
	Souris	2169997
	mammarenavirus
	Sourvirus sour	2560509
	South African	63723
	cassava mosaic
	virus
	Southern bean	12139
	mosaic virus
	Southern cowpea	196398
	mosaic virus
	Southern	1159195
	elephant seal
	virus
	Southern rice	519497
	black-streaked
	dwarf virus
	Southern tomato	591166
	virus
	Sowbane mosaic	378833
	virus
	Soybean	1985413
	associated
	gemycircularvirus 1
	Sophora yellow	2169837
	stunt
	alphasatellite
5
	Sorex araneus	2734504
	coronavirus T14
	Sorex araneus	2560769
	polyomavirus 1
	Sorex coronatus	2560770
	polyomavirus 1
	Sorex minutus	2560771
	polyomavirus 1
	Sorghum	107804
	chlorotic spot
	virus
	Sorghum mosaic	32619
	virus
	Sororoca	2560772
	orthobunyavirus
	Sortsnevirus	2734190
	IME279
	Switchgrass	2049938
	mosaic-
	associated virus
	Symapivirus A
	Synechococcus	2734100
	virus SRIM12-08
	Synedrella leaf	1544378
	curl alphasatellite
	Synedrella	1914900
	yellow vein
	clearing virus
	Synetaeris
	tenuifemur
	ichnovirus
	Syngnathid	2734305
	ichthamaparvovirus 1
	Synodus	2749934
	synodonvirus
	Tabernariusvirus	2560691
	tabernarius
	Tacaiuma	611707
	orthobunyavirus
	Tacaribe	11631
	mammarenavirus
	Tacheng	2734606
	uukuvirus
	Tahyna	2560796
	orthobunyavirus
	Tangaroavirus	2733962
	tv951510a
	Tankvirus tank	1982567
	Tapara	2734474
	phlebovirus
	Tapirape	2560798
	pacuvirus
	Tapwovirus cesti	2509383
	Taranisvirus	2734146
	taranis
	Taro bacilliform	1634914
	CH virus
	Taro bacilliform	178354
	virus
	Tarumizu	2734340
	coltivirus
	Tataguine	2560799
	orthobunyavirus
	Taterapox virus	28871
	Taupapillomavirus 1	1176148
	Taupapillomavirus 2	1513274
	Taupapillomavirus 3	1961786
	Taupapillomavirus 4	2170222
	Taura syndrome	142102
	virus
	Tawavirus JSF7	2733965
	Tea plant	2419939
	necrotic ring
	blotch virus
	Tefnutvirus	2734147
	siom18
	Tegunavirus r1rt	1921705
	Tegunavirus	1921706
	yenmtg1
	Tehran	2734475
	phlebovirus
	Telfairia golden	2169737
	mosaic virus
	Telfairia mosaic	1859135
	virus
	Tellina virus	359995
	Tellina virus 1	321302
	Telosma mosaic	400394
	virus
	Tembusu virus	64293
	Tensaw	2560800
	orthobunyavirus
	Tent-making bat	1508712
	hepatitis B virus
	Teseptimavirus	2733885
	YpsPG
	Testudine
	orthoreovirus
	Testudinid	2560801
	alphaherpesvirus 3
	Tete	35319
	orthobunyavirus
	Tetterwort vein	1712389
	chlorosis virus
	Teviot	2560803
	pararubulavirus
	Thailand	1980492
	orthohantavirus
	Thalassavirus	2060093
	thalassa
	Thaumasvirus	2734148
	stim4
	Thermoproteus	292639
	tenax spherical
	virus
1
	Thermoproteus	10479
	tenax virus 1
	Thermus virus	1714273
	IN93
	Thermus virus	1714272
	P23-77
	Thetaarterivirus	2501999
	kafuba
	Thetaarterivirus	2502000
	mikelba l
	Thetapapilloma-	197772
	virus 1
	Thetapolyomavirus	1891755
	censtriata
	Thetapolyomavirus	2218588
	trebernacchii
	Thetapolyomavirus	2170103
	trepennellii
	Thetisvirus ssm1	2734149
	Thiafora	1980529
	orthonairovirus
	Thimiri	1819305
	orthobunyavirus
	Thin paspalum	1352511
	asymptomatic
	virus
	Thistle mottle
	virus
	Thogotovirus	11318
	dhoriense
	Thogotovirus	11569
	thogotoense
	Thomixvirus	2560804
	OH3
	Thornevirus	2560336
	SP15
	Thosea asigna	83810
	virus
	Thottopalayam	2501370
	thottimvirus
	Thunberg	299200
	fritillary mosaic
	virus
	Thysanoplusia	101850
	orichalcea
	nucleopolyhedro
	virus
	Tiamatvirus	268748
	PSSP7
	Tibetan frog	2169919
	hepatitis B virus
	Tibrovirus	1987018
	alphaekpoma
	Tibrovirus	2170224
	beatrice
	Tibrovirus	1987019
	betaekpoma
	Tibrovirus	1972586
	coastal
	Tibrovirus congo	1987017
	Tibrovirus	1987013
	sweetwater
	Tibrovirus	1972584
	tibrogargan
	Tick associated	2560805
	circovirus 1
	Tick associated	2560806
	circovirus 2
	Tick-borne	11084
	encephalitis virus
	Tico phebovirus	2734476
	Tidunavirus	2560834
	pTD1
	Tidunavirus	2560833
	VP4B
	Tiger puffer	43764
	nervous necrosis
	virus
	Tigray	2560807
	orthohantavirus
	Tigrvirus E122	431892
	Tigrvirus E202	431893
	Tobacco leaf curl	439423
	Comoros virus
	Tobacco leaf curl	336987
	Cuba virus
	Tobacco leaf curl	2528965
	Dominican
	Republic virus
	Tobacco leaf curl	2010326
	Japan
	betasatellite
	Tobacco leaf curl	2010327
	Patna
	betasatellite
	Tobacco leaf curl	905054
	Pusa virus
	Tobacco leaf curl	409287
	Thailand virus
	Tobacco leaf curl	211866
	Yunnan virus
	Tobacco leaf curl	223337
	Zimbabwe virus
	Tobacco leaf	196691
	rugose virus
	Veracruzvirus	1032892
	heldan
	Veracruzvirus	2003502
	rockstar
	Verbena latent	134374
	virus
	Verbena virus Y	515446
	Vernonia crinkle	1925153
	virus
	Vernonia yellow	666635
	vein betasatellite
	Vernonia yellow	2169908
	vein Fujian
	alphasatellite
	Vernonia yellow	2050589
	vein Fujian
	betasatellite
	Vernonia yellow	1001341
	vein Fujian virus
	Vernonia yellow	367061
	vein virus
	Versovirus	2011076
	VfO3K6
	Verticillium	759389
	dahliae
	chrysovirus 1
	Vesicular	35612
	exanthema of
	swine virus
	Vesiculovirus	1972579
	alagoas
	Vesiculovirus	1972567
	bogdanovac
	Whitefly-	2169744
	associated
	begomovirus 7
	White-tufted-ear	2170205
	marmoset simian
	foamy virus
	Whitewater	46919
	Arroyo
	mammarenavirus
	Wifcevirus	2734154
	ECML117
	Wifcevirus	2734155
	FEC19
	Wifcevirus WFC	2734156
	Wifcevirus WFH	2734157
	Wigeon	1159908
	coronavirus
	HKU20
	Wild cucumber	70824
	mosaic virus
	Wild melon
	banding virus
	Wild onion	1862127
	symptomless
	virus
	Wild potato	187977
	mosaic virus
	Wild tomato	400396
	mosaic virus
	Wild Vitis latent	2560839
	virus
	Wilnyevirus	2560486
	billnye
	Wilsonroadvirus	2734007
	Sd1
	Winged bean	2169693
	alphaendornavirus 1
	Winklervirus	2560752
	chi14
	Wiseana signata	65124
	nucleopolyhedro
	virus
	Wissadula golden	51673
	mosaic virus
	Wissadula yellow	1904884
	mosaic virus
	Wisteria	1973265
	badnavirus 1
	Wisteria vein	201862
	mosaic virus
	Witwatersrand	2560841
	orthobunyavirus
	Wizardvirus	2170253
	twister6
	Wizardvirus	2170254
	wizard
	Woesvirus woes	1982751
	Wolkberg	2170059
	orthobunyavirus
	Wongorr virus	47465
	Wongtaivirus	2169922
	HK542
	Woodchuck	35269
	hepatitis virus
	Woodruffvirus	1982746
	TP1604
	Woodruffvirus	1982747
	YDN12
	Woolly monkey	68416
	hepatitis B virus
	Woolly monkey	11970
	sarcoma virus
	Wound tumor	10987
	virus
	Wphvirus	2560329
	BPS10C
	Wphvirus BPS13	1987727
	Wphvirus hakuna	1987729
	Wphvirus	1987728
	megatron
	Wphvirus WPh	1922328
	Wuchang	1980542
	cockroach
	orthophasmavirus
1
	Wuhan mivirus	2507319
	Wuhan mosquito	1980543
	orthophasmavirus 1
	Wuhan mosquito	1980544
	orthophasmavirus
	2
	Wuhan virus	2733969
	PHB01
	Wuhanvirus	2733970
	PHB02
	Wumivirus	2509286
	millepedae
	Wumpquatrovirus	400567
	WMP4
	Wumptrevirus	440250
	WMP3
	Wutai mosquito	1980612
	phasivirus
	Wyeomyia	273350
	orthobunyavirus
	Xanthophyllomyces	1167690
	dendrorhous
	virus L1A
	Xanthophyllomyces	1167691
	dendrorhous
	virus L1B
	Xapuri	2734417
	mammarenavirus
	Xestia c-nigrum	51677
	granulovirus
	Xiamenvirus	1982373
	RDJL1
	Xiamenvirus	1982374
	RDJL2
	Xilang striavirus	2560844
	Xinzhou mivirus	2507320
	Xipapillomavirus 1	10561
	Xipapillomavirus 2	1513273
	Yokohamavirus	1980942
	PEi21
	Yokose virus	64294
	Yoloswagvirus	2734158
	yoloswag
	Yongjia	2734607
	uukuvirus
	Youcai mosaic	228578
	virus
	Yunnan orbivirus	306276
	Yushanvirus	2733978
	Spp001
	Yushanvirus	2733979
	SppYZU05
	Yuyuevirus	2508254
	beihaiense
	Yuyuevirus	2508255
	shaheense
	Zaire ebolavirus	186538
	Zaliv Terpeniya	2734608
	uukuvirus
	Zantedeschia	270478
	mild mosaic virus
	Zarhavirus	2734410
	zahedan
	Zika virus	64320

The cascade assays described herein are particularly well-suited for simultaneous testing of multiple targets. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., pools of 2-1000, 2-100, 2-50, or 2-10 target nucleic acids of interest. Further testing may be used to identify the specific member of the pool, if warranted.
While the methods described herein do not require the target nucleic acid of interest to be DNA (and in fact it is specifically contemplated that the target nucleic acid of interest may be RNA), it is understood by those in the field that a reverse transcription step to convert target RNA to cDNA may be performed prior to or while contacting the biological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in the reaction mix, either provided as a protein, a coding sequence for the protein, or, in many embodiments, in a ribonucleoprotein (RNP) complex. In some embodiments, the one or more nucleic acid-guided nucleases in the reaction mix may be, for example, a Cas nucleic acid-guided nuclease. Any nucleic acid-guided nuclease having both cis- and trans-cleavage activity may be employed, and the same nucleic acid-guided nuclease may be used for both RNP complexes or different nucleic acid-guided nucleases may be used in RNP1 and RNP2. For example, RNP1 and RNP2 may both comprise Cas12a nucleic acid-guided nucleases, or RNP1 may comprise a Cas13 nucleic acid-guided nuclease and RNP2 may comprise a Cas12a nucleic acid-guided nuclease or vice versa. In embodiments where a variant nucleic acid-guided nuclease is employed, only RNP2 will comprise the variant, and RNP1 may comprise either a Cas12a or Cas13 nucleic acid-guided nuclease. In embodiments where a variant nucleic acid-guided nuclease is not employed, either or both RNP1 and RNP2 can comprise a Cas13 nucleic acid-guided nuclease. Note that trans-cleavage activity is not triggered unless and until cis-cleavage activity (i.e., sequence specific activity) is initiated. Nucleic acid-guided nucleases include Type V and Type VI nucleic acid-guided nucleases, as well as nucleic acid-guided nucleases that comprise a RuvC nuclease domain or a RuvC-like nuclease domain but lack an HNH nuclease domain. Nucleic acid-guided nucleases with these properties are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly-discovered systems include BioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.paris-saclay.fr).
The type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA nucleic acid-guided nuclease (e.g., a Cas12a, Cas14a, or Cas3) should be utilized if the target nucleic acid of interest is a DNA molecule, and an RNA nucleic acid-guided nuclease (e.g., Cas13a or Cas12g) should be utilized if the target nucleic acid of interest is an RNA molecule. Exemplary nucleic acid-guided nucleases include, but are not limited to, Cas RNA-guided DNA nucleic acid-guided nucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNA nucleic acid-guided nucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g; and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as Cas12a, Cas13a, or Cas14a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleic acid-guided nucleases may also be employed.
In an RNP with a single crRNA (i.e., lacking/without a tracrRNA), Cas12a nucleases and related homologs and orthologs interact with a PAM (protospacer adjacent motif) sequence in a target nucleic acid for dsDNA unwinding and R-loop formation. Cas12a nucleases employ a multistep mechanism to ensure accurate recognition of spacer sequences in the target nucleic acid. The WED, REC1 and PAM-interacting (PI) domains of Cas12a nucleases are responsible for PAM recognition and for initiating invasion of the crRNA in the target dsDNA and for R-loop formation. It has been hypothesized that a conserved lysine residue is inserted into the dsDNA duplex, possibly initiating template strand/non-template strand unwinding. (See Jinek, et al, Mol. Cell, 73(3):589-600.e4 (2019).) PAM binding further introduces a kink in the target strand, which further contributes to local strand separation and facilitates base paring of the target strand to the seed segment of the crRNA while the displaced non-target strand is stabilized by interactions with the PAM-interacting domains. (Id.) The variant nucleic acid-guided nucleases disclosed herein and discussed in detail below have been engineered to disrupt one or both of the WED and PI domains to reconfigure the site of unwinding and R-loop formation to, e.g., sterically obstruct dsDNA target nucleic acids from binding to the variant nucleic acid-guided nuclease and/or to minimize strand separation and/or stabilization of the non-target strand. Though contrary to common wisdom, engineering the variant nucleic acid-guided nucleases in this way contributes to a robust and high-fidelity cascade assay.
The variant nucleic acid-guided nucleases disclosed herein are variants of wildtype Type V nucleases LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas 12a (Flavobacteriales bacterium Cas12a), Lb4Cas 12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a), BoCas 12a (Bacteroidetes bacterium Cas12a), CMaCas 12a (Candidatus Methanomethylophilus alvus CMx1201 Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guided nucleases and homologs and orthologs of these nucleic acid-guided nucleases (and other nucleic acid-guided nucleases that exhibit both cis-cleavage and trans-cleavage activity), where mutations have been made to the PAM interacting domains such that double-stranded DNA (dsDNA) substrates are bound much more slowly to the variant nucleic acid-guided nucleases than to their wildtype nucleic acid-guided nuclease counterpart, yet single-stranded DNA (ssDNA) substrates are bound at the same rate or nearly so as their wildtype nucleic acid-guided nuclease counterpart. The variant nucleic acid-guided nucleases comprise reconfigured domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules to achieve this phenotype and are described in detail below.
Guide RNA (gRNA)
The present disclosure detects a target nucleic acid of interest via a reaction mixture containing at least two guide RNAs (gRNAs) each incorporated into a different RNP complex (i.e., RNP1 and RNP2). Suitable gRNAs include at least one crRNA region to enable specificity in every reaction. The gRNA of RNP1 is specific to a target nucleic acid of interest and the gRNA of RNP2 is specific to an unblocked nucleic acid or a synthesized activating molecule (both described in detail below). As will be clear given the description below, an advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (i.e., the gRNA specific to the target nucleic acid of interest), the cascade assay components can stay the same (i.e., are identical or substantially identical) no matter what target nucleic acid(s) of interest are being detected, and the gRNA in RNP1 is easily reprogrammable.
Like the nucleic acid-guided nuclease, the gRNA may be provided in the cascade assay reaction mix in a preassembled RNP, as an RNA molecule, or may also be provided as a DNA sequence to be transcribed, in, e.g., a vector backbone. Providing the gRNA in a pre-assembled RNP complex (i.e., RNP1 or RNP2) is preferred if rapid kinetics are preferred. If provided as a gRNA molecule, the gRNA sequence may include multiple endoribonuclease recognition sites (e.g., Csy4) for multiplex processing. Alternatively, if provided as a DNA sequence to be transcribed, an endoribonuclease recognition site may be encoded between neighboring gRNA sequences such that more than one gRNA can be transcribed in a single expression cassette. Direct repeats can also serve as endoribonuclease recognition sites for multiplex processing. Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementary to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest.
The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease of RNP1 to perform cis-cleavage of a target nucleic acid of interest (e.g., a DNA or RNA), which triggers non-sequence specific trans-cleavage of other molecules in the reaction mix. Guide RNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest (or target sequences generated by unblocking blocked nucleic acid molecules or target sequences generated by synthesizing synthesized activating molecules as described below). Target nucleic acids of interest (describe in detail above) preferably include a protospacer-adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target nucleic acid of interest.
In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistant circular molecule that can include several DNA bases between the 5′ end and the 3′ end of a natural guide RNA and is capable of binding a target sequence. The length of the circularized guide for RNP1 can be such that the circular form of guide can be complexed with a nucleic acid-guided nuclease to form a modified RNP1 which can still retain its cis-cleavage i.e., (specific) and trans-cleavage (i.e., non-specific) nuclease activity.
In any of the foregoing embodiments, the gRNA may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the cascade assay “reaction mix” may comprise separate nucleic acid-guided nucleases and gRNAs (or coding sequences therefor), the cascade assays preferably comprise preassembled ribonucleoprotein complexes (RNPs) in the reaction mix, allowing for faster detection kinetics. The present cascade assay employs at least two types of RNP complexes—RNP1 and RNP2—each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the same nucleic acid-guided nuclease or may comprise different nucleic acid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 μM of a given RNP1, or about 1 pM to about 1 μM of a given RNP1, or about 10 pM to about 500 pM of a given RNP1. In some embodiments the reaction mixture contains about 6×10⁴to about 6×10¹²complexes per microliter (μl) of a given RNP1, or about 6×10⁶to about 6×10¹⁰complexes per microliter (μl) of a given RNP1. In some embodiments, the reaction mixture contains about 1 fM to about 500 μM of a given RNP2, or about 1 pM to about 250 μM of a given RNP2, or about 10 pM to about 100 μM of a given RNP2. In some embodiments the reaction mixture contains about 6×10⁴to about 6×10¹²complexes per microliter (μl) of a given RNP2 or about 6×10⁶to about 6×10¹²complexes per microliter (μl) of a given RNP2. See Example II below describing preassembling RNPs and Examples V and VI below describing various cascade assay conditions where the relative concentrations of RNP2 and the blocked nucleic acid molecules is adjusted as described below.
In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,0000 or more RNP1s), where different RNPls comprise a different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for environmental or oncology testing comprises more than one unique RNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose of detecting more than one target nucleic acid of interest. That is, more than one RNP1 may also be present for the purpose of targeting one target nucleic acid of interest from many sources or for targeting more than one target nucleic acid of interest from a single source.
In any of the foregoing embodiments, the gRNA of RNP1 may be homologous or heterologous, relative to the gRNA of other RNP1(s) present in the reaction mixture. A homologous mixture of RNP1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP1 gRNAs has multiple gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and/or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twenty heterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty-four heterologous gRNAs, or more than twenty-five heterologous gRNAs. Such a heterologous mixture of RNP1 gRNAs in a single reaction enables multiplex testing.
As a first non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNPls (RNP1-1s) having a gRNA targeting parainfluenza virus 1; a number of RNP1s (RNP1-2s) having a gRNA targeting human metapneumovirus; a number of RNP1s (RNP1-3s) having a gRNA targeting human rhinovirus; a number of RNP1s (RNP1-4s) having a gRNA targeting human enterovirus; and a number of RNP1s (RNP1-5s) having a gRNA targeting coronavirus HKU1. As a second non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNPls containing a gRNA targeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariants thereof.
As another non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain RNP1s targeting two or more target nucleic acids of interest from organisms that infect grapevines, such as Guignardia bidwellii (RNP1-1), Uncinula necator (RNP1-2), Botrytis cincerea (RNP1-3), Plasmopara viticola (RNP1-4), and Botryotinis fuckleina (RNP1-5).

Reporter Moieties

The cascade assay detects a target nucleic acid of interest via detection of a signal generated in the reaction mix by a reporter moiety. In some embodiments the detection of the target nucleic acid of interest occurs virtually instantaneously. For example, see the results reported in Example VI for assays comprising 3e4 or 30 copies of MRSA target and within 1 minute or less at 3 copies of MRSA target (see, e.g., FIGS. 10B-10H). Reporter moieties can comprise DNA, RNA, a chimera of DNA and RNA, and can be single stranded, double stranded, or a moiety that is a combination of single stranded portions and double stranded portions.
Depending on the type of reporter moiety used, trans- and/or cis-cleavage by the nucleic acid-guided nuclease in RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone reporter moieties (e.g., not bound to any blocked nucleic acid molecules or blocked primer molecules) may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown in FIG. 1B and at top of FIG. 4 ). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal. In alternative embodiments and preferably, the reporter moiety may be bound to the blocked nucleic acid molecule, where trans-cleavage of the blocked nucleic acid molecule (or blocked primer molecule) and conversion to an unblocked nucleic acid molecule (or unblocked primer molecule) may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time, thus allowing for real time reporting of results (shown at FIG. 4 , center). In yet another embodiment, the reporter moiety may be bound to a blocked nucleic acid molecule such that cis-cleavage following the binding of the RNP2 to an unblocked nucleic acid molecule releases a PAM distal sequence, which in turn generates a signal at rates that are proportional to the cleavage rate (shown at FIG. 4 , bottom). In this case, activation of RNP2 by cis- (target specific) cleavage of the unblocked nucleic acid molecule directly produces a signal, rather than producing a signal via indiscriminate trans-cleavage activity. Alternatively or in addition, a reporter moiety may be bound to the gRNA.
The reporter moiety may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TaqMan probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The reporter and quencher may be about 20-30 bases apart or less (i.e., 10-11 nm apart or less) for effective quenching via fluorescence resonance energy transfer (FRET). Alternatively, signal generation may occur through different mechanisms. Other detectable moieties, labels, or reporters can also be used to detect a target nucleic acid of interest as described herein. Reporter moieties can be labeled in a variety of ways, including direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, or colorimetric moiety.
Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, and protein-protein binding pairs, e.g., protein-antibody binding pairs. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, and phycoerythrin. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers also include radioactive elements such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H. Reporters can also include a change in pH or charge of the cascade assay reaction mix.
The methods used to detect the generated signal will depend on the reporter moiety or moieties used. For example, a radioactive label can be detected using a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Fluorescent labels can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. When pairs of fluorophores are used in an assay, fluorophores are chosen that have distinct emission patterns (wavelengths) so that they can be easily distinguished. In some embodiments, the signal can be detected by lateral flow assays (LFAs). Lateral flow tests are simple devices intended to detect the presence or absence of a target nucleic acid of interest in a sample. LFAs can use nucleic acid molecules conjugated nanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detection probe, which hybridizes to a complementary target sequence. (See FIG. 9 and the description thereof below.) The classic example of an LFA is the home pregnancy test.
Single-stranded, double-stranded or reporter moieties comprising both single- and double-stranded portions can be introduced to show a signal change proportional to the cleavage rate, which increases with every new activated RNP2 complex over time. In some embodiments and as described in detail below, reporter moieties can also be embedded into the blocked nucleic acid molecules (or blocked primer molecules) for real time reporting of results.
For example, the method of detecting a target nucleic acid molecule in a sample using a cascade assay as described herein can involve contacting the reaction mix with a labeled detection ssDNA containing a fluorescent resonance energy transfer (FRET) pair, a quencher/phosphor pair, or both. A FRET pair consists of a donor chromophore and an acceptor chromophore, where the acceptor chromophore may be a quencher molecule. FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used. EDANS is (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid); DABCYL is 4-(4- dimethylaminophenyl) diazenylbenzoic acid. Useful quenchers include, but are not limited to, BHQ, DABCYL, QSY 7 and QSY 33.
In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, template molecules, synthesized activating molecules, and reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked nucleic acid molecules, gRNAs, template molecules, reporter moieties, and blocked primer molecules described herein are introduced to optimize the molecule's biophysical properties (e.g., increasing nucleic acid-guided nuclease resistance and/or increasing thermal stability). Modifications typically are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages.
For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, reporter molecules, synthesized activating molecules, and template molecules) may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in U.S. Pat. No. 3,687,808; Kroschwitz, ed., The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302.
In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2′ sugar modifications, including 2′-O-methyl (2′ -O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, and/or 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify the nucleic acid molecules described herein (i.e., blocked nucleic acid molecules, gRNAs, synthesized activating molecules, reporter molecules, and blocked primer molecules) may include all possible orientations of OH; F; O-, S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- or di-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Finally, modifications to the cascade assay components may comprise internucleoside modifications such as phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Signal Boosting Cascade Assay Employing Blocked Nucleic Acid Molecules

Before getting to the details relating to addressing undesired unwinding of the blocked nucleic acid molecules (or blocked primer molecules), understanding the cascade assay itself is key. FIG. 1B, described above, depicts the cascade assay generally. A specific embodiment of the cascade assay utilizing blocked nucleic acid molecules is depicted in FIG. 2A and described in detail below. In this embodiment, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method in FIG. 2A begins with providing the cascade assay components RNP1 (201), RNP2 (202) and blocked nucleic acid molecules (203). RNP1 (201) comprises a gRNA specific for a target nucleic acid of interest and a nucleic acid-guided nuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid of interest or a Cas 13a for an RNA target nucleic acid of interest) and RNP2 (202) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease (again, e.g., Cas 12a or Cas 14 for a DNA unblocked nucleic acid molecule or a Cas 13a for an RNA unblocked nucleic acid molecule). As described above, the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same or different depending on the type of target nucleic acid of interest and unblocked nucleic acid molecule. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following initiation of cis-cleavage activity.
In a first step, a sample comprising a target nucleic acid of interest (204) is added to the cascade assay reaction mix. The target nucleic acid of interest (204) combines with and activates RNP1 (205) but does not interact with or activate RNP2 (202). Once activated, RNP1 binds the target nucleic acid of interest (204) and cuts the target nucleic acid of interest (204) via sequence-specific cis-cleavage, activating non-specific trans-cleavage of other nucleic acids present in the reaction mix, including the blocked nucleic acid molecules (203). At least one of the blocked nucleic acid molecules (203) becomes an unblocked nucleic acid molecule (206) when the blocking moiety (207) is removed. As described below, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and/or structural modifications.
Once at least one of the blocked nucleic acid molecules (203) is unblocked, the unblocked nucleic acid molecule (206) can then bind to and activate an RNP2 (208). Because the nucleic acid-guided nucleases in the RNP1s (205) and RNP2s (208) have both cis- and trans-cleavage activity, the trans-cleavage activity causes more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering activation of even more RNP2s (208) and more trans-cleavage activity in a cascade. FIG. 2A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (205) and RNP2 (208). The intact reporter moieties (209) become activated reporter moieties (212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases rapidly as more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering cis-cleavage activity of more RNP2s (208) and thus more trans-cleavage activity of the reporter moieties (209). Again, the reporter moieties are shown here as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNA1), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected.
FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule (220) and an exemplary technique for unblocking the blocked nucleic acid molecules described herein. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (220) comprising a target strand (222) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (224) containing unhybridized and cleavable secondary loop structures (226) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNP1s or RNP2s, generates short strand nucleotide sequences or regions (228) which, because of the short length and low melting temperature T_mcan dehybridize at room temperature (e.g., 15°-25° C.), thereby unblocking the blocked nucleic acid molecule (220) to create an unblocked nucleic acid molecule (230), enabling the internalization of the unblocked nucleic acid molecule (230) (target strand) into an RNP2, leading to RNP2 activation.
A blocked nucleic acid molecule may be single-stranded or double-stranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleic acid molecules typically have a low binding affinity, or high dissociation constant (K_d) in relation to binding to RNP2 and may be referred to herein as a high K_dnucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_dvalues range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_dvalues are in the range of 100 nM to about 10-100 10 mM and thus are about 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold or higher as compared to low K_dvalues. Of course, the ideal blocked nucleic acid molecule would have an “infinite K_d.”
The blocked nucleic acid molecules (high K_dmolecules) described herein can be converted into unblocked nucleic acid molecules (low K_dmolecules—also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNP1s and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked nucleic acid molecule, although, as described below, there is some “leakiness” where some blocked nucleic acid molecules are able to interact with the gRNA in the RNP2 triggering undesired unwinding.
Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules, resulting in a positive feedback loop or cascade.
In embodiments where blocked nucleic acid molecules are linear and/or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments) or on separate nucleic acid molecules (e.g., for double-strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single-strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-cleavage activity of RNP2.
In some embodiments, the second nucleotide sequence at least partially hybridizes to the first nucleotide sequence, resulting in a secondary structure containing at least one loop (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Such loops block the nucleic acid molecule from binding or incorporating into an RNP complex thereby initiating cis- or trans-cleavage (see, e.g., the exemplary structures in FIGS. 2C-2F).
In some embodiments, the blocked nucleic acid molecule may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence, positioned between the first and second nucleotide sequences, where the first sequence is 5′ to the PAM sequence, or partial PAM sequence, (see FIG. 2G). Inclusion of a PAM sequence may increase the reaction kinetics internalizing the unblocked nucleic acid molecule into RNP2 and thus decrease the time to detection. In other embodiments, the blocked nucleic acid molecule does not contain a PAM sequence.
In some embodiments, the blocked nucleic acid molecules (i.e., high K_dnucleic acid molecules in relation to binding to RNP2) of the disclosure may include a structure represented by Formula I (e.g., FIG. 2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or Formula IV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3′ direction:
A-(B-L)J-C-M-T-D (Formula I);

- wherein A is 0-15 nucleotides in length;
- B is 4-12 nucleotides in length;
- L is 3-25 nucleotides in length;
- J is an integer between 1 and 10;
- C is 4-15 nucleotides in length;
- M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)J-C and T-D are separate nucleic acid strands;
- T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25) and comprises a sequence complementary to B and C; and
- D is 0-10 nucleotides in length and comprises a sequence complementary to A;

D-T-T′-C-(L-B)J-A (Formula II);

- wherein D is 0-10 nucleotides in length;
- T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
- T′ is 1-10 nucleotides in length and does not hybridize with T;
- C is 4-15 nucleotides in length and comprises a sequence complementary to T;
- L is 3-25 nucleotides in length and does not hybridize with T;
- B is 4-12 nucleotides in length and comprises a sequence complementary to T;
- J is an integer between 1 and 10;
- A is 0-15 nucleotides in length and comprises a sequence complementary to D;

T-D-M-A-(B-L)J-C (Formula III);

- wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
- D is 0-10 nucleotides in length;
- M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)J-C are separate nucleic acid strands;
- A is 0-15 nucleotides in length and comprises a sequence complementary to D;
- B is 4-12 nucleotides in length and comprises a sequence complementary to T;
- L is 3-25 nucleotides in length;
- J is an integer between 1 and 10; and
- C is 4-15 nucleotides in length;

T-D-M-A-Lp-C (Formula IV);

- wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25);
- D is 0-15 nucleotides in length;
- M is 1-25 nucleotides in length;
- A is 0-15 nucleotides in length and comprises a sequence complementary to D; and
- L is 3-25 nucleotides in length;
- p is 0 or 1;
- C is 4-15 nucleotides in length and comprises a sequence complementary to T.
In alternative embodiments of any of these molecules, T (or T-T′) can have a maximum length of 1000 nucleotides, e.g., at most 750, at most 500, at most 400, at more 300, at most 250, at most 200, at most 150, at most 135, at most 100, at most 75, at most 50, or at most 25 nucleotides.

Nucleotide mismatches can be introduced in any of the above structures containing double-strand segments (for example, where M is absent in Formula I or Formula III) to reduce the melting temperature (T_m) of the segment such that once the loop (L) is cleaved, the double-strand segment is unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary between 0% and 50%; however, the maximum number of nucleotide mismatches is limited to a number where the secondary loop structure still forms. “Segments” in the above statement refers to A, B, and C. In other words, the number of hybridized bases can be less than or equal to the length of each double-strand segment and vary based on number of mismatches introduced.
In any blocked nucleic acid molecule having the structure of Formula I, III, or IV, T will have sequence complementarity to a nucleotide sequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotide sequence of T is to be designed such that hybridization of T to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. In any blocked nucleic acid molecule having structure of Formula II, T-T′ will have sequence complementarity to a sequence (e.g., a spacer sequence) within the gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed such that hybridization of T-T′ to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. For T or T-T′, full complementarity to the gRNA is not necessarily required, provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of RNP2.
In any of the foregoing embodiments, the blocked nucleic acid molecules of the disclosure may and preferably do further contain a reporter moiety attached thereto such that cleavage of the blocked nucleic acid releases a signal from the reporter moiety. (See FIG. 4 , mechanisms depicted at center and bottom.)
Also, in any of the foregoing embodiments, the blocked nucleic acid molecule may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked nucleic acid molecules of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blocked nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof.
FIG. 2G at left shows an exemplary single-strand blocked nucleic acid molecule and how the configuration of this blocked nucleic acid molecule is able to prevent (or significantly prevent) undesired unwinding of the blocked nucleic acid molecule (or blocked primer molecule) and R-loop formation with an RNP complex, thereby blocking activation of the trans-cleavage activity of RNP2. The single-strand blocked nucleic acid molecule is self-hybridized and comprises: a target strand (TS) sequence complementary to the gRNA (e.g., crRNA) of RNP2; a cleavable non-target strand (NTS) sequence that is partially hybridized (e.g., it contains secondary loop structures) to the TS sequence; and a protospacer adjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that is specifically located at the 3′ end of the TS sequence. An RNP complex with 3′→5′ diffusion (e.g., 1D diffusion) initiates R-loop formation upon PAM recognition. R-loop formation is completed upon a stabilizing >17 base hybridization of the TS to the gRNA of RNP2; however, because of the orientation of the PAM sequence relative to the secondary loop structure(s), the blocked nucleic acid molecule sterically prevents the target strand from hybridizing with the gRNA of RNP2, thereby blocking the stable R-loop formation required for the cascade reaction.
FIG. 2G at right shows the blocked nucleic acid molecule being unblocked via trans-cleavage (e.g., by RNP1) and subsequent dehybridization of the non-target strand's secondary loop structures, followed by binding of the target strand to the gRNA of RNP2, thereby completing stable R-loop formation and activating the trans-cleavage activity of the RNP2 complex.
In some embodiments, the blocked nucleic acid molecules provided herein are circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), and the blocked nucleic acid molecules may include different base compositions depending on the Cas enzyme used for RNP1 and RNP2. For the circular design of blocked nucleic acid molecules, the 5′ and 3′ ends are covalently linked together. This configuration makes internalization of the blocked nucleic acid molecule into RNP2—and subsequent RNP2 activation—sterically unfavorable, thereby blocking the progression of the cascade assay. Thus, RNP2 activation (e.g., trans-cleavage activity) happens after cleavage of a portion of the blocked nucleic acid molecule followed by linearization and internalization of unblocked nucleic acid molecule into RNP2.
In some embodiments, the blocked nucleic acid molecules are topologically circular molecules with 5′ and 3′ portions hybridized to each other using DNA, RNA, LNA, BNA, or PNA bases which have a very high melting temperature (Tm). The high Tm causes the structure to effectively behave as a circular molecule even though the 5′ and 3′ ends are not covalently linked. The 5′ and 3′ ends can also have base non-naturally occurring modifications such as phosphorothioate bonds to provide increased stability.
In embodiments where the blocked nucleic acid molecules are circularized (e.g., circular or topologically circular), as illustrated in FIG. 2H, each blocked nucleic acid molecule includes a first region, which is a target sequence specific to the gRNA of RNP2, and a second region, which is a sequence that can be cleaved by nuclease enzymes of activated RNP1 and/or RNP2. The first region may include a nuclease-resistant nucleic acid sequence such as, for example, a phosphorothioate group or other non-naturally occurring nuclease-resistant base modifications, for protection from trans-nucleic acid-guided nuclease activity. In some embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence. In other embodiments, when the Cas enzyme in RNP1 is Cas12a and the Cas enzyme in RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In yet other embodiments, when the Cas enzyme in RNP1 is Cas13a and the Cas enzyme in RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In some other embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable RNA sequence.

The Signal Boosting Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acid molecules described above may also be blocked primer molecules. Blocked primer molecules include a sequence complementary to a primer binding domain (PBD) on a template molecule (see description below in reference to FIGS. 3A and 3B) and can have the same general structures as the blocked nucleic acid molecules described above. A PBD serves as a nucleotide sequence for primer hybridization followed by primer polymerization by a polymerase. In any of Formulas I, II, or III described above, the blocked primer nucleic acid molecule may include a sequence complementary to the PBD on the 5′ end of T. The unblocked primer nucleic acid molecule can bind to a template molecule at the PBD and copy the template molecule via polymerization by a polymerase.
Specific embodiments of the cascade assay which utilize blocked primer molecules and are depicted in FIGS. 3A and 3B. In the embodiments using blocked nucleic acid molecules described above, activation of RNP1 by binding of N nucleotides of the target nucleic acid molecules or cis-cleavage of the target nucleic acid molecules initiates trans-cleavage of the blocked nucleic acid molecules which were used to activate RNP2—that is, the unblocked nucleic acid molecules are a target sequence for the gRNA in RNP2. In contrast, in the embodiments using blocked primers activation of RNP1 and trans-cleavage unblocks a blocked primer molecule that is then used to prime a template molecule for extension by a polymerase, thereby synthesizing synthesized activating molecules that are the target sequence for the gRNA in RNP2.
FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules. At left of FIG. 3A is a cascade assay reaction mix comprising 1) RNP 1 s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) linear template molecules (330) (which is the non-target strand); 4) a circular blocked primer molecule (334) (i.e., a high K_dmolecule); and 5) a polymerase (338), such as a 129 polymerase. The linear template molecule (330) (non-target strand) comprises a PAM sequence (331), a primer binding domain (PBD) (332) and, optionally, a nucleoside modification (333) to protect the linear template molecule (330) from 3′→5′ exonuclease activity. Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the linear template molecule (330).
Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) is bound by with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334) (i.e., a high K_dmolecule, where high K_drelates to binding to RNP2) upon cleavage becomes an unblocked linear primer molecule (344) (a low K_dmolecule, where low K_drelates to binding to RNP2), which has a region (336) complementary to the PBD (332) on the linear template molecule (330) and can bind to the linear template molecule (330).
Once the unblocked linear primer molecule (344) and the linear template molecule (330) are hybridized (i.e., hybridized at the PBD (332) of the linear template molecule (330) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the end of the unblocked primer molecule (344) and the polymerase (338) can copy the linear template molecule (330) to produce a synthesized activating molecule (346) which is a complement of the non-target strand, which is the target strand. The synthesized activating molecule (346) is capable of activating RNP2 (302→308). As described above, because the nucleic acid-guided nuclease in the RNP2 (308) complex exhibits (that is, possesses) both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. As stated above in relation to blocked and unblocked nucleic acid molecules (both linear and circular), the unblocked primer molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked primer molecule, although there may be some “leakiness” where some blocked primer molecules are able to interact with the gRNA in RNP2. However, an unblocked primer molecule has a substantially higher likelihood than a blocked primer molecule to hybridize with the gRNA of RNP2.
FIG. 3A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorophore emits a fluorescent signal (313). Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating molecules (346) and triggering activation of more RNP2 (308) complexes and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also, as with the cascade assay embodiment utilizing blocked nucleic acid molecules that are not blocked primers, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.
FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules. The cascade assay of FIG. 3B differs from that depicted in FIG. 3A by the configuration of the template molecule. Where the template molecule in FIG. 3A was linear, in FIG. 3B the template molecule is circular. At left of FIG. 3B is a cascade assay reaction mix comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) a circular template molecule (352) (non-target strand); 4) a circular blocked primer molecule (334); and 5) a polymerase (338), such as a Φ29 polymerase. The circular template molecule (352) (non-target strand) comprises a PAM sequence (331) and a primer binding domain (PBD) (332). Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the circular template molecule (352).
Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) binds to and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334), upon cleavage, becomes an unblocked linear primer molecule (344), which has a region (336) complementary to the PBD (332) on the circular template molecule (352) and can hybridize with the circular template molecule (352).
Once the unblocked linear primer molecule (344) and the circular template molecule (352) are hybridized (i.e., hybridized at the PBD (332) of the circular template molecule (352) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the 3′ end of the unblocked primer molecule (344). The polymerase (338) can now use the circular template molecule (352) (non-target strand) to produce concatenated activating nucleic acid molecules (360) (which are concatenated target strands), which will be cleaved by the trans-cleavage activity of activated RNP1. The cleaved regions of the concatenated synthesized activating molecules (360) (target strand) are capable of activating the RNP2 (302→308) complex.
As described above, because the nucleic acid-guided nuclease in RNP2 (308) comprises both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. FIG. 3B at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorescent signal (313) is unquenched and can be detected. Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating nucleic acid molecules and triggering activation of more RNP2s (308) and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also note that as with the other embodiments of the cascade assay, in this embodiment, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.
The polymerases used in the “blocked primer molecule” embodiments serve to polymerize a reverse complement strand of the template molecule (non-target strand) to generate a synthesized activating molecule (target strand) as described above. In some embodiments, the polymerase is a DNA polymerase, such as a BST, T4, or Therminator polymerase (New England BioLabs Inc., Ipswich Mass., USA). In some embodiments, the polymerase is a Klenow fragment of a DNA polymerase. In some embodiments the polymerase is a DNA polymerase with 5′→3′ DNA polymerase activity and 3′→5′ exonuclease activity, such as a Type I, Type II, or Type III DNA polymerase. In some embodiments, the DNA polymerase, including the Phi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent® DNA polymerases (New England BioLabs Inc., Ipswich Mass., USA), or any active portion or variant thereof. Also, a 3′ to 5′ exonuclease can be separately used if the polymerase lacks this activity.
FIG. 4 depicts three mechanisms in which a cascade assay reaction can release a signal from a reporter moiety. FIG. 4 at top shows the mechanism discussed in relation to FIGS. 2A, 3A and 3B. In this embodiment, a reporter moiety 409 is a separate molecule from the blocked nucleic acid molecules present in the reaction mix. Reporter moiety (409) comprises a quencher (410) and a fluorophore (411). An activated reporter moiety (412) emits a signal from the fluorophore (411) once it has been physically separated from the quencher (410).

Reporter Moiety Configurations

FIG. 4 at center shows a blocked nucleic acid molecule (403), which is also a reporter moiety. In addition to quencher (410) and fluorophore (411), a blocking moiety (407) can be seen (see also blocked nucleic acid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reporter moiety (403) comprises a quencher (410) and a fluorophore (411). In this embodiment of the cascade assay, when the blocked nucleic acid molecule (403) is unblocked due to trans-cleavage initiated by the target nucleic acid of interest binding to RNP1, the unblocked nucleic acid molecule (406) also becomes an activated reporter moiety with fluorophore (411) separated from quencher (410). Note both the blocking moiety (407) and the quencher (410) are removed. In this embodiment, reporter signal is directly generated as the blocked nucleic acid molecules become unblocked. Embodiments of this schema can be used to supply the bulky modifications to the blocked nucleic acid molecules described below.
FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleic acid molecule or a synthesized activating molecule at a PAM distal sequence by RNP2 generates a signal. Shown are activated RNP2 (408), unblocked nucleic acid molecule (461), quencher (410), and fluorophore (411) forming an activated RNP2 with the unblocked nucleic acid/reporter moiety intact (460). Cis-cleavage of the unblocked nucleic acid/reporter moiety (461) results in an activated RNP2 with the reporter moiety activated (462), comprising the activated RNP2 (408), the unblocked nucleic acid molecule with the reporter moiety activated (463), quencher (410) and fluorophore (411). Embodiments of this schema also can be used to supply the bulky modifications to the blocked nucleic acid molecules described below, and in fact a combination of the configurations of reporter moieties shown in FIG. 4 at center and at bottom may be used.

Preventing Undesired Blocked Nucleic Acid Molecule Unwinding

The present disclosure improves upon the signal cascade assay described in U.S. Ser. Nos. 17/861,207; 17/861,208; and 17/861,209 by addressing the problem with undesired “unwinding” of the blocked nucleic acid molecule. As described above in detail in relation to FIGS. 1B, 2A, 2B, 2G, 3A, 3B, and 4 , the cascade assay is initiated when a target nucleic acid of interest binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1). The gRNA of RNP1 (gRNA1), comprising a sequence complementary to the target nucleic acid of interest, guides RNP1 to the target nucleic acid of interest. Upon binding of the target nucleic acid of interest to RNP1, RNP1 becomes activated, and the target nucleic acid of interest is cleaved in a sequence specific manner (i.e., cis-cleavage) while also triggering non-sequence specific, indiscriminate trans-cleavage activity which unblocks the blocked nucleic acid molecules in the reaction mix. The unblocked nucleic acid molecules can then activate a second pre-assembled ribonucleoprotein complex (RNP2), where RNP2 comprises a second gRNA (gRNA2) comprising a sequence complementary to the unblocked nucleic acid molecules, and at least one of the unblocked nucleic acid molecules is cis-cleaved in a sequence specific manner. Binding of the unblocked nucleic acid molecule to RNP2 leads to cis-cleavage of the unblocked nucleic acid molecule and non-sequence specific, indiscriminate trans-cleavage activity by RNP2, which in turn unblocks more blocked nucleic acid molecules (and reporter moieties) in the reaction mix activating more RNP2s. Each newly activated RNP2 activates more RNP2s, which in turn cleave more blocked nucleic acid molecules and reporter moieties in a reaction cascade, where all or most of the signal generated comes from the trans-cleavage activity of RNP2.
The improvement to the signal boost cascade assay described herein is drawn to preventing undesired unwinding of the blocked nucleic acid molecules in the reaction mix before the blocked nucleic acid molecules are unblocked via trans-cleavage; that is, preventing undesired unwinding that happens not as a result of unblocking due to trans-cleavage subsequent to cis-cleavage of the target nucleic acid of interest or trans-cleavage of unblocked nucleic acid molecules, but due to other factors. For a description of undesired unwinding, please see FIG. 1C and the attendant description herein. Minimizing undesired unwinding serves two purposes. First, preventing undesired unwinding that happens not as a result of designed or engineered unblocking leads to a “leaky” cascade assay system, which in turn leads to non-specific signal generation and false positives.
Second, preventing undesired unwinding limits non-specific interactions between the nucleic acid-guided nucleases (here, the RNP2s) and blocked nucleic acid molecules (i.e., the target nucleic acids for RNP2) such that only blocked nucleic acid molecules that become unblocked due to trans-cleavage activity react with the nucleic acid-guided nucleases. This “fidelity” in the cascade assay leads primarily to desired interactions and limits “wasteful” interactions where the nucleic acid-guided nucleases are essentially interacting with blocked nucleic acid molecules rather than interacting with unblocked nucleic acid molecules. That is, if unwinding is minimized the nucleic acid-guided nucleases are focused on desired interactions which then leads to immediate signal generation in the cascade assay. Preventing undesired unwinding leads to a more efficient cascade assay system providing more accurate quantification yet with the rapid results characteristic of the cascade assay (see FIGS. 10A-10H and 12 below).

Ratio of RNP2 to Blocked Nucleic Acid Molecules or Blocked Primers

In one modality to prevent undesired unwinding, the present disclosure describes using an unconventional ratio of blocked nucleic acid molecule (i.e., the target molecule for RNP2) and an RNP complex, here RNP2. The unconventional ratio may be used along with the blocked nucleic acid molecules and RNP2s described above as a primary method for minimizing unwinding or may be used in combination with the other modalities described below to minimize unwinding even more. For example, if one were to design an ideal blocked nucleic acid molecule having an “infinite K_d” such as, e.g., through design of the blocked nucleic acid molecule (or blocked primer molecule) and/or inclusion of bulky modifications on the blocked nucleic acid molecule (or blocked primer molecule), the ratio of blocked nucleic acid molecules to RNP2s would not affect the reaction mix to any discernable degree. The common wisdom of the ratio of enzyme to target (here, RNP2 to blocked nucleic acid molecule) is that results are achieved—a signal is generated—when there is a high concentration of nucleic acid-guided nuclease (i.e., RNP complex) and a lower concentration of target or, stated another way, when there is a significant excess of nucleic acid-guided nuclease to target. As described above, in CRISPR detection/diagnostic assay protocols known to date, the CRISPR enzyme (i.e., nucleic acid-guided nuclease) is far in excess of blocked nucleic acid molecules (see, Sun, et al., J. of Translational Medicine, 12:74 (2021); Broughton, et al., Nat. Biotech., 38:870-74 (2020); and Lee, et al., PNAS, 117(41):25722-31 (2020)). However, in a cascade assay system where the nucleic acid-guided nuclease (or RNP complex) is in excess of the targets (here, the blocked nucleic acid molecules), the nucleic acid-guided nucleases encounter the blocked nucleic acid molecules repeatedly, probing the blocked nucleic acid molecules and subjecting them to unwinding. If the blocked nucleic acid molecules are probed and unwound repeatedly, they finally unwind which then triggers activation of RNP2 and cis-cleavage of the blocked nucleic acid molecule even in the absence of a target nucleic acid of interest and the trans-cleavage activity generated thereby.
However, by adjusting the ratio of RNP2 to blocked nucleic acid molecules such that there is an excess of blocked nucleic acid molecules to RNP2, any one blocked nucleic acid molecule may be probed by RNP2; however, the likelihood that any one blocked nucleic acid molecule will be probed repeatedly (and thus unwound) is much lower. If a blocked nucleic acid molecule is probed but then has time to re-hybridize or “recover”, that blocked nucleic acid molecule will stay blocked, will not be subject to non-specific unwinding, and will not trigger activation of RNP2. That is, how often any one blocked nucleic acid molecule is probed is important. As long as an improperly probed blocked nucleic acid has time to re-hybridize after unwinding, there is far less chance that the blocked nucleic acid will be unblocked (i.e., unwound) and will trigger signal generation. That is, preventing non-specific unwinding of the blocked nucleic acid molecules makes the nucleic acid-guided nuclease available for desired unwinding interactions.
In order to prevent non-specific unwinding as described herein, the ratio of blocked nucleic acid molecules to RNP2 should be about 50:1, or about 40:1, or about 35:1, or about 30:1, or about 25:1, or about 20:1, or about 15:1, or about 10:1, or about 7.5:1, or about 5:1, or about 4:1, or about 3:1, or about 2.5:1, or about 2:1, or about 1.5:1, or at least where the molar concentration of blocked nucleic acid molecules is equal to or greater than the molar concentration of RNP2s. As noted above, the signal amplification cascade assay reaction mixture typically contains about 1 fM to about 1 mM of a given RNP2, or about 1 pM to about 500 μM of a given RNP2, or about 10 pM to about 100 μM of a given RNP2; thus, the signal amplification cascade assay reaction mixture typically contains about 2.5 fM to about 2.5 mM blocked nucleic acid molecules, or about 2.5 pM to about 1.25 mM blocked nucleic acid molecules, or about 25 pM to about 250 μM blocked nucleic acid molecules. That is, the reaction mixture contains about 6×10⁴to about 6×10¹⁴RNP2s per microliter (μl) or about 6×10⁶to about 6×10¹²RNP2s per microliter (μl) and thus about 6×10⁴to about 6×10¹⁴RNP2s per microliter (μl) or about 6×10⁶to about 6×10¹²blocked nucleic acid molecules per microliter (μl). Note, the ratios may be used along with the blocked nucleic acid molecules and RNP2s described above as a primary method for minimizing unwinding or the ratios of blocked nucleic acid molecules to RNP2s may be used in combination with the other modalities described below to further minimize unwinding. Again, if one were to design an ideal blocked nucleic acid molecule having an “infinite K_d”, the ratio of blocked nucleic acid molecules to RNP2s would not affect the reaction mix to any discernable degree and the ratios of blocked nucleic acid molecules to RNP2s would not necessarily be within these ranges.
Variant Engineered Nucleic Acid-Guided Nucleases
In some embodiments, the protein sequence of the Cas12a nucleic acid-guided nuclease is modified, with e.g., mutations to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules (see Shin et al., Front. Genet., 11:1577 (2021); doi: 10.3389/fgene.2020.571591, herein incorporated by reference; and Yamano et al., Mol. Cell, 67(4): 633-645 (2017); doi: 10.1016/j.molcel.2017.06.035, herein incorporated by reference) such that the variant engineered nucleic acid-guided nuclease has reduced (or absent) PAM specificity, relative to the unmodified or wildtype nucleic acid-guided nuclease and reduced cleavage activity in relation to double strand DNA with or without a PAM. Such enzymes are referred to herein as single-strand-specific Cas12a nucleic acid-guided nucleases or variant engineered nucleic acid-guided nucleases.
FIG. 5 is a simplified block diagram of an exemplary method 500 for designing, synthesizing and screening variant nucleic acid-guided nucleases. In a first step, mutations or modifications to a nucleic acid-guided nuclease are designed 502, based on, e.g., homology to related nucleic acid-guided nucleases, predicted protein structure and active site configuration, and mutagenesis modeling. For assessment of homologies to other nucleic acid-guided nucleases, amino acid sequences may be found in publicly available databases known to those with skill in the art, including, e.g., Protein DataBank Europe (PDBe), Protein Databank Japan (PDBj), SWISS-PROT, GenBank, RefSeq, TrEMBL, PROSITE, DisProt, InterPro, PIR-International, and PRF/SEQDB. Amino acid homology alignments for purposes of determining similarities to known nucleic acid-guided nucleases can be performed using CUSTALW, CUSTAL OMEGA, COBALT: Multiple Alignment Tool; SIM; and PROBCONS.
For protein engineering and amino acid substitution model predictions for each of the desired mutations, protein modeling software such as SWISS-MODEL, HHpred, I-TASSER, IntFOLD, RaptorX, FoldX, Rosetta, and trRosetta may be used to simulate the structural change(s) and to calculate various parameters due to the structural changes as a result of the amino acid substitution(s), including root mean square deviation (RMSD) value in Angstrom units (i.e., a measurement of the difference between the backbones of the initial nucleic acid-guided nuclease and the mutated nucleic acid nucleic acid-guided nuclease) and changes to the number of hydrogen bonds and conformation in the active site. For the methods used to generate the variant engineered nucleic acid-guided nucleases described herein, see Example VII below.
Following modelling, coding sequences for the variant nucleic acid-guided nucleases that appear to deliver desired properties are synthesized and inserted into an expression vector 504. Methods for site-directed mutagenesis are known in the art, including PCR-based methods such as traditional PCR, where primers are designed to include the desired change; primer extension, involving incorporating mutagenic primers in independent nested PCR before combining them in the final product; and inverse PCR. Additionally, CRISPR gene editing may be performed to introduce the desired mutation or modification to the nucleic acid-guided nuclease coding sequence. The mutated (variant) coding sequences are inserted into an expression vector backbone comprising regulatory sequences such as enhancer and promoter regions. The type of expression vector (e.g., plasmid or viral vector) will vary depending on the type of cells to be transformed.
At step 506, cells of choice are transformed with the variant expression vectors. A variety of delivery systems may be used to introduce (e.g., transform or transfect) the expression vectors into a host cell, including the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Once cells are transformed (or transfected), the transformants are allowed to recover and grow.
Following transformation, the cells are screened for expression of nucleic acid-guided nucleases with desired properties 508, such as cut activity or lack thereof, paste activity or lack thereof, PAM recognition or changes thereto, stability and the ability to form RNPs at various temperatures, and/or cis- and trans-cleavage activity at various temperatures. The assays used to screen the variant nucleic acid-guided nucleases will vary depending on the desired properties, but may include in vitro and in vivo PAM depletion, assays for editing efficiency such as a GFP to BFP assay, and, as used to assess the variant nucleic acid-guided nucleases described herein, in vitro transcription/translation (IVTT) assays were used to measure in vitro trans cleavage with both dsDNA and ssDNA and with and without the presence of a PAM in the blocked nucleic acid molecules, where dsDNA should not activate trans-cleavage regardless of the presence of PAM sequence.
After screening the variant nucleic acid-guided nucleases via the IVTT assays, variants with the preferred properties are identified and selected 510. At this point, a variant may be chosen 512 to go forward into production for use in, e.g., the CRISPR cascade systems described herein; alternatively, promising mutations and/or modifications may be combined 514 and the construction, screening and identifying process is repeated.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease may not recognize one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN wherein “A” represents adenine, “C” represents cytosine, “T” represents thymine, “G” represents guanine, “V” represents guanine or cytosine or adenine, “Y” represents guanine or adenine, and “N” represents any nucleotide. In some embodiments, the Cas12a nucleic acid-guided nuclease may have reduced recognition for one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN. The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may have at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, such as about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) reduced recognition (i.e., specificity) for one or more of the following PAM or partial PAM sequences (listed from 5′ to 3′) : TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN.
Exemplary wild type (WT) Cas12a protein sequences are described in Table 7 below. FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacterium Cas12a), and FIG. 6B shows the result of example mutations on the LbCas12a protein structure prediction using Rosetta and SWISS modeling of LbCas12a and indicating the PAM regions (described in more detail in relation to Example VII). Any of these sequences (e.g., SEQ ID NOs: 1-15 and homologs or orthologs thereof) may be modified, as described herein, to generate a single-strand-specific nucleic acid-guided nuclease.

TABLE 7

Exemplary wild type Cas12a nucleic acid-guided nucleases

Species	SEQ
Name	ID
Reference ID	NO:	Protein Sequence

Lachnospiraceae	SEQ	MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAED
bacterium Cas12a	ID	YKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENK
(LbCas12a)	NO: 1	ELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIAL
PDD: 6KL9_A		VNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNM
		DIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGI
		DVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQV
		LSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKN
		FDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKK
		KAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQ
		KVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKS
		FENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYV
		TQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYL
		AIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSK
		KWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISR
		YPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEV
		DKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHG
		QIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLS
		YDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPY
		VIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSL
		LDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAV
		IALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPC
		ATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVN
		LLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADY
		IKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGI
		NYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDF
		LISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAI
		GQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH

Acidaminococcus	SEQ	MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
sp. Cas12a	ID	YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR
(AsCas12a)	NO: 2	NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN
NCBI Ref.:		GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI
WP_021736722.1		STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFV
		STSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
		NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVI
		QSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSAL
		CDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIIS
		AAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQL
		DSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
		RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNG
		LYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIP
		KCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKK
		FQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP
		SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY
		NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP
		KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLS
		HDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAA
		NSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
		LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVI
		HEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLID
		KLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVP
		APYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGD
		FILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGK
		RIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN
		DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDS
		RFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN
		QDWLAYIQELRN

Candidatus	SEQ	MNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEK
Methanoplasma	ID	YKILKEAIDEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDK
termitum	NO: 3	KVFLSEQKRMRQEIVSEFKKDDRFKDLFSKKLFSELLKEEIYKKGN
(CtCas12a)		HQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEITAISNRIVNENFP
NCBI Gene ID:		KFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNK
24818655		VLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKG
		RIHMTPLFKQILSEKESFSYIPDVFTEDSQLLPSIGGFFAQIENDKDG
		NIFDRALELISSYAEYDTERIYIRQADINRVSNVIFGEWGTLGGLMR
		EYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNNDA
		FNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQ
		FLHFFNLFKARQDIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLT
		KNNLNTKKIKLNFKNPTLANGWDQNKVYDYASLIFLRDGNYYLGI
		INPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVFLTSTKG
		KKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDW
		SKFNFYFSPTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKG
		DLFLFQIYNKDFVKAATGKKDMHTIYWNAAFSPENLQDVVVKLN
		GEAELFYRDKSDIKEIVHREGEILVNRTYNGRTPVPDKIHKKLTDY
		HNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLT
		LNFKANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSG
		KIIDQQSLNVIDGFDYREKLNQREIEMKDARQSWNAIGKIKDLKEG
		YLSKAVHEITKMAIQYNAIVVMEELNYGFKRGRFKVEKQIYQKFE
		NMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQTGI
		LFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSA
		KDGGIFAFAFDYRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRN
		ELFDPSKEIKEALTSSGIKYDGGQNILPDILRSNNNGLIYTMYSSFIA
		AIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDADANGAYNI
		ALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGD

Eubacterium	SEQ	MNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDEL
eligens	ID	RQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSP
(EeCas12a)	NO: 4	SKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLLKEILPDFI
NCBI Gene ID:		KNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVS
41356122		TSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGD
		WELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNN
		YNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETE
		KGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGC
		VENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDE
		KERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEM
		KKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVP
		LYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILI
		RDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGA
		NKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLI
		DYFKNSIEKHAEWRKYEFKFSATDSYSDISEFYREVEMQGYRIDW
		TYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFS
		EENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQL
		DNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRT
		AQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQN
		DDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVE
		KEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLIVEYNAIIAM
		EDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDEPGG
		LLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNF
		KSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGK
		TQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYA
		DGHDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNSYTEAEEQE
		NGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYC
		IALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE

Moraxella	SEQ	MLFQDFTHLYPLSKTVRFELKPIGKTLEHIHAKNFLNQDETMADM
bovoculi Cas12a	ID	YQKVKAILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKD
(Mb3Cas12a)	NO: 5	DGLQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDG
GenBank:		KELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMY
AKG12737.1		SDEDKHTAIAYRLIHENLPRFIDNLQILATIKQKHSALYDQIINELTA
		SGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSRKIQGINE
		LINSHHNQHCHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEV
		CQAVNEFYRHYADVFAKVQSLFDGFDDYQKDGIYVEYKNLNELS
		KQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTK
		EKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLA
		GVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSDKSPEIRQLKEL
		LDNALNVAHFAKLLTTKTTLHNQDGNFYGEFGALYDELAKIATLY
		NKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQK
		DGCYYLALLDKAHKKVFDNAPNTGKSVYQKMIYKLLPGPNKMLP
		KVFFAKSNLDYYNPSAELLDKYAQGTHKKGDNFNLKDCHALIDFF
		KAGINKHPEWQHFGFKFSPTSSYQDLSDFYREVEPQGYQVKFVDIN
		ADYINELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDN
		LVNPIYKLNGEAEIFYRKASLDMNETTIHRAGEVLENKNPDNPKKR
		QFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSI
		QQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGT
		QMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQIS
		QLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHL
		VLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTS
		KIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADRGYFEFHIDY
		AKFNDKAKNSRQIWKICSHGDKRYVYDKTANQNKGATIGVNVND
		ELKSLFTRYHINDKQPNLVMDICQNNDKEFHKSLMYLLKTLLALR
		YSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIA
		LKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR

Francisella	SEQ	MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY
novicida Cas12a	ID	KKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL
(FnCas12a)	NO: 6	QKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL
UniProtKB/Swiss-		WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK
Prot: A0Q7Q2.1		NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK
		KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN
		TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQIL
		SDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSL
		LFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYIT
		QQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI
		DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQ
		ASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFY
		LVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANG
		WDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGE
		GYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTK
		NGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQR
		YNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDF
		SAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIP
		KKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPIT
		INFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDG
		KGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNI
		KEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQ
		VYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKK
		MGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFD
		KICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDK
		NHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFA
		KLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMP
		QDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQ
		NRNN

Francisella	SEQ	MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY
tularensis subsp.	ID	KKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL
novicida FTG	NO: 7	QKDFKSAKDTIKKQISKYINDSEKFKNLFNQNLIDAKKGQESDLIL
Cas12a		WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK
(FnoCas12a)		NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK
NCBI Gene ID:		KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN
60806594		TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQIL
		SDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSL
		LFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYIT
		QQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKHRD
		IDKQCRFEEILSNFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLL
		QASAEEDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHF
		YLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLASG
		WDKNKESANTAILFIKDDKYYLGIMDKKHNKIFSDKAIEENKGEG
		YKKIVYKQIADASKDIQNLMIIDGKTVCKKGRKDRNGVNRQLLSL
		KRKHLPENIYRIKETKSYLKNEARFSRKDLYDFIDYYKDRLDYYDF
		EFELKPSNEYSDFNDFTNHIGSQGYKLTFENISQDYINSLVNEGKLY
		LFQIYSKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAE
		LFYRKQSIPKKITHPAKETIANKNKDNPKKESVFEYDLIKDKRFTED
		KFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLA
		YYTLVDGKGNIIKQDNFNIIGNDRMKTNYHDKLAAIEKDRDSARK
		DWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRG
		RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTA
		PFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKS
		QEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRL
		INFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICG
		ESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDS
		RQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIK
		NEEYFEFVQNRNN

Flavobacteriales	SEQ	MKNNNMLNFTNKYQLSKTLRFELKPIGKTKENIIAKNILKKDEERA
bacterium	ID	ESYQLMKKTIDGFHKHFIELAMQEVQKTKLSELEEFAELYNKSAEE
(FbCas12a)	NO: 8	KKKDDKFDDKFKKVQEALRKEIVKGFNSEKVKYYYSNIDKKILFT
NCBI Gene ID:		ELLKNWIPNEKMITELSEWNAKTKEEKEHLVYLDKEFENFTTYFG
MBE7442138.1		GFHKNRENMYTDKEQSTAIAYRLIHENLPKFLDNINIYKKVKEIPV
		LREECKVLYKEIEEYLNVNSIDEVFELSYYNKTLTQKDIDVYNLIIG
		GRTLEEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQILSDRDS
		ISWLPESFEDDNEKTASQKVLEAINLYYRDNLLCFQPKDKKDTENV
		LEETKKLLAGLSTSDLSKIYIRNDRAITDISQALFKDYGVIKDALKF
		QFIQSFTIGKNGLSKKQEEAIEKHLKQKYFSIAEIENALFTYQSETDA
		LKELKENSHPVVDYFINHFKAKKKEETDKDFDLIANIDAKYSCIKG
		LLNTPYPKDKKLYQRSKGDNDIDNIKAFLDALMELLHFVKPLALS
		NDSTLEKDQNFYSHFEPYYEQLELLIPLYNKVRNFAAKKPYSTEKF
		KLNFDNATLLNGWDKNKETDNTSVILRKDGLYYLAIMPQDNKNV
		FKDSPDLKANENCFEKMDYKQMALPMGFGAFVRKCFGTASQLG
		WNCPESCKNEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYK
		EYGFDFKESNKYESLREFFIDVEQQGYKITFQNISENYINQLVEDGK
		LYLFQIYNKDFSPYSKGKPNMHTMYWKALFDSENLKDVVYKLNG
		QAEVFYRKKSIEQKNIVTHKANEPIDNKNPKAKKKQSTFEYDLIKD
		KRYTVDKFQFHVPITLNFKATGNDYINQDVLTYLKNNPEVNIIGLD
		RGERHLIYLTLINQKGEILLQESLNTIVNKKYDIETPYHTLLQNKED
		ERAKARENWGVIENIKELKEGYISQVVHKIAKLMVEYNAIVVMED
		LNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVFKDKDPSEVGGL
		YHALQLTNKFENFSKIGKQSGFLFYVPAWNTSKIDPTTGFVNLFNT
		KYESVPKAQEFFKKFKSIKFNSAENYFEFAFDYNDFTTRAEGTKTD
		WIVCTYGDRIKTFRNPDKVNQWDNQEVNLTEQFEDFFGKNNLIYG
		DGNCIKNQIILHDKKEFFEGLLHLLKLTLQMRNSITNSEVDYLISPV
		KNNKGEFYDSRKANNTLPKDADANGAYHIAKKGLVLLNRLKENE
		VEEFEKSKKVKDGKSQWLPNKDWLDFVQRNVEDMVVV

Lachnospira	SEQ	MNGNRSIVYREFVGVTPVAKTLRNELRPVGHTQEHIIQNGLIQEDE
eligens	ID	LRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSS
(Lb4Cas12a)	NO: 9	PSKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLFKEILPDFI
NCBI Gene ID:		KNYNQYDVKDKAGKLETVALFNGFSTYFTDFFEKRKNVFTKEAV
MBS6299380.1		STSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGD
		WELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTRNN
		YNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETE
		KGNIIVKLKDIVNKYDELDEKRIYISKDFYETLSCFISGNWNLITGC
		VENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDE
		KERNEFKNSNAKQYIREISNIITDTETAHLEYDEHISLIESEEKADEM
		KKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVP
		LYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILI
		RDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGA
		NKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLI
		DYFKNSIEKHAEWRKYEFKFSATDSYNDISEFYREVEMQGYRIDW
		TYISEADINKLDEEGKIYLFQIYNKYFAENSTGKENLHTMYFKNIFS
		EENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQL
		DNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRT
		AQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMAVKYIAQN
		DDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVE
		KEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLMVEYNAIIA
		MEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKGKSVDEP
		GGLLRGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAF
		NFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITM
		GKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLKDNKIN
		YADGHDVRIDMEKMDEDKNSEFFAQLLSLYKLTVQMRNSYTEAE
		EQEKGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANG
		AYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRY
		E

Moraxella	SEQ	MLFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMADMY
bovoculi	ID	QKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDD
(MbCas12a)	NO:	GLQKQLKDLQAVLRKESVKPIGSGGKYKTGYDRLFGAKLFKDGK
NCBI Gene ID:	10	ELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYS
WP_046697655.1		DEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTAS
		GLDVSLASHLDGYHKLLTQEGITAYNRIIGEVNGYTNKHNQICHKS
		ERIAKLRPLHKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYT
		DVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGR
		VLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSL
		ASLEQAIEHHTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNN
		HSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNV
		AHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYL
		SQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLA
		LLDKAHKKVFDNAPNTGKNVYQKMVYKLLPGPNKMLPKVFFAK
		SNLDYYNPSAELLDKYAKGTHKKGDNFNLKDCHALIDFFKAGINK
		HPEWQHFGFKFSPTSSYRDLSDFYREVEPQGYQVKFVDINADYIDE
		LVEQGKLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIY
		KLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYD
		IIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDE
		VNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQVTTP
		YHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQINQLMLK
		YNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDK
		ADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPET
		GFVDLLKPRYENIAQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTD
		KAKNSRQKWAICSHGDKRYVYDKTANQNKGAAKGINVNDELKS
		LFARYHINDKQPNLVMDICQNNDKEFHKSLMCLLKTLLALRYSNA
		SSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGL
		WLLNELKNSDDLNKVKLAIDNQTWLNFAQNR

Prevotella bryantii	SEQ	MKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDQHRADSY
(Pb2Cas12a)	ID	KKVKKIIDEYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKR
NCBI Gene ID:	NO:	IEKTEKDKFAKIQDNLRKQIADHLKGDESYKTIFSKDLIRKNLPDFV
WP_039871282.1	11	KSDEERTLIKEFKDFTTYFKGFYENRENMYSAEDKSTAISHRIIHEN
		LPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYF
		SMVMTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKL
		PKLKLLFKQILSDRIAISWLPDNFKDDQEALDSIDTCYKNLLNDGN
		VLGEGNLKLLLENIDTYNLKGIFIRNDLQLTDISQKMYASWNVIQD
		AVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCLR
		AYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTP
		YPENANLAQDKETVALIKNLLDSLKRLQRFIKPLLGKGDESDKDER
		FYGDFTPLWETLNQITPLYNMVRNYMTRKPYSQEKIKLNFENSTLL
		GGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKDKLDNSGD
		CYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHK
		KGANFNLADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDF
		YREVEQQGYSISFCDVSVEYINKMVEKGDLYLFQIYNKDFSEFSKG
		TPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFRKKSLNYKRPTHP
		AHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFK
		STGNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVE
		QFTLNEIVNEYGGNIYRTNYHDLLDTREQNREKARESWQTIENIKE
		LKEGYISQVIHKITDLMQKYHAVVVLEDLNMGFMRGRQKVEKQV
		YQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQKLG
		KQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSI
		RYNADKDWFEFAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQA
		KNSQWDNEEIDLTKAYKAFFAKHGINIYDNIKEAIAMETEKSFFED
		LLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRICDNSLPAN
		ADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFAQEK
		PYLND

Candidatus	SEQ	MENKNNQTQSIWSVFTKKYSLQKTLRFELKPVGETKKWLEENDIF
Parcubacteria	ID	KKDLNIDKSYNQAKFYFDKLHQDFIKESLSVENGIRNIDFEKFAKIF
bacterium	NO:	ESNKEKIVSLKKKNKEVKDKNKKNWDEISKLEKEIEGQRENLYKEI
(PgCas12a)	12	RELFDKRAEKWKKEYQDKEIERGGKKEKIKFSSADLKQKGVNFLT
NCBI Gene ID:		AAGIINILKYKFPAEKDEEFRKEGYPSLFINDELNPGKKIYIFESFDK
BCX15829.1		FTTYLSKFQQTRENLYKDDGTSTAVATRIVSNFERFLENKSLFEEK
		YKNKAKDVGLTKEEEKVFEINYYYDCLIQEGIDKYNKIIGEINRKT
		KEYRDKNKIDKKDLPLFLNLEKQILGEVKKERVFIEAKDEKTEEEV
		FIDRFQEFIKRNKIKIYGDEKEEIEGAKKFIEDFTSGIFENDYQSIYLK
		KNVINEIVNKWFSNPEEFLMKLTGVKSEEKIKLKKFTSLDEFKNAIL
		SLEGDIFKSRFYKNEVNPEAPLEKEEKSNNWENFLKIWRFEFESLFK
		DKVEKGEIKKDKNGEPIQIFWGYTDKLEKEAEKIKFYSAEKEQIKTI
		KNYCDAALRINRMMRYFNLSDKDRKDVPSGLSTEFYRLVDEYFN
		NFEFNKYYNGIRNFITKKPSDENKIKLNFESRSLLDGWDVSKEKDN
		LGLIFIKNNKYYLGVLRKENSKLFDYQITEKDNQKEKERKNNLKNE
		ILANDNEDFYLKMNYWQIADPAKDIFNLVLMPDNTVKRFTKLEEK
		NKHWPDEIKRIKEKGTYKREKVNREDLVKIINYFRKCALIYWKKF
		DLKLLPSEEYQTFKDFTDHIALQGYKINFDKIKASYIEKQLNDGNL
		YLFEVSNKDFYKYKKPDSRKNIHTLYWEHIFSKENLEEIKYPLIRLN
		GKAEIFYRDVLEMNEEMRKPVILERLNGAKQAKREDKPVYHYQR
		YLKPTYLFHCPITLNADKPSSSFKNFSSKLNHFIKDNLGKINIIGIDR
		GEKNLLYYCVINQNQEILDYGSLNKINLNKVNNVNYFDKLVEREK
		QRQLERQSWEPVAKIKDLKQGYISYVVRKICDLIINHNAIVVLEDLS
		RRFKQIRNGISERTVYQQFEKALIDKLNYLIFKDNRDVFSPGGVLN
		GYQLAAPFTSFKDIEKAKQTGVLFYTSAEYTSQTDPLTGFRKNIYIS
		NSASQEKIKELINKLKKFGWDDTEESYFIEYNQVDFAEKKKKPLSK
		DWTIWTKVPRVIRWKESKSSYWSYKKINLNEEFRDLLEKYGFEAQ
		SNDILSNLKKRIAENDKLLVEKKEFDGRLKNFYERFIFLFNIVLQVR
		NTYSLSVEIDKTEKKLKKIDYGIDFFASPVKPFFTTFGLREIGIEKDG
		KVVKDNAREEIASENLAEFKDRLKEYKPEEKFDADGVGAYNIARK
		GLIILEKIKNNPNKPDLSISKEEWDKFVQR

Acidaminococcus	SEQ	MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
sp.	ID	YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR
(AaCas12a)	NO:	NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN
NCBI Gene ID:	13	GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI
WP_021736722.1		STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFV
		STSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
		NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVI
		QSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSAL
		CDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIIS
		AAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQL
		DSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
		RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNG
		LYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIP
		KCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKK
		FQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP
		SSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY
		NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRP
		KSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLS
		HDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAA
		NSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
		LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVI
		HEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLID
		KLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVP
		APYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGD
		FILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGK
		RIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN
		DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDS
		RFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN
		QDWLAYIQELRN

Bacteroidetes	SEQ	MESPTTQLKKFTNLYQLSKTLRFELKPVGKTKEHIETKGILKKDEE
bacterium	ID	RAVNYKLIKKIIDGFHKHFIELAMQQVKLSKLDELAELYNASAERK
(BoCas12a)	NO:	KEESYKKELEQVQAALRKEIVKGFNIGEAKEIFSKIDKKELFTELLD
NCBI Gene ID:	14	EWVKNLEEKKLVDDFKTFTTYFTGFHENRKNMYTDKAQSTAIAY
PKP47250.1		RLVHENLPKFLDNTKIFKQIETKFEASKIEEIETKLEPIIQGTSLSEIFT
		LDYYNHALTQAGIDFINNIIGGYTEDEGKKKIQGLNEYINLYNQKQ
		EKKNRIPKLKILYKQILSDRDSISFLPDAFEDSQEVLNAIQNYYQTN
		LIDFKPKDKEETENVLEETKKLLTELFSNELSKIYIRNDKAITDISQA
		LFNDWGVFKSALEYKFIQDLELGTKELSKKQENEKEKYLKQAYFSI
		AEIENALFAYQNETDVLNEIKENSHPIADYFTKHFKAKKKVDTSTS
		SVEKDFDLIANIDAKYSCIKGILNTDYPKDKKLNQEKKTIDDLKVFL
		DSLMELLHFVKPLALPNDSILEKDENFYSHFESYYEQLELLIPLYNK
		VRNYAAKKPYSTEKFKLNFENATLLKGWDKNKEIDNTSVILRKRG
		LYYLAIMPQDNKNVFKKSPNLKNNESCFEKMDYKQMALPMGFGA
		FVRKCFGTAFQLGWNCPKSCINEEDKIIIKEDEVKNNRAEIIDCYKD
		FLNIYEKDGFQYKEYGFNFKESKEYESLREFFIDVEQKGYKIEFQNI
		SENYIHQLVNEGKLYLFQIYNKDFSSYSKGKPNMHTMYWKALFDP
		ENLKDVVYKLNGQAEVFYRKKSIEDKNIITHKANEPIENKNPKAKK
		TQSTFEYDLIKDKRYTVDKFHFHVPITINFKATGNNYINQQVLDHL
		KNNTDVNIIGLDRGERHLIYLTLINQKGEILLQESLNTIVNKKFDIET
		PYHTLLQNKEDERAKARENWGVIENIKELKEGYLSQVVHKIAKLM
		VDYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVF
		KDKDPNEVGGLYNALQLTNKFESFSKMGKQSGFLFYVPAWNTSKI
		DPTTGFVNLFYAKYESIPKAQDFFTKFKSIRYNSDENYFEFAFDYN
		DFTTRAEGTKSDWTVCTYGDRIKTFRNPEKNNQWDNQEVNLIEQF
		EAFFGKHNITYGDGNCIKKQLIEQDKKEFFEELFHLFKLTLQMRNSI
		TNSEIDYLISPVKNSKKEFYDSRKADSTLPKDADANGAYHIAKKGL
		MWLEKINSFKGSDWKKLDLDKTNKTWLNFVQETASEKHKKLQTV

Candidatus	SEQ	MDAKEFTGQYPLSKTLRFELRPIGRTWDNLEASGYLAEDRHRAEC
Methanomethyl-	ID	YPRAKELLDDNHRAFLNRVLPQIDMDWHPIAEAFCKVHKNPGNK
ophilus alvus	NO:	ELAQDYNLQLSKRRKEISAYLQDADGYKGLFAKPALDEAMKIAKE
Mx1201
	15	NGNESDIEVLEAFNGFSVYFTGYHESRENIYSDEDMVSVAYRITED
(CMaCas12a)		NFPRFVSNALIFDKLNESHPDIISEVSGNLGVDDIGKYFDVSNYNNF
NCBI Gene ID:		LSQAGIDDYNHIIGGHTTEDGLIQAFNVVLNLRHQKDPGFEKIQFK
15139718		QLYKQILSVRTSKSYIPKQFDNSKEMVDCICDYVSKIEKSETVERAL
		KLVRNISSFDLRGIFVNKKNLRILSNKLIGDWDAIETALMHSSSSEN
		DKKSVYDSAEAFTLDDIFSSVKKFSDASAEDIGNRAEDICRVISETA
		PFINDLRAVDLDSLNDDGYEAAVSKIRESLEPYMDLFHELEIFSVG
		DEFPKCAAFYSELEEVSEQLIEIIPLFNKARSFCTRKRYSTDKIKVNL
		KFPTLADGWDLNKERDNKAAILRKDGKYYLAILDMKKDLSSIRTS
		DEDESSFEKMEYKLLPSPVKMLPKIFVKSKAAKEKYGLTDRMLEC
		YDKGMHKSGSAFDLGFCHELIDYYKRCIAEYPGWDVFDFKFRETS
		DYGSMKEFNEDVAGAGYYMSLRKIPCSEVYRLLDEKSIYLFQIYN
		KDYSENAHGNKNMHTMYWEGLFSPQNLESPVFKLSGGAELFFRK
		SSIPNDAKTVHPKGSVLVPRNDVNGRRIPDSIYRELTRYFNRGDCRI
		SDEAKSYLDKVKTKKADHDIVKDRRFTVDKMMFHVPIAMNFKAI
		SKPNLNKKVIDGIIDDQDLKIIGIDRGERNLIYVTMVDRKGNILYQD
		SLNILNGYDYRKALDVREYDNKEARRNWTKVEGIRKMKEGYLSL
		AVSKLADMIIENNAIIVMEDLNHGFKAGRSKIEKQVYQKFESMLIN
		KLGYMVLKDKSIDQSGGALHGYQLANHVTTLASVGKQCGVIFYIP
		AAFTSKIDPTTGFADLFALSNVKNVASMREFFSKMKSVIYDKAEG
		KFAFTFDYLDYNVKSECGRTLWTVYTVGERFTYSRVNREYVRKV
		PTDIIYDALQKAGISVEGDLRDRIAESDGDTLKSIFYAFKYALDMR
		VENREED YIQSPVKNASGEFFCSKNAGKSLPQDSDANGAYNIALK
		GILQLRMLSEQYDPNAESIRLPLITNKAWLTFMQSGMKTWKN

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with LbCas12a): K538A, K538D, K538E, Y542A, Y542D, Y542E, or K595A, K595D, K595E relative to the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with AsCas12a): K548A, K548D, K548E, N552A, N552D, N552E, or K607A, K607D, K607 relative to the amino acid sequence of SEQ ID NO: 2.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with CtCas12a): K534A, K534D, K534E, Y538A, Y538D, Y538E, or R591A, R591D, R591E relative to the amino acid sequence of SEQ ID NO: 3.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with EeCas12a): K542A, K541D, K541E, N545A, N545D, N545E or K601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 4.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Mb3Cas12a): K579A, K579D, K579E, N583A, N583D, N583E or K635A, K635D, K635E relative to the amino acid sequence of SEQ ID NO: 5.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FnCas12a): K613A, K613D, K613E, N617A, N617D, N617E or K671A, K671D, K671E relative to the amino acid sequence of SEQ ID NO: 6.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FnoCas12a): K613A, K613D, K613E, N617A, N617D, N617E or N671A, N671D, N671E relative to the amino acid sequence of SEQ ID NO: 7.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with FbCas12a): K617A, K617D, K617E, N621A, N621D, N621E or K678A, K678D, K678E relative to the amino acid sequence of SEQ ID NO: 8.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Lb4Cas12a): K541A, K541D, K541E, N545A, N545D, N545E or K601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 9.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with MbCas12a): K569A, K569D, K569E, N573A, N573D, N573E or K625A, K625D, K625E relative to the amino acid sequence of SEQ ID NO: 10.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with Pb2Cas12a): K562A, K562D, K562E, N566A, N566D, N566E or K619A, K619D, K619E relative to the amino acid sequence of SEQ ID NO: 11.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with PgCas12a): K645A, K645D, K645E, N649A, N649D, N649E or K732A, K732D, K732E relative to the amino acid sequence of SEQ ID NO: 12.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with AaCas12a): K548A, K548D, K548E, N552A, N552D, N552E or K607A, K607D, K607E relative to the amino acid sequence of SEQ ID NO: 13.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with BoCas12a): K592A, K592D, K592E, N596A, N596D, N596E or K653A, K653D, K653E relative to the amino acid sequence of SEQ ID NO: 14.
In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease contains one or more of the following substitutions (aligned with CMaCas12a): K521A, K521D, K521E, K525A, K525D, K525E or K577A, K577D, K577E relative to the amino acid sequence of SEQ ID NO: 15.
The mutations described herein may be described in the context of a natural Cas12a (any one of SEQ ID NOs: 15) sequence and mutational positions can be carried out by aligning the amino acid sequence of a Cas12a nucleic acid-guided nuclease with, for example, SEQ ID NO: 1 and making the equivalent modification (e.g., substitution) at the equivalent position. By way of example, Table 8 illustrates the equivalent amino acid positions of fifteen orthologous Cas12a nucleic acid-guided nucleases (SEQ ID NOs: 1-15). Any one of the amino acids indicated in Table 8 may be mutated (i.e., via a comparable amino acid substitution).

TABLE 8

Equivalent amino acid positions in homologous Cas12a nucleic
acid-guided nuclease

Cas

12a	AA	AA	AA	AA
WT SEQ ID NO	Ortholog	position	position	position	position

SEQ ID NO: 1	LbCas12a	G532	K538	Y542	K595
SEQ ID NO: 2	AsCas12a	S542	K548	N552	K607
SEQ ID NO: 3	CtCas12a	N528	K534	Y538	R591
SEQ ID NO: 4	EeCas12a	N535	K541	N545	K601
SEQ ID NO: 5	Mb3Cas12a	N573	K579	N583	K635
SEQ ID NO: 6	FnCas12a	N607	K613	N617	K671
SEQ ID NO: 7	FnoCas12a	N607	K613	N617	N671
SEQ ID NO: 8	FbCas12a	N611	K617	N621	K678
SEQ ID NO: 9	Lb4Cas12a	N535	K541	N545	K601
SEQ ID NO: 10	MbCas12a	N563	K569	N573	K625
SEQ ID NO: 11	Pb2Cas12a	G556	K562	N566	K619
SEQ ID NO: 12	PgCas12a	D639	K645	N649	K732
SEQ ID NO: 13	AaCas12a	S542	K548	N552	K607
SEQ ID NO: 14	BoCas12a	K586	K592	N596	K653
SEQ ID NO: 15	CMaCas12a	D515	K521	N525	K577

The variant single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-15 (excluding the residues listed in Table 8) and contain any conservative mutation one or more residues indicated in Tables 9-13.
It should be appreciated that any of the amino acid mutations described herein, (e.g., K595A) from a first amino acid residue (e.g., K, an amino acid with a basic side chain) to a second amino acid residue (e.g., A, an amino acid with an aliphatic side chain) may also include mutations from the first amino acid residue, lysine, to an amino acid residue that is similar to (e.g., conserved) the second amino acid residue, alanine, such as valine or glycine. As another example, mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may be a mutation to a second amino acid with an acidic side chain (e.g., glutamic acid or aspartic acid). As another example, mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may be a mutation to a second amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine). The skilled artisan would recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and are likely to be well tolerated without compromising function. That is, a mutation from one amino acid to a threonine may be an amino acid mutation to a serine; a mutation from one amino acid to an arginine may be an amino acid mutation to a lysine; a mutation from one amino acid to an isoleucine, may be an amino acid mutation to an alanine, valine, methionine, or leucine; a mutation from one amino acid to a lysine may be an amino acid mutation to an arginine; a mutation from one amino acid to an aspartic acid may be an amino acid mutation to a glutamic acid or asparagine; a mutation from one amino acid to a valine may be an amino acid mutation to an alanine, isoleucine, methionine, or leucine; a mutation from one amino acid to a glycine may be an amino acid mutation to an alanine. It should be appreciated, however, that additional conserved amino acid residues would be recognized by the skilled artisan and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.
Exemplary variant Cas12a orthologs are shown in tables 9-13.

TABLE 9

Exemplary Variant Ortholog Cas12a’s

	Variant LbCas12a		Variant AsCas12a		Variant CtCas12a
SEQ	(in relation to wt	SEQ	(in relation to wt	SEQ	(in relation to wt
ID	LbCas12a SEQ ID	ID	AsCas12a SEQ ID	ID	CtCas12a SEQ ID
NO:	NO: 1)	NO:	NO: 2)	NO:	NO: 3)

16	K595A	55	K607A	94	R591A
17	K595D	56	K607D	95	R591D
18	K595E	57	K607E	96	R591E
19	K538A/K595A	58	K548A/K607A	97	K534A/R591A
20	K538A/K595D	59	K548A/K607D	98	K534A/R591D
21	K538A/K595E	60	K548A/K607E	99	K534A/R591E
22	K538D/K595A	61	K548D/K607A	100	K534D/R591A
23	K538D/K595D	62	K548D/K607D	101	K534D/R591D
24	K538D/K595E	63	K548D/K607E	102	K534D/R591E
25	K538E/K595A	64	K548E/K607A	103	K534E/R591A
26	K538E/K595D	65	K548E/K607D	104	K534E/R591D
27	K538E/K595E	66	K548E/K607E	105	K534E/R591E
28	K538A/Y542A/K595A	67	K548A/N552A/K607A	106	K534A/Y538A/R591A
29	K538A/Y542D/K595A	68	K548A/N552D/K607A	107	K534A/Y538D/R591A
30	K538A/Y542E/K595A	69	K548A/N552E/K607A	108	K534A/Y538E/R591A
31	K538A/Y542A/K595D	70	K548A/N552A/K607D	109	K534A/Y538A/R591D
32	K538A/Y542D/K595D	71	K548A/N552D/K607D	110	K534A/Y538D/R591D
33	K538A/Y542E/K595D	72	K548A/N552E/K607D	111	K534A/Y538E/R591D
34	K538A/Y542A/K595E	73	K548A/N552A/K607E	112	K534A/Y538A/R591E
35	K538A/Y542D/K595E	74	K548A/N552D/K607E	113	K534A/Y538D/R591E
36	K538A/Y542E/K595E	75	K548A/N552E/K607E	114	K534A/Y538E/R591E
37	K538D/Y542A/K595A	76	K548D/N552A/K607A	115	K534D/Y538A/R591A
38	K538D/Y542D/K595A	77	K548D/N552D/K607A	116	K534D/Y538D/R591A
39	K538D/Y542E/K595A	78	K548D/N552E/K607A	117	K534D/Y538E/R591A
40	K538D/Y542A/K595D	79	K548D/N552A/K607D	118	K534D/Y538A/R591D
41	K538D/Y542D/K595D	80	K548D/N552D/K607D	119	K534D/Y538D/R591D
42	K538D/Y542E/K595D	81	K548D/N552E/K607D	120	K534D/Y538E/R591D
43	K538D/Y542A/K595E	82	K548D/N552A/K607E	121	K534D/Y538A/R591E
44	K538D/Y542D/K595E	83	K548D/N552D/K607E	122	K534D/Y538D/R591E
45	K538D/Y542E/K595E	84	K548D/N552E/K607E	123	K534D/Y538E/R591E
46	K538E/Y542A/K595A	85	K548E/N552A/K607A	124	K534E/Y538A/R591A
47	K538E/Y542D/K595A	86	K548E/N552D/K607A	125	K534E/Y538D/R591A
48	K538E/Y542E/K595A	87	K548E/N552E/K607A	126	K534E/Y538E/R591A
49	K538E/Y542A/K595E	88	K548E/N552A/K607D	127	K534E/Y538A/R591D
50	K538E/Y542D/K595E	89	K548E/N552D/K607D	128	K534E/Y538D/R591D
51	K538E/Y542E/K595E	90	K548E/N552E/K607D	129	K534E/Y538E/R591D
52	K538E/Y542A/K595E	91	K548E/N552A/K607E	130	K534E/Y538A/R591E
53	K538E/Y542D/K595E	92	K548E/N552D/K607E	131	K534E/Y538D/R591E
54	K538E/Y542E/K595E	93	K548E/N552E/K607E	132	K534E/Y538E/R591E

TABLE 10

Exemplary Variant Ortholog Cas12a’s

	Variant EeCas12a
SEQ	(in relation to wt
ID	EeCas12a SEQ ID
NO:	NO: 4)

133	K601A
134	K601D
135	K601E
136	K541A/K601A
137	K541A/K601D
138	K541A/K601E
139	K541D/K601A
140	K541D/K601D
141	K541D/K601E
142	K541E/K601A
143	K541E/K601D
144	K541E/K601E
145	K541A/N545A/K601A
146	K541A/N545D/K601A
147	K541A/N545E/K601A
148	K541A/N545A/K601D
149	K541A/N545D/K601D
150	K541A/N545E/K601D
151	K541A/N545A/K601E
152	K541A/N545D/K601E
153	K541A/N545E/K601E
154	K541D/N545A/K601A
155	K541D/N545D/K601A
156	K541D/N545E/K601A
157	K541D/N545A/K601D
158	K541D/N545D/K601D
159	K541D/N545E/K601D
160	K541D/N545A/K601E
161	K541D/N545D/K601E
162	K541D/N545E/K601E
163	K541E/N545A/K601A
164	K541E/N545D/K601A
165	K541E/N545E/K601A
166	K541E/N545A/K601D
167	K541E/N545D/K601D
168	K541E/N545E/K601D
169	K541E/N545A/K601E
170	K541E/N545D/K601E
171	K541E/N545E/K601E
172	K635A
173	K635D
174	K635E
175	K579A/K635A
176	K579A/K635D
177	K579A/K635E
178	K579D/K635A
179	K579D/K635D
180	K579D/K635E
181	K579E/K635A
182	K579E/K635D
183	K579E/K635E
184	K579A/N583A/K635A
185	K579A/N583D/K635A
186	K579A/N583E/K635A
187	K579A/N583A/K635D
188	K579A/N583D/K635D
189	K579A/N583E/K635D
190	K579A/N583A/K635E
191	K579A/N583D/K635E
192	K579A/N583E/K635E
193	K579D/N583A/K635A
194	K579D/N583D/K635A
195	K579D/N583E/K635A
196	K579D/N583A/K635D
197	K579D/N583D/K635D
198	K579D/N583E/K635D
199	K579D/N583A/K635E
200	K579D/N583D/K635E
201	K579D/N583E/K635E
202	K579E/N583A/K635A
203	K579E/N583D/K635A
204	K579E/N583E/K635A
205	K579E/N583A/K635D
206	K579E/N583D/K635D
207	K579E/N583E/K635D
208	K579E/N583A/K635E
209	K579E/N583D/K635E
210	K579E/N583E/K635E
211	K671A
212	K671D
213	K671E
214	K613A/K671A
215	K613A/K671D
216	K613A/K671E
217	K613D/K671A
218	K613D/K671D
219	K613D/K671E
220	K613E/K671A
221	K613E/K671D
222	K613E/K671E
223	K613A/N617A/K671A
224	K613A/N617D/K671A
225	K613A/N617E/K671A
226	K613A/N617A/K671D
227	K613A/N617D/K671D
228	K613A/N617E/K671D
229	K613A/N617A/K671E
230	K613A/N617D/K671E
231	K613A/N617E/K671E
232	K613D/N617A/K671A
233	K613D/N617D/K671A
234	K613D/N617E/K671A
235	K613D/N617A/K671D
236	K613D/N617D/K671D
237	K613D/N617E/K671D
238	K613D/N617A/K671E
239	K613D/N617D/K671E
240	K613D/N617E/K671E
241	K613E/N617A/K671A
242	K613E/N617D/K671A
243	K613E/N617E/K671A
244	K613E/N617A/K671D
245	K613E/N617D/K671D
246	K613E/N617E/K671D
247	K613E/N617A/K671E
248	K613E/N617D/K671E
249	K613E/N617E/K671E

TABLE 11

Exemplary Variant Ortholog Cas12a’s

SEQ	Variant FnoCas12a
ID	(in relation to wt
NO:	FnoCas12a SEQ ID NO: 7)

250	N671A
251	N671D
252	N671E
253	K613A/N671A
254	K613A/N671D
255	K613A/N671E
256	K613D/N671A
257	K613D/N671D
258	K613D/N671E
259	K613E/N671A
260	K613E/N671D
261	K613E/N671E
262	K613A/N617A/N671A
263	K613A/N617D/N671A
264	K613A/N617E/N671A
265	K613A/N617A/N671D
266	K613A/N617D/N671D
267	K613A/N617E/N671D
268	K613A/N617A/N671E
269	K613A/N617D/N671E
270	K613A/N617E/N671E
271	K613D/N617A/N671A
272	K613D/N617D/N671A
273	K613D/N617E/N671A
274	K613D/N617A/N671D
275	K613D/N617D/N671D
276	K613D/N617E/N671D
277	K613D/N617A/N671E
278	K613D/N617D/N671E
279	K613D/N617E/N671E
280	K613E/N617A/N671A
281	K613E/N617D/N671A
282	K613E/N617E/N671A
283	K613E/N617A/N671D
284	K613E/N617D/N671D
285	K613E/N617E/N671D
286	K613E/N617A/N671E
287	K613E/N617D/N671E
288	K613E/N617E/N671E
289	K678A
290	K678D
291	K678E
292	K617A/K678A
293	K617A/K678D
294	K617A/K678E
295	K617D/K678A
296	K617D/K678D
297	K617D/K678E
298	K617E/K678A
299	K617E/K678D
300	K617E/K678E
301	K617A/N621A/K678A
302	K617A/N621D/K678A
303	K617A/N621E/K678A
304	K617A/N621A/K678D
305	K617A/N621D/K678D
306	K617A/N621E/K678D
307	K617A/N621A/K678E
308	K617A/N621D/K678E
309	K617A/N621E/K678E
310	K617D/N621A/K678A
311	K617D/N621D/K678A
312	K617D/N621E/K678A
313	K617D/N621A/K678D
314	K617D/N621D/K678D
315	K617D/N621E/K678D
316	K617D/N621A/K678E
317	K617D/N621D/K678E
318	K617D/N621E/K678E
319	K617E/N621A/K678A
320	K617E/N621D/K678A
321	K617E/N621E/K678A
322	K617E/N621A/K678D
323	K617E/N621D/K678D
324	K617E/N621E/K678D
325	K617E/N621A/K678E
326	K617E/N621D/K678E
327	K617E/N621E/K678E
328	K601A
329	K601D
330	K601E
331	K541A/K601A
332	K541A/K601D
333	K541A/K601E
334	K541D/K601A
335	K541D/K601D
336	K541D/K601E
337	K541E/K601A
338	K541E/K601D
339	K541E/K601E
340	K541A/N545A/K601A
341	K541A/N545D/K601A
342	K541A/N545E/K601A
343	K541A/N545A/K601D
344	K541A/N545D/K601D
345	K541A/N545E/K601D
346	K541A/N545A/K601E
347	K541A/N545D/K601E
348	K541A/N545E/K601E
349	K541D/N545A/K601A
350	K541D/N545D/K601A
351	K541D/N545E/K601A
352	K541D/N545A/K601D
353	K541D/N545D/K601D
354	K541D/N545E/K601D
355	K541D/N545A/K601E
356	K541D/N545D/K601E
357	K541D/N545E/K601E
358	K541E/N545A/K601A
359	K541E/N545D/K601A
360	K541E/N545E/K601A
361	K541E/N545A/K601D
362	K541E/N545D/K601D
363	K541E/N545E/K601D
364	K541E/N545A/K601E
365	K541E/N545D/K601E
366	K541E/N545E/K601E

TABLE 12

Exemplary Variant Ortholog Cas12a’s

SEQ	Variant MbCas12a
ID	(in relation to wt
NO:	MbCas12a SEQ ID NO: 10)

367	K625A
368	K625D
369	K625E
370	K569A/K625A
371	K569A/K625D
372	K569A/K625E
373	K569D/K625A
374	K569D/K625D
375	K569D/K625E
376	K569E/K625A
377	K569E/K625D
378	K569E/K625E
379	K569A/N573A/K625A
380	K569A/N573D/K625A
381	K569A/N573E/K625A
382	K569A/N573A/K625D
383	K569A/N573D/K625D
384	K569A/N573E/K625D
385	K569A/N573A/K625E
386	K569A/N573D/K625E
387	K569A/N573E/K625E
388	K569D/N573A/K625A
389	K569D/N573D/K625A
390	K569D/N573E/K625A
391	K569D/N573A/K625D
392	K569D/N573D/K625D
393	K569D/N573E/K625D
394	K569D/N573A/K625E
395	K569D/N573D/K625E
396	K569D/N573E/K625E
397	K569E/N573A/K625A
398	K569E/N573D/K625A
399	K569E/N573E/K625A
400	K569E/N573A/K625D
401	K569E/N573D/K625D
402	K569E/N573E/K625D
403	K569E/N573A/K625E
404	K569E/N573D/K625E
405	K569E/N573E/K625E
406	K619A
407	K619D
408	K619E
409	K562A/K619A
410	K562A/K619D
411	K562A/K619E
412	K562D/K619A
413	K562D/K619D
414	K562D/K619E
415	K562E/K619A
416	K562E/K619D
417	K562E/K619E
418	K562A/N566A/K619A
419	K562A/N566D/K619A
420	K562A/N566E/K619A
421	K562A/N566A/K619D
422	K562A/N566D/K619D
423	K562A/N566E/K619D
424	K562A/N566A/K619E
425	K562A/N566D/K619E
426	K562A/N566E/K619E
427	K562D/N566A/K619A
428	K562D/N566D/K619A
429	K562D/N566E/K619A
430	K562D/N566A/K619D
431	K562D/N566D/K619D
432	K562D/N566E/K619D
433	K562D/N566A/K619E
434	K562D/N566D/K619E
435	K562D/N566E/K619E
436	K562E/N566A/K619A
437	K562E/N566D/K619A
438	K562E/N566E/K619A
439	K562E/N566A/K619D
440	K562E/N566D/K619D
441	K562E/N566E/K619D
442	K562E/N566A/K619E
443	K562E/N566D/K619E
444	K562E/N566E/K619E
445	K732A
446	K732D
447	K732E
448	K645A/K732A
449	K645A/K732D
450	K645A/K732E
451	K645D/K732A
452	K645D/K732D
453	K645D/K732E
454	K645E/K732A
455	K645E/K732D
456	K645E/K732E
457	K645A/N649A/K732A
458	K645A/N649D/K732A
459	K645A/N649E/K732A
460	K645A/N649A/K732D
461	K645A/N649D/K732D
462	K645A/N649E/K732D
463	K645A/N649A/K732E
464	K645A/N649D/K732E
465	K645A/N649E/K732E
466	K645D/N649A/K732A
467	K645D/N649D/K732A
468	K645D/N649E/K732A
469	K645D/N649A/K732D
470	K645D/N649D/K732D
471	K645D/N649E/K732D
472	K645D/N649A/K732E
473	K645D/N649D/K732E
474	K645D/N649E/K732E
475	K645E/N649A/K732A
476	K645E/N649D/K732A
477	K645E/N649E/K732A
478	K645E/N649A/K732D
479	K645E/N649D/K732D
480	K645E/N649E/K732D
481	K645E/N649A/K732E
482	K645E/N649D/K732E
483	K645E/N649E/K732E

TABLE 13

Exemplary Variant Ortholog Cas12a’s

SEQ	Variant AaCas12a
ID	(in relation to wt
NO:	AaCas12a SEQ ID NO: 13)

484	K607A
485	K607D
486	K607E
487	K548A/K607A
488	K548A/K607D
489	K548A/K607E
490	K548D/K607A
491	K548D/K607D
492	K548D/K607E
493	K548E/K607A
494	K548E/K607D
495	K548E/K607E
496	K548A/N552A/K607A
497	K548A/N552D/K607A
498	K548A/N552E/K607A
499	K548A/N552A/K607D
500	K548A/N552D/K607D
501	K548A/N552E/K607D
502	K548A/N552A/K607E
503	K548A/N552D/K607E
504	K548A/N552E/K607E
505	K548D/N552A/K607A
506	K548D/N552D/K607A
507	K548D/N552E/K607A
508	K548D/N552A/K607D
509	K548D/N552D/K607D
510	K548D/N552E/K607D
511	K548D/N552A/K607E
512	K548D/N552D/K607E
513	K548D/N552E/K607E
514	K548E/N552A/K607A
515	K548E/N552D/K607A
516	K548E/N552E/K607A
517	K548E/N552A/K607D
518	K548E/N552D/K607D
519	K548E/N552E/K607D
520	K548E/N552A/K607E
521	K548E/N552D/K607E
522	K548E/N552E/K607E
523	K653A
524	K653D
525	K653E
526	K592A/K653A
527	K592A/K653D
528	K592A/K653E
529	K592D/K653A
530	K592D/K653D
531	K592D/K653E
532	K592E/K653A
533	K592E/K653D
534	K592E/K653E
535	K592A/N596A/K653A
536	K592A/N596D/K653A
537	K592A/N596E/K653A
538	K592A/N596A/K653D
539	K592A/N596D/K653D
540	K592A/N596E/K653D
541	K592A/N596A/K653E
542	K592A/N596D/K653E
543	K592A/N596E/K653E
544	K592D/N596A/K653A
545	K592D/N596D/K653A
546	K592D/N596E/K653A
547	K592D/N596A/K653D
548	K592D/N596D/K653D
549	K592D/N596E/K653D
550	K592D/N596A/K653E
551	K592D/N596D/K653E
552	K592D/N596E/K653E
553	K592E/N596A/K653A
554	K592E/N596D/K653A
555	K592E/N596E/K653A
556	K592E/N596A/K653D
557	K592E/N596D/K653D
558	K592E/N596E/K653D
559	K592E/N596A/K653E
560	K592E/N596D/K653E
561	K592E/N596E/K653E
562	K577A
563	K577D
564	K577E
565	K521A/K577A
566	K521A/K577D
567	K521A/K577E
568	K521D/K577A
569	K521D/K577D
570	K521D/K577E
571	K521E/K577A
572	K521E/K577D
573	K521E/K577E
574	K521A/N525A/K577A
575	K521A/N525D/K577A
576	K521A/N525E/K577A
577	K521A/N525A/K577D
578	K521A/N525D/K577D
579	K521A/N525E/K577D
580	K521A/N525A/K577E
581	K521A/N525D/K577E
582	K521A/N525E/K577E
583	K521D/N525A/K577A
584	K521D/N525D/K577A
585	K521D/N525E/K577A
586	K521D/N525A/K577D
587	K521D/N525D/K577D
588	K521D/N525E/K577D
589	K521D/N525A/K577E
590	K521D/N525D/K577E
591	K521D/N525E/K577E
592	K521E/N525A/K577A
593	K521E/N525D/K577A
594	K521E/N525E/K577A
595	K521E/N525A/K577D
596	K521E/N525D/K577D
597	K521E/N525E/K577D
598	K521E/N525A/K577E
599	K521E/N525D/K577E
600	K521E/N525E/K577E

In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 70% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 75% identical to any one of SEQ ID NOs: 16-600 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 80% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 85% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 90% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 95% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 16-600. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is any one of SEQ ID NOs: 16-600.
The mutations described herein are described in the context of the WT LbCas12a (e.g., SEQ ID NO: 1) sequence and mutational positions can be carried out by aligning the amino acid sequence of a Cas12a nucleic acid-guided nuclease with SEQ ID NO: 1 and making the equivalent modification (e.g., substitution) at the equivalent position. By way of example, the mutations described herein may be applied to a Cas12a enzyme shown in Table 7, or any other homolog Cas12a thereof by aligning the amino acid sequence of the Cas12a to SEQ ID NO: 1 and making the modifications described in Tables 9-13 (changes to the wildtype residue to alanine, aspartic acid or glutamic acid or conservative equivalents at the Cas12a ortholog's equivalent position (e.g., see Table 8 for an example of equivalent residue positions).
For example, in addition to the variant LbCas12a sequences in Table 9 (variant sequences SEQ ID Nos: 16-54), like variants are envisioned for AsCas12a (variant sequences SEQ ID Nos: 55-93), CtCas12a (variant sequences SEQ ID Nos: 94-132), EeCas12a (variant sequences SEQ ID Nos: 133-171), Mb3Cas12a (variant sequences SEQ ID Nos: 172-210), FnCas12a (variant sequences SEQ ID Nos: 211-249), FnoCas12a (variant sequences SEQ ID Nos: 250-288), FbCas12a (variant sequences SEQ ID Nos: 289-327), Lb4Cas12a (variant sequences SEQ ID Nos: 328-366), MbCas12a (variant sequences SEQ ID Nos: 367-405), Pb2Cas12a (variant sequences SEQ ID Nos: 406-444), PgCas12a (variant sequences SEQ ID Nos: 445-483), AaCas12a (variant sequences SEQ ID Nos: 484-522), BoCas12a (variant sequences SEQ ID Nos: 523-561), and CmaCas12a (variant sequences SEQ ID Nos: 562-600). In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 70% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 75% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 80% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 85% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 90% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least 95% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is at least %, 97%, 98% or 99% identical to any one of SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listed in Tables 9-13 or the equivalent in a different ortholog. In some embodiments, the single-strand-specific Cas12a nucleic acid-guided nuclease is any one of SEQ ID NOs: 16-600.
The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may be any Cas12a nucleic acid-guided nuclease that largely prevents double-stranded nucleic acid unwinding and R-loop formation. The single-strand-specific Cas12a nucleic acid-guided nucleases described herein may also be any Cas12a nucleic acid-guided nuclease that lacks cis-cleavage activity yet maintains trans-nucleic acid-guided nuclease activity on single-stranded nucleic acid molecules. Such single-strand-specific Cas12a nucleic acid-guided nucleases may be generated via the mutations described herein.
Additionally, or alternatively, such single-strand-specific Cas12a nucleic acid-guided nucleases may be generated via post-translational modifications (e.g., acetylation). The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an acetylated Cas12a enzyme. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an LbCas12a (i.e., SEQ ID NO: 1) with an acetylated K595 (K595K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an AsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a CtCas12a (i.e., SEQ ID NO: 3) with an acetylated R591 (R591R^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an EeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635 (K635K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FnCas12a (i.e., SEQ ID NO: 6) with an acetylated K671 (K671K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylated N671 (N671K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an FbCas12a (i.e., SEQ ID NO: 8) with an acetylated K678 (K678K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylated K601 (K601K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an MbCas12a (i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732 (K732K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an AaCas12a (i.e., SEQ ID NO: 13) with an acetylated K607 (K607K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an BoCas12a (i.e., SEQ ID NO: 14) with an acetylated K653 (K653K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylated K577 (K577K^Ac) residue. The single-strand-specific Cas12a nucleic acid-guided nucleases of the disclosure may be a Cas12a ortholog acetylated at the amino acid of the ortholog equivalent to K595 of SEQ ID NO:1. Acetylation of Cas12a can be carried out with any suitable acetyltransferase. For a discussion and methods for disabling of Cas12a by ArVA5, see Dong, et al., Nature Structural and Molecular Bio., 26(4):308-14 (2019). For example, LbCas12a can be incubated with AcrVA5 in order to acetylate the K595 residue, thereby deactivating the dsDNA activity (e.g., FIG. 7 ). In addition to acetylation, phosphorylation and methylation of select amino acid residues may be employed.

Bulky Modifications

In addition to the modalities of adjusting the ratio of the concentration of the blocked nucleic acid molecules to the concentration of the RNP2 and altering the domains of the variant nucleic acid-guided nuclease of RNP2 that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules to vary dsDNA vs. ssDNA recognition properties as described in detail above, the present disclosure additionally contemplates use of “bulky modifications” at the 5′ and/or 3′ ends and/or at internal nucleic acid bases of the blocked nucleic acid molecule and/or using modifications between internal nucleic acid bases. FIG. 8A is an illustration of the steric hindrance at the PAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by 5′ and 3′ modifications to a blocked nucleic acid molecule. At top in FIG. 8A is an illustration of the target stand and non-target strand, and below this is an illustration of a self-hybridized blocked nucleic acid molecule comprising three loop regions, as well as bulky modifications on the 5′ and 3′ ends of the blocked nucleic acid molecule. Example “bulky modifications” include a fluorophore and quencher pair (as shown here) or biotin, but in general encompass molecules with a size of about 1 nm or less, or 0.9 nm or less, or 0.8 nm or less, or 0.7 nm or less, or 0.6 nm or less, or 0.5 nm or less, or 0.4 nm or less, or 0.3 nm or less, or 0.2 nm or less, or 0.1 nm or less, or 0.05 nm or less, or as small as 0.025 nm or less.
In the illustration at center, the blocked nucleic acid molecule with the 5′ and 3′ ends comprising a fluorophore and a quencher is shown being cleaved at the loop regions. Note that the bulky modifications in this embodiment also allow the blocked nucleic acid molecule to act as a reporter moiety; that is, when the loop regions of the blocked nucleic acid molecule are cleaved, the short nucleotide segments of the non-target strand dehybridize from the target strand due to low T_m, thereby separating the fluorophore and quencher such that fluorescence from the fluorophore is no longer quenched and can be detected. In the illustration at bottom, the intact blocked nucleic acid molecule with the bulky modifications (at left) sterically hinders interaction with the PAM-interacting (PI) domain of the nucleic acid-guided nuclease in RNP2 such that the intact blocked nucleic acid molecule cannot be cleaved via cis-cleavage by the nucleic acid-guided nuclease. However, once the loop regions of the blocked nucleic acid molecule are cleaved (via, e.g., trans-cleavage from RNP1 (at right)) and the short nucleotide segments of the non-target strand dehybridize from the target strand, leaving the 3′ end of the now single-stranded target strand is now free to initiate R-loop formation with RNP2. R-loop formation leads to cis-cleavage of the single-strand target strand, and subsequent activation of trans-cleavage of RNP2.
FIG. 8B illustrates five exemplary variations of blocked nucleic acid molecules with bulky modifications, including at the 5′ and/or 3′ ends of a self-hybridizing blocked nucleic acid molecule and/or at internal nucleic acid bases of the blocked nucleic acid molecule. Embodiment (i) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore at its 5′ end and a quencher at its 3′ end. Embodiment (ii) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore and a quencher at internal nucleic acid bases flanking a loop sequence. Embodiment (iii) illustrates a self-hybridizing blocked nucleic acid molecule having a fluorophore at its 5′ end and a quencher at its 3′ end as well as having a fluorophore and a quencher at internal nucleic acid bases where the internal fluorophore and quencher flank a loop sequence. The fluorophore/quencher embodiments work as long as the fluorophore and quencher are at a distance of about 10-11 nm or less apart. Embodiment (iv) illustrates a self-hybridizing blocked nucleic acid molecule having a biotin molecule at its 5′ end, and embodiment (v) illustrates a self-hybridizing blocked nucleic acid molecule having a biotin at an internal nucleic acid base. Note that bulky modifications of internal nucleic acid bases often are made at or near a loop region of a blocked nucleic acid molecule (or blocked target molecule). The loop regions are regions of the blocked nucleic acid molecules—in addition to the 5′ and 3′ ends—that may be vulnerable to unwinding.
Modifications can be used in self-hybridized blocked nucleic acid molecules lacking a PAM or those comprising a PAM, partially self-hybridized blocked nucleic acid molecules lacking a PAM or those comprising a PAM, or reverse PAM molecules. Other variations include using RNA loops instead of DNA loops if a Cas 13 nucleic acid-guided nuclease is used as the nucleic acid-guided nuclease in RNP1, or entire RNA molecules if a Cas 13 nucleic acid-guided nuclease is used as the nucleic acid-guided nuclease in RNP1 and RNP2.
FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, and internal positions in blocked nucleic acid molecules, and Table 14 below lists sequences of exemplary self-hybridizing blocked nucleic acid molecules. 56-FAM stands for 5′6-FAM (6-fluorescein amidite); and 3BHQ stands for 3′ BLACK HOLE QUENCHER®-1.

TABLE 14

Bulky Modifications

	SEQ
	ID	Molecule
No.	NO:	Name	Molecule Sequence (5′→3′)

5' FAM + 3' BHQ

1	601	5’F_U29_Q	/56-
			FAM/GATCCATTTTATTTTAGATCATATATATACATGATCGG
			ATC/3BHQ_1/

2	602	5’F_1C	/56-
		armor_	FAM/CGATCCATTTTATTTTAGATCATATATATACATGATCG
		U29_Q	GATCG/3BHQ_1/

3	603	5’F_2CC	/56-
		armor_	FAM/CCGATCCATTTTATTTTAGATCATATATATACATGATC
		U29_Q	GGATCGG/3BHQ_1/

4	604	5’F_1A	/56-
		armor_	FAM/AGATCCATTTTATTTTAGATCATATATATACATGATCG
		U29_Q	GATCT/3BHQ_1/

5	605	5’F_2AT	/56-
		armor_	FAM/ATGATCCATTTTATTTTAGATCATATATATACATGATC
		U29_Q	GGATCAT/3BHQ_1/

6	606	5’F_U250_	/56-
		Q	FAM/GATATATAAAAAAAAAAAGATCATATACATATATGAT
			CATATATC/3BHQ_1/

7	607	5’F_1C	/56-
		armor_	FAM/CGATATATAAAAAAAAAAAGATCATATACATATATGA
		U250_Q	TCATATATCG/3BHQ_1/

8	608	5’F_2CC	/56-
		armor_	FAM/CCGATATATAAAAAAAAAAAGATCATATACATATATG
		U250_Q	ATCATATATCGG/3BHQ_1/

9	609	5’F_1A	/56-
		armor_	FAM/AGATATATAAAAAAAAAAAGATCATATACATATATGA
		U250_Q	TCATATATCT/3BHQ_1/

10	610	5’F_2AT	/56-
		armor_	FAM/ATGATATATAAAAAAAAAAAGATCATATACATATATG
		U250_Q	ATCATATATCAT/3BHQ_1/

5' Fluorsceine (modification on base) + 3' BHQ

11	611	5’FdT_	/SFluorT/GATCCATTTTATTTTAGATCATATATATACATGATC
		U29_Q	GGATCA/3BHQ_1/

12	612	5’FdT_1C	/SFluorT/CGATCCATTTTATTTTAGATCATATATATACATGAT
		armor_	CGGATCGA/3BHQ_1/
		U29_Q

13	605	5’FdT_1A	A/iFluorT/GATCCATTTTATTTTAGATCATATATATACATGAT
		armor_	CGGATCAT/3BHQ_1/
		U29_Q

14	613	5’FdT_	/SFluorT/GATATATAAAAAAAAAAAGATCATATACATATATG
		U250_Q	ATCATATATCA/3BHQ_1/

15	614	5’FdT_1C	/SFluorT/CGATATATAAAAAAAAAAAGATCATATACATATAT
		armor_	GATCATATATCGA/3BHQ_1/
		U250_Q

16	610	5’FdT_1A	A/iFluorT/GATATATAAAAAAAAAAAGATCATATACATATAT
		armor_	GATCATATATCAT/3BHQ_1/
		U250_Q

5' FAM + Internal Fluorsceine (modification on base) + 3' BHQ

17	601	5’F_IntFdt_	/56-
		U29_Q	FAM/GA/iFluorT/CCATTTTATTTTAGATCATATATATACATG
			ATCGGATC/3BHQ_1/

18	606	5’F_IntFdt_	/56-
		U250_Q	FAM/GA/iFluorT/ATATAAAAAAAAAAAGATCATATACATAT
			ATGATCATATATC/3BHQ_1/

19	602	5’F_1C	/56-
		armor_	FAM/CGA/iFluorT/CCATTTTATTTTAGATCATATATATACAT
		IntFdt_U29_Q	GATCGGATCG/3BHQ_1/

20	604	5’F_1A	/56-
		armor_	FAM/AGA/iFluorT/CCATTTTATTTTAGATCATATATATACAT
		IntFdt_U29_Q	GATCGGATCT/3BHQ_1/

21	607	5’F_1C	/56-
		armor_	FAM/CGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA
		IntFdt_U250_Q	TATGATCATATATCG/3BHQ_1/

22	609	5’F_1A	/56-
		armor_	FAM/AGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA
		IntFdt_U250_Q	TATGATCATATATCT/3BHQ_1/

23	603	5’F_2CC	/56-
		armor_	FAM/CCGA/iFluorT/CCATTTTATTTTAGATCATATATATACA
		IntFdt_U29_Q	TGATCGGATCGG/3BHQ_1/

24	605	5’F_2AT	/56-
		armor_	FAM/ATGA/iFluorT/CCATTTTATTTTAGATCATATATATACA
		IntFdt_U29_Q	TGATCGGATCAT/3BHQ_1/

25	608	5'F_2CC	/56-
		armor_	FAM/CCGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT
		dIntFt_U250_Q	ATATGATCATATATCGG/3BHQ_1/

26	610	5’F_2AT	/56-
		armor_	FAM/ATGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT
		IntFdt_U250_Q	ATATGATCATATATCAT/3BHQ_1/

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a target nucleic acid of interest in a sample that provide instantaneous or nearly instantaneous results even at ambient temperatures at 16° C. and above, allow for massive multiplexing and minimum workflow, yet provide accurate results at low cost. Moreover, the various embodiments of the cascade assay are notable in that, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected and RNP1 is easily reprogrammed. Moreover, the cascade assay can be massively multiplexed for detecting several to many to target nucleic acid molecules simultaneously. For example, the assay may be designed to detect one to several to many different pathogens (e.g., testing for many different pathogens in one assay), or the assay may be designed to detect one to several to many different sequences from the same pathogen (e.g., to increase specificity and sensitivity), or a combination of the two.
As described above, early and accurate identification of, e.g., infectious agents, microbe contamination, and variant nucleic acid sequences that indicate the present of such diseases such as cancer or contamination by heterologous sources is important in order to select correct therapeutic treatment, identify tainted food, pharmaceuticals, cosmetics and other commercial goods; and to monitor the environment. The cascade assay described herein can be applied in diagnostics for, e.g., infectious disease (including but not limited to Covid, HIV, flu, the common cold, Lyme disease, STDs, chicken pox, diptheria, mononucleosis, hepatitis, UTIs, pneumonia, tetanus, rabies, malaria, dengue fever, Ebola, plague; see Table 1), for rapid liquid biopsies and companion diagnostics (biomarkers for cancers, early detection, progression, monitoring; see Table 4), prenatal testing (including but not limited to chromosomal abnormalities and genetic diseases such as sickle cell, including over-the-counter versions of prenatal testing assays), rare disease testing (achondroplasia, Addison's disease, α1-antitrypsin deficiency, multiple sclerosis, muscular dystrophy, cystic fibrosis, blood factor deficiencies), SNP detection/DNA profiling/epigenetics, genotyping, low abundance transcript detection, labeling for cell or droplet sorting, in situ nucleic acid detection, sample prep, library quantification of NGS, screening biologics (including engineered therapeutic cells for genetic integrity and/or contamination), development of agricultural products, food compliance testing and quality control (e.g., detection of genetically modified products, confirmation of source for high value commodities, contamination detection), infectious disease in livestock, infectious disease in cash crops, livestock breeding, drug screening, personal genome testing including clinical trial stratification, personalized medicine, nutrigenomics, drug development and drug therapy efficacy, transplant compatibility and monitoring, environmental testing and forensics, and bioterrorism agent monitoring.
Target nucleic acids of interest are derived from samples as described in more detail above. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or microbe. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample may be any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus.
For example, a biological sample can be a biological fluid obtained from a human or non-human (e.g., livestock, pets, wildlife) animal, and may include but is not limited to blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab).
In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In other embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in U.S. Pat. No. 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those (see, e.g., Example I below). In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HCl. Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519 and 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain cell-debris free supernatant before applying the reagents. Suitable methods are contemplated in U.S. Pat. No. 8,809,519, among other references. In still other embodiments, minimal processing may include performing DNA/RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents.
Table 15 below lists exemplary commercial sample processing kits, and Table 16 below lists point of care processing techniques.

TABLE 15

Exemplary Commercial Sample and Nucleic Acid Processing Kits

Manufacturer	Kit	Sample Type	Output	Lysing and extraction methods

Qiagen ®	DNeasy ™ Blood	small volumes	genomic	Isolation of Genomic DNA from Small
	& Tissue Kits	of blood	DNA	Volumes of Blood
		dried blood		1. Uses Chemical and
		spots		Biological/Enzymatic lysis methods
		urine
		2. Uses SPE with Column Purification
		tissues		Isolation of Genomic DNA from Tissues
		laser-		1. Uses Chemical and
		microdissected		Biological/Enzymatic lysis methods
		tissues
		2. Used to dissolve and lyse tissue sections
				completely, higher temperature and
				longer time incubations up to 24 hours are
				used
Qiagen ®	QIAamp ® UCP	whole blood	microbial	Specific pretreatment protocols are
	Pathogen	swabs	DNA	suggested depending on sample type with
	Mini Handbook	cultures—		or without the use of kits for Mechanical
	microbial DNA	pelleted		Lysis Method before downstream
	purification	microbial cells		applications.
		body fluids		Downstream applications contain:
				1. Chemical and Biological/Enzymatic
				lysis methods

				2. SPE with Column Purification
Qiagen ®	QIAamp ® Viral	plasma and	viral DNA	1. Uses Chemical lysis methods
	RNA Kits	serum			2. Uses SPE with Column Purification
		CSF
		urine
		other cell-free
		body fluids
		cell-culture
		supernatants
		swabs
Zymo	Quick-	whole blood	genomic	1. Uses chemical lysis methods
Research ™	DNA ™Microprep	plasma	DNA		2. Uses SPE with column purification
	Kit	serum
		body fluids
		buffy coat
		lymphocytes
		swabs
		cultured cells
Zymo	Quick-DNA ™	A. fumigatus	Microbial	Uses Bead lysis and pretreatment with:
Research ™	Fungal/Bacterial	C. albicans	DNA	1. Chemical lysis methods with
	Miniprep Kit	N. crassa		chaotropic salts
		S. cerevisiae
		2. NAE with SPE with silica matrices
		S. pombe
		mycelium
		Gram positive
		bacteria
		Gram negative
		bacteria

TABLE 16

Point of Care Sample Processing Techniques

Steps	Protocol Example 1	Protocol Example 2	Protocol Example 3

	Field-deployable viral	Streamlined	Lucira Health ™
	diagnostics using	inactivation,
	CRISPR-Cas13	amplification, and
	Science,	Cas13-based detection
	27; 360(6387):444-448	of SARS-CoV-2
	(2018)	NatCommun, 11: 5921
		(2020)
1. Cell disruption	Samples were thermally	A NP swab or saliva	Lucira Health uses a
(lysis) and	treated at ~40° C. for ~15	sample was lysed and	single buffer that lyses
inactivation of	minutes for nuclease	inactivated for 10	and inactivates
nucleases	deactivation, thereafter	minutes with thermal	nucleases and/or
In POC setting, cell	at 90° C. for 5 minutes	treatment. These	inhibitors.
disruption and	for viral deactivation.	samples were incubated	A nasal swab is directly
inactivation of	Sample Types:	for 5 min at 40° C.,	added to a single
nucleases is done	Urine	followed by 5 min at	lysing/reaction buffer
commonly through	Saliva	70° C. (or 5 min at 95° C.,	and vigorously stirred
thermal lysis.	Diluted blood	if saliva)	to release the viral
	(1:3 with PBS)		particulates from the
	Targets: Viruses		swab.
			Target: SARS-Cov-2
2. Assay on crude	Thermally treated	Thermally treated	Processed biological
sample	biological	biological	sample is used in an
This is usually a direct	samples(above) were	samples(above) were	isothermal reaction for
assay on the crude	used directly for	used directly for	pathogenic nucleic acid
sample post cell	amplification and	amplification and	detection.
disruption and	detection of pathogenic	detection of pathogenic
inactivation of	nucleic acid.	nucleic acid.
nucleases. No
extraction is usually
performed.

FIG. 9 shows a lateral flow assay (LFA) device that can be used to detect the cleavage and separation of a signal from a reporter moiety. For example, the reporter moiety may be a single-stranded or double-stranded oligonucleotide with terminal biotin and fluorescein amidite (FAM) modifications; and, as described above, the reporter moiety may also be part of a blocked nucleic acid. The LFA device may include a pad with binding particles, such as gold nanoparticles functionalized with anti-FAM antibodies; a control line with a first binding moiety attached, such as avidin or streptavidin; a test line with a second binding moiety attached, such as antibodies; and an absorption pad. After completion of a cascade assay (see FIGS. 2A, 3A, and 3B), the assay reaction mix is added to the pad containing the binding particles, (e.g., antibody labeled gold nanoparticles). When the target nucleic acid of interest is present, a reporter moiety is cleaved, and when the target nucleic acid of interest is absent, the reporter is not cleaved.
A moiety on the reporter binds to the binding particles and is transported to the control line. When the target nucleic acid of interest is absent, the reporter moiety is not cleaved, and the first binding moiety binds to the reporter moiety, with the binding particles attached. When the target nucleic acid of interest is present, one portion of the cleaved reporter moiety binds to the first binding moiety, and another portion of the cleaved reporter moiety bound to the binding particles via the moiety binds to the second binding moiety. In one example, anti-FAM gold nanoparticles bind to a FAM terminus of a reporter moiety and flow sequentially toward the control line and then to the test line. For reporters that are not trans-cleaved, gold nanoparticles attach to the control line via biotin-streptavidin and result in a dark control line. In a negative test, since the reporter has not been cleaved, all gold conjugates are trapped on control line due to attachment via biotin-streptavidin. A negative test will result in a dark control line with a blank test line. In a positive test, reporter moieties have been trans-cleaved by the cascade assay, thereby separating the biotin terminus from the FAM terminus. For cleaved reporter moieties, nanoparticles are captured at the test line due to anti-FAM antibodies. This positive test results in a dark test line in addition to a dark control line.
The components of the cascade assay may be provided in various kits for testing at, e.g., point of care facilities, in the field, pandemic testing sites, and the like. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), blocked nucleic acid molecules, and reporter moieties. The first complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid(s) of interest. Binding of the first complex (RNP1) to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The blocked nucleic acid molecule comprises a sequence complementary to the second gRNA, where trans-cleavage of the blocked nucleic acid molecule results in an unblocked nucleic acid molecule and the unblocked nucleic acid molecule can bind to the second complex (RNP2), thereby activating the trans-cleavage activity of the second nucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked nucleic acid molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and/or are operably linked to the blocked nucleic acid molecules and produce a detectable signal upon cleavage by RNP2.
In a second aspect, the kit for detecting a target nucleic acid molecule in sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), template molecules, blocked primer molecules, a polymerase, NTPs, and reporter moieties. The first ribonucleoprotein complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid of interest and where binding of RNP1 to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The template molecules comprise a primer binding domain (PBD) sequence as well as a sequence corresponding to a spacer sequence of the second gRNA. The blocked primer molecules comprise a sequence that is complementary to the PBD on the template nucleic acid molecule and a blocking moiety.
Upon binding to the target nucleic acid of interest, RNP1 becomes active triggering trans-cleavage activity that cuts at least one of the blocked primer molecules to produce at least one unblocked primer molecule. The unblocked primer molecule hybridizes to the PBD of one of the template nucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′ exonuclease activity of the polymerase and is then extended by the polymerase and NTPs to form a synthesized activating molecule with a sequence that is complementary to the second gRNA of RNP2 (i.e., the synthesized activating molecule is the target strand). Upon activating RNP2, additional trans-cleavage activity is initiated, cleaving at least one additional blocked primer molecule. Continued cleavage of blocked primer molecules and subsequent activation of more RNP2s proceeds at an exponential rate. A signal is generated upon cleavage of a reporter molecule by active RNP2 complexes; therefore, a change in signal production indicates the presence of the target nucleic acid molecule.
Any of the kits described herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and/or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

Example I

Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by various methods depending on the cell type and source (e.g., tissue, blood, saliva, environmental sample, etc.). Mechanical lysis is a widely used cell lysis method and may be used to extract nucleic acids from bacterial, yeast, plant and mammalian cells. Cells are disrupted by agitating a cell suspension with “beads” at high speeds (beads for disrupting various types of cells can be sourced from, e.g., OPS Diagnostics (Lebanon N.J., US) and MP Biomedicals (Irvine, Calif., USA)). Mechanical lysis via beads begins with harvesting cells in a tissue or liquid, where the cells are first centrifuged and pelleted. The supernatant is removed and replaced with a buffer containing detergents as well as lysozyme and protease. The cell suspension is mixed to promote breakdown of the proteins in the cells and the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that are mixed (e.g., vortexed) with the cell suspension at high speeds. The beads collide with the cells, breaking open the cell membrane with shear forces. After “bead beating”, the cell suspension is centrifuged to pellet the cellular debris and beads, and the supernatant may be purified via a nucleic acid binding column (such as the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham, Mass., USA) and others from Qiagen (Hilden, Germany), TakaraBio (San Jose, Calif., USA), and Biocomma (Shenzen, China)) to collect the nucleic acids (see the discussion of solid phase extraction below).
Solid phase extraction (SPE): Another method for capturing nucleic acids is through solid phase extraction. SPE involves a liquid and stationary phase, which selectively separates the target analyte (here, nucleic acids) from the liquid in which the cells are suspended based on specific hydrophobic, polar, and/or ionic properties of the target analyte in the liquid and the stationary solid matrix. Silica binding columns and their derivatives are the most commonly used SPE techniques, having a high binding affinity for DNA under alkaline conditions and increased salt concentration; thus, a highly alkaline and concentrated salt buffer is used. The nucleic acid sample is centrifuged through a column with a highly porous and high surface area silica matrix, where binding occurs via the affinity between negatively charged nucleic acids and positively charged silica material. The nucleic acids bind to the silica matrices, while the other cell components and chemicals pass through the matrix without binding. One or more wash steps typically are performed after the initial sample binding (i.e., the nucleic acids to the matrix), to further purify the bound nucleic acids, removing excess chemicals and cellular components non-specifically bound to the silica matrix. Alternative versions of SPE include reverse SPE and ion exchange SPE, and use of glass particles, cellulose matrices, and magnetic beads.
Thermal lysis: Thermal lysis involves heating a sample of mammalian cells, virions, or bacterial cells at high temperatures thereby damaging the cellular membranes by denaturizing the membrane proteins. Denaturizing the membrane proteins results in the release of intracellular DNA. Cells are generally heated above 90° C., however time and temperature may vary depending on sample volume and sample type. Once lysed, typically one or more downstream methods, such as use of nucleic acid binding columns for solid phase extraction as described above, are required to further purify the nucleic acids.
Physical lysis: Common physical lysis methods include sonication and osmotic shock. Sonication involves creating and rupturing of cavities or bubbles to release shockwaves, thereby disintegrating the cellular membranes of the cells. In the sonication process, cells are added into lysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibit proteases. The cell samples are then placed in a water bath and a sonication wand is placed directly into the sample solution. Sonication typically occurs between 20-50 kHz, causing cavities to be formed throughout the solution as a result of the ultrasonic vibrations; subsequent reduction of pressure then causes the collapse of the cavity or bubble resulting in a large amount of mechanical energy being released in the form of a shockwave that propagates through the solution and disintegrates the cellular membrane. The duration of the sonication pulses and number of pulses performed varies depending on cell type and the downstream application. After sonication, the cell suspension typically is centrifuged to pellet the cellular debris and the supernatant containing the nucleic acids may be further purified by solid phase extraction as described above.
Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in DI/distilled water with no salt added. Because the salt concentration is lower in the solution than in the cells, water is forced into the cell causing the cell to burst, thereby rupturing the cellular membrane. The sample is typically purified and extracted by techniques such as e.g., solid phase extraction or other techniques known to those of skill in the art.
Chemical lysis: Chemical lysis involves rupturing cellular and nuclear membranes by disrupting the hydrophobic-hydrophilic interactions in the membrane bilayers via detergents. Salts and buffers (such as, e.g., Tris-HCl pH 8) are used to stabilize pH during extraction, and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors (e.g., Proteinase K) are also added to preserve the integrity of the nucleic acids and protect against degradation. Often, chemical lysis is used with enzymatic disruption methods (see below) for lysing bacterial cell walls. In addition, detergents are used to lyse and break down cellular membranes by solubilizing the lipids and membrane proteins on the surface of cells. The contents of the cells include, in addition to the desired nucleic acids, inner cellular proteins and cellular debris. Enzymes and other inhibitors are added after lysis to inactivate nucleases that may degrade the nucleic acids. Proteinase K is commonly added after lysis, destroying DNase and RNase enzymes capable of degrading the nucleic acids. After treatment with enzymes, the sample is centrifuged, pelleting cellular debris, while the nucleic acids remain in the solution. The nucleic acids may be further purified as described above.
Another form of chemical lysis is the widely used procedure of phenol-chloroform extraction. Phenol-chloroform extraction involves the ability for nucleic acids to remain soluble in an aqueous solution in an acidic environment, while the proteins and cellular debris can be pelleted down via centrifugation. Phenol and chloroform ensure a clear separation of the aqueous and organic (debris) phases. For DNA, a pH of 7-8 is used, and for RNA, a more acidic pH of 4.5 is used.
Enzymatic lysis: Enzymatic disruption methods are commonly combined with other lysis methods such as those described above to disrupt cellular walls (bacteria and plants) and membranes. Enzymes such as lysozyme, lysostaphin, zymolase, and protease are often used in combination with other techniques such as physical and chemical lysis. For example, one can use cellulase to disrupt plant cell walls, lysosomes to disrupt bacterial cell walls and zymolase to disrupt yeast cell walls.

Example II

RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein was incubated with 375 nM of a target specific gRNA in 1× Buffer (10 mM Tris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂at 25° C. for 20 minutes. The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease to gRNAs were tested, including 1:1, 1:2 and 1:5. The incubation temperature ranged from 16° C.-37° C., and the incubation time ranged from 10 minutes to 4 hours.

Example III

Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6 ° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to 37° C. at 0.015 ° C./second to form the desired secondary structure.
Snap cooling: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6 ° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by removing the heat source to form the desired secondary structure.
Snap cooling on ice: For formation of the secondary structure of blocked nucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6 ° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by placing the reaction tube on ice to form the desired secondary structure.

Example IV

Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-9 bases in length (e.g., with sequences of TTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5′ and 3′ ends, respectively. In one example using a Cas12a cascade, the fluorophore was FAM-6 and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, Iowa). In another example using a Cas13 cascade, the reporter moieties were single-stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n).

Example V

Cascade Assay

Format I (final reaction mix components added at the same time): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the Methicillin resistant Staphylococcus aureus (MRSA) DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1× NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a:112.5 nM gRNA. Thereafter, the final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1× ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a:22.5 nM gRNA RNP1, 20 nM LbCas12a:35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. 1 μL of MRSA DNA target (with samples having as low as three copies and as many as 30000 copies—see FIGS. 6-14 ) was added to make a final volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.
Format II (RNP1 and MRSA target pre-incubated before addition to final reaction mix): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to RNP formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1× NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a:112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 16° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1× ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a:35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.
Format III (RNP1 and MRSA target pre-incubated before addition to final reaction mix and blocked nucleic acid molecule added to final reaction mix last): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1× NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a:112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 16° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1× ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a:35 nM gRNA RNP2 in a total volume of 9 μL. Once the reaction mix was made, 1 μL (50 nM) blocked nucleic acid molecule (any one of Formula I-IV) was added for a total volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Example VI

Detection of MRSA and Test Reaction Conditions

To detect the presence of Methicillin resistant Staphylococcus aureus (MRSA) and determine the sensitivity of detection with the cascade assay, titration experiments with a MRSA DNA target nucleic acid of interest were performed. The MRSA DNA sequence (NCBI Reference Sequence NC: 007793.1) is as follows.

	SEQ ID NO: 615:
	ATGAAAAAGATAAAAATTGTTCCACTTATTTTAAT

	AGTTGTAGTTGTCGGGTTTGGTATATATTTTTATG

	CTTCAAAAGATAAAGAAATTAATAATACTATTGAT

	GCAATTGAAGATAAAAATTTCAAACAAGTTTATAA

	AGATAGCAGTTATATTTCTAAAAGCGATAATGGTG

	AAGTAGAAATGACTGAACGTCCGATAAAAATATAT

	AATAGTTTAGGCGTTAAAGATATAAACATTCAGGA

	TCGTAAAATAAAAAAAGTATCTAAAAATAAAAAAC

	GAGTAGATGCTCAATATAAAATTAAAACAAACTAC

	GGTAACATTGATCGCAACGTTCAATTTAATTTTGT

	TAAAGAAGATGGTATGTGGAAGTTAGATTGGGATC

	ATAGCGTCATTATTCCAGGAATGCAGAAAGACCAA

	AGCATACATATTGAAAATTTAAAATCAGAACGTGG

	TAAAATTTTAGACCGAAACAATGTGGAATTGGCCA

	ATACAGGAACAGCATATGAGATAGGCATCGTTCCA

	AAGAATGTATCTAAAAAAGATTATAAAGCAATCGC

	TAAAGAACTAAGTATTTCTGAAGACTATATCAAAC

	AACAAATGGATCAAAATTGGGTACAAGATGATACC

	TTCGTTCCACTTAAAACCGTTAAAAAAATGGATGA

	ATATTTAAGTGATTTCGCAAAAAAATTTCATCTTA

	CAACTAATGAAACAGAAAGTCGTAACTATCCTCTA

	GGAAAAGCGACTTCACATCTATTAGGTTATGTTGG

	TCCCATTAACTCTGAAGAATTAAAACAAAAAGAAT

	ATAAAGGCTATAAAGATGATGCAGTTATTGGTAAA

	AAGGGACTCGAAAAACTTTACGATAAAAAGCTCCA

	ACATGAAGATGGCTATCGTGTCACAATCGTTGACG

	ATAATAGCAATACAATCGCACATACATTAATAGAG

	AAAAAGAAAAAAGATGGCAAAGATATTCAACTAAC

	TATTGATGCTAAAGTTCAAAAGAGTATTTATAACA

	ACATGAAAAATGATTATGGCTCAGGTACTGCTATC

	CACCCTCAAACAGGTGAATTATTAGCACTTGTAAG

	CACACCTTCATATGACGTCTATCCATTTATGTATG

	GCATGAGTAACGAAGAATATAATAAATTAACCGAA

	GATAAAAAAGAACCTCTGCTCAACAAGTTCCAGAT

	TACAACTTCACCAGGTTCAACTCAAAAAATATTAA

	CAGCAATGATTGGGTTAAATAACAAAACATTAGAC

	GATAAAACAAGTTATAAAATCGATGGTAAAGGTTG

	GCAAAAAGATAAATCTTGGGGTGGTTACAACGTTA

	CAAGATATGAAGTGGTAAATGGTAATATCGACTTA

	AAACAAGCAATAGAATCATCAGATAACATTTTCTT

	TGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAAT

	TTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAA

	GATATACCAAGTGATTATCCATTTTATAATGCTCA

	AATTTCAAACAAAAATTTAGATAATGAAATATTAT

	TAGCTGATTCAGGTTACGGACAAGGTGAAATACTG

	ATTAACCCAGTACAGATCCTTTCAATCTATAGCGC

	ATTAGAAAATAATGGCAATATTAACGCACCTCACT

	TATTAAAAGACACGAAAAACAAAGTTTGGAAGAAA

	AATATTATTTCCAAAGAAAATATCAATCTATTAAC

	TGATGGTATGCAACAAGTCGTAAATAAAACACATA

	AAGAAGATATTTATAGATCTTATGCAAACTTAATT

	GGCAAATCCGGTACTGCAGAACTCAAAATGAAACA

	AGGAGAAACTGGCAGACAAATTGGGTGGTTTATAT

	CATATGATAAAGATAATCCAAACATGATGATGGCT

	ATTAATGTTAAAGATGTACAAGATAAAGGAATGGC

	TAGCTACAATGCCAAAATCTCAGGTAAAGTGTATG

	ATGAGCTATATGAGAACGGTAATAAAAAATACGAT

	ATAGATGAATAA

Briefly, a RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target a 20 bp region of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 616). An RNP2 was preassembled with a gRNA sequence designed to target the unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) blocked nucleic acid molecule U29 (FIG. 10A). The reaction mix contained the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂and 101 mM NaCl.
FIG. 10A shows the structure and segment parameters of molecule U29. Note molecule U29 has a secondary structure free energy value of −5.84 kcal/mol and relatively short self-hybridizing, double-stranded regions of 5 bases and 6 bases. FIGS. 10B-10H show the results achieved for detection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecA gene of MRSA (n=3) at 25° C. with varying concentrations of blocked nucleic acid, RNP2 and reporter moiety. FIG. 10B shows the results achieved when 100 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 10:1. Note first that with 3E4 copies, nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 28.06 at 0 minutes, a signal-to-noise ratio of 24.23 at 5 minutes, and a signal-to-noise ratio of 21.01 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 12.45 at 0 minutes, 14.07 at 5 minutes and 16.16 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.79 at 0 minutes, 1.64 at 5 minutes and is 2.04 at 10 minutes. Note the measured fluorescence at 0 copies increases only slightly over the 10- and 30-minutes intervals, resulting in a flat negative. A flat negative (the results obtained over the time period for 0 copies) demonstrates that there is very little non-specific or undesired signal generation in the system. Note that the negative when the ratio of blocked nucleic acid molecules to RNP2s is 10:1 is flatter than those in FIGS. 10C through 10H.
FIG. 10C shows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. Note first that with 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 12.85, a signal-to-noise ratio of 10.51 at 5 minutes, and a signal-to-noise ratio of 8.18 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.85 at 0 minutes, 6.44 at 5 minutes and 6.48 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.54 at 0 minutes, 1.61 at 5 minutes and is 1.71 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2.
FIG. 10D shows the results achieved when 50 nM blocked nucleic acid molecules, 10 nM RNP2s and 2500 nM reporter moieties are used. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 34.92, a signal-to-noise ratio of 30.62 at 5 minutes, and a signal-to-noise ratio of 25.81 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 7.97 at 0 minutes, 1.73 at 5 minutes and 10.50 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.65 at 0 minutes, 1.73 at 5 minutes and is 1.82 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s, but likely due to the 5× increase in the concentration of reporter moieties; however, note also that a higher concentration of reporter moieties allows for a higher signal-to-noise ratio for 3E4 and 30 copies of MRSA target.
FIG. 10E shows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and 4 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1 but double the concentration of both of these molecules than that shown in FIGS. 10C and 10D. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 11.89, a signal-to-noise ratio of 8.97 at 5 minutes, and a signal-to-noise ratio of 6.53 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.46 at 0 minutes, 5.85 at 5 minutes and 5.43 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.58 at 0 minutes, 1.65 at 5 minutes and is 1.80 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the ratio of blocked nucleic acid molecules to RNP2s (5:1) appears to be more important than the ultimate concentration (100 nM/20 nM) by comparison to FIG. 10D where the ratio of blocked nucleic acid molecules to RNP2s was also 5:1 however the concentration of blocked nucleic acid molecules was 50 nM and the concentration of RNP2 was 10 nM.
FIG. 1OF shows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 4 mM NaCl. In this experiment the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 25.85, a signal-to-noise ratio of 21.36 at 5 minutes, and a signal-to-noise ratio of 16.24 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.28 at 0 minutes, 6.19 at 5 minutes and 7.02 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.53 at 5 minutes and is 1.73 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was reduced at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s.
FIG. 10G shows the results achieved when 50 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 12.75, a signal-to-noise ratio of 7.78 at 5 minutes, and a signal-to-noise ratio of 3.66 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 6.09 at 0 minutes, 6.23 at 5 minutes and 3.58 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is very low at 0 minutes, 1.40 at 5 minutes and is 1.62 at 10 minutes. Note the measured fluorescence at 0 copies increases, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was reduced substantially at the 2.5:1 ratio of blocked nucleic acid molecules to RNP2s and that the NaCl concentration at 10 mM vs. 4 mM (FIG. 10F) did not make much of a difference.
FIG. 10H shows the results achieved when 100 nM blocked nucleic acid molecules, 20 nM RNP2s and 500 nM reporter moieties are used and using a concentration of 10 mM NaCl. Thus, in this experiment, the ratio of blocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of 77.38, a signal-to-noise ratio of 74.18 at 5 minutes, and a signal-to-noise ratio of 67.90 at 10 minutes. Additionally, the signal-to-noise ratios for detection with 30 copies of MRSA target is 5.94 at 0 minutes, 7,45 at 5 minutes and 9.73 at 10 minutes; and the signal-to-noise ratios for detection with 3 copies of MRSA target is 1.66 at 0 minutes, 2.13 at 5 minutes and is 2.38 at 10 minutes. Note the measured fluorescence at 0 copies increases slightly, resulting in less of a flat negative than the 10:1 ratio of blocked nucleic acid molecules to RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio for all concentrations was increased substantially at the 5:1 ratio of blocked nucleic acid molecules to RNP2s as compared to the 2.5:1 ration of blocked nucleic acid molecules to RNP2s. In summary, the results shown in FIGS. 10B-10H indicate that a 5:1 ratio of blocked nucleic acid molecules to RNP2s or greater leads to higher signal-to-noise ratios for all concentrations of MRSA target.

Example VII

Homology Modeling and Mutation Structure Analysis

The variant nucleic acid-guided nucleases presented herein were developed in the following manner: For protein engineering and amino acid substitution model predictions, a first Protein Data Bank (pdb) file with the amino acid sequence and structure information for the RNP comprising the base nucleic acid-guided nuclease to be mutated, the gRNA and a bound dsDNA target nucleic acid was obtained. (For structural information for RNPs comprising AsCas12s and LbCas12a, see, e.g., Yamano, et al., Molecular Cell, 67:633-45 (2017).) Desired and/or random amino acid substitutions were then “made” to the base nucleic acid-guided nuclease (LbCas12a)., the resulting structural change to the base nucleic acid-guided nuclease due to each amino acid substitution was used to generate updated files for the resulting RNPs comprising each of the variant nucleic acid-guided nucleases using SWISS-MODEL and the original pdf file as a reference template. SWISS-MODEL worked well in the present case as the amino acid sequences of wildtype LbCas12a was known, as were the planned amino acid substitutions. The output of the updated files for each variant nucleic acid-guided nuclease included a root mean square deviation (RMSD) value for the structural changes compared to the RNP complex for wt LbCas12a in Angstrom units (i.e., a measurement of the difference between the backbones of wt LbCas12a and the variant nucleic acid-guided nuclease) and the updated pdb files of the variant nucleic acid-guided nucleases are further assessed at the point of mutations for changes in the hydrogen bonds compared to the reference original pdb file of the nuclease.
After SWISS modeling, an independent step for calculating free energy was performed using, e.g., a Flex ddG module based on the program Rosetta CM to extract locally destabilizing mutations. This was used as a proxy for amino acid interference with PAM regions of the DNA to assess the probability of unwinding of the target nucleic acid. (See, e.g., Shanthirabalan, et al., Proteins: Structure, Function, and Bioinformatics 86(8):853-867 (2018); and Barlow, et al., J. Physical Chemistry B, 122(21):5389-99 (2018).)
Generally, the results of the SWISS-Model and Rosetta analysis indicated that stable enzyme function related to the PAM domain would require a global RMSD value range from 0.1 to 2.1 angstroms, and the following ΔΔG Flex Values: for stabilizing mutations ΔΔG≤−1.0 kcal/mol; for neutral mutations: −1.0 kcal/mol<ΔΔG<1.0 kcal/mol; and for destabilizing mutations: ΔΔG≥1.0 kcal/mol. Sixteen single mutations were identified that, singly or in combination, met the calculated criteria. Structural modeling for mutations at four exemplary amino acid residues are described below.
FIG. 6A shows the result of protein structure prediction using Rosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacterium Cas12a). Protein structure prediction using Rossetta and SWISS modeling of exemplary variants of wildtype LbCas12a are shown below.
Mutation 1, G532A: The structure of an RNP comprising the G532A variant nucleic acid-guided nuclease is shown in FIG. 11A. Modeling indicated the following changes to the wildtype LbCas12a structure with the G532A substitution (seen in FIG. 11A as a red residue): loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595; no addition or loss of a hydrogen bond at amino acid residue 532. Per simulations, mutation G532A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 17.

TABLE 17

Mutation 1: G532A

Global RMSD: 0.976
PIRMSD: 0.361
REC1 RMSD: 0.289 (235 to 235 atoms)
WED RMSD: 0.306 (198 to 198 atoms)
ΔΔG Flex Value: −1.13

PI = PAM-interacting domain of the G532A variant
REC1 = REC1 domain of the G532A variant
WED = WED domain of the G532A variant

Mutation 2, K538A: The structure of an RNP comprising the K538A variant nucleic acid-guided nuclease is shown at left in FIG. 11B. Modeling indicated the following changes to the wildtype LbCas12a structure with the K538A substitution (seen in FIG. 11B as a pink residue): loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595. Per simulations, mutation K538A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 18.

TABLE 18

Mutation 2: K538A

Global RMSD: 0.990
PI RMSD: 0.376
REC1 RMSD: 0.305 (236 to 236 atoms)
WED RMSD: 0.324 (194 to 194 atoms)
ΔΔG Flex Value: 0.06

PI = PAM-interacting domain of the K538A variant
REC1 = REC1 domain of the K538A variant
WED = WED domain of the K538A variant

Mutation 3, Y542A: The structure of an RNP comprising the Y542A variant nucleic acid-guided nuclease is shown in FIG. 11C. Modeling indicated the following changes to the wildtype LbCas12a structure with the Y542A substitution (seen in FIG. 11C as a blue residue): loss of two hydrogen bonds with TS-PAM (target strand PAM) at amino acid residue 542; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538; loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595. Per simulations, mutation Y542A is a structurally stabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 19.

TABLE 19

Mutation 3: Y542A

Global RMSD: 0.989
PI RMSD: 0.377
REC1 RMSD: 0.306 (237 to 237 atoms)
WED RMSD: 0.338 (199 to 199 atoms)
ΔΔG Flex Value: −2.06

PI = PAM-interacting domain of the Y542A variant
REC1 = REC1 domain of the Y542A variant
WED = WED domain of the Y542A variant

Mutation 4, K595A: The structure of an RNP comprising the K595A variant nucleic acid-guided nuclease is shown in FIG. 11D. Modeling indicated the following changes to the wildtype LbCas12a structure with the K595A substitution (seen in FIG. 11D as an orange residue): loss of two hydrogen bonds with TS-PAM (target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) at amino acid residue 538. Per simulations, mutation K595A is a structurally destabilizing mutation. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 20.

TABLE 20

Mutation 4: K595A

Global RMSD: 0.976
PI RMSD: 0.361
REC1 RMSD: 0.289 (235 to 235 atoms)
WED RMSD: 0.306 (198 to 198 atoms)
ΔΔG Flex Value: 1.26

PI = PAM-interacting domain of the K595A variant
REC1 = REC1 domain of the K595A variant
WED = WED domain of the K595A variant

Mutation 5, Combination G532A, K538A, Y542A, and K595A: The structure of an RNP comprising the combination G532A/K538A/Y542A/K595A variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11E. Modeling indicated the following changes to the wildtype LbCas12a structure with the four substitutions: loss of five hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM (non-target strand PAM). Per simulations, the combination variant is structurally stable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 21.

TABLE 21

Mutation 5: G532A/K538A/Y542A/K595A

Global RMSD: 0.966
PI RMSD: 0.351
REC1 RMSD: 0.261 (226 to 226 atoms)
WED RMSD: 0.288 (200 to 200 atoms)
ΔΔG Flex Value: −3.31

PI = PAM-interacting domain of the combination variant
REC1 = REC1 domain of the combination variant
WED = WED domain of the combination variant

Mutation 6, K595D: The structure of an RNP comprising the K595D variant nucleic acid-guided nuclease is shown in FIG. 11F. Modeling indicated the following changes to the wildtype LbCas12a structure at location 595 with this substitution: loss of two hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM (non-target strand PAM); and gain of one hydrogen bond with NTS-PAM. Per simulations, the K595D variant is structurally unstable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 22.

TABLE 22

Mutation 6: K595D

Global RMSD: 1.001
PI RMSD: 0.367 (89 to 89 atoms)
REC1 RMSD: 0.296 (235 to 235 atoms)
WED RMSD: 0.320 (197 to 197 atoms)
ΔΔG Flex Value: 2.04

PI = PAM-interacting domain of the combination variant
REC1 = REC1 domain of the combination variant
WED = WED domain of the combination variant

Mutation 7, K595E: The structure of an RNP comprising the K595E variant nucleic acid-guided nuclease is shown in FIG. 11G. Modeling indicated the following changes to the wildtype LbCas12a structure at location 595 with this substitution: loss of two hydrogen bonds with TS-PAM (target strand PAM); loss of one hydrogen bond with NTS; and no gain of hydrogen bonds. Per simulations, the K595E variant is structurally unstable. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 23.

TABLE 23

Mutation 6: K595E

Global RMSD: 0.975
PI RMSD: 0.352 (89 to 89 atoms)
REC1 RMSD: 0.264 (226 to 226 atoms)
WED RMSD: 0.290 (198 to 198 atoms)
ΔΔG Flex Value: 1.37

PI = PAM-interacting domain of the combination variant
REC1 = REC1 domain of the combination variant
WED = WED domain of the combination variant

Mutation 8, Combination K538A, Y542A, K595D: The structure of an RNP comprising the combination K538A/Y542A/K595D variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11H. Modeling indicated the following changes to the wildtype LbCas12a structure with the three substitutions: loss of two hydrogen bonds with TS (target strand) at position 595; loss of one hydrogen bond with NTS (non-target); combined loss of three hydrogen bonds at 532/242 positions; and gain of one hydrogen bond at 595. Per simulations, the combination variant is structurally destabilizing. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 24.

TABLE 24

Mutation 6: K538A, Y542A, K595D

Global RMSD: 0.976
PI RMSD: 0.351 (89 to 89 atoms)
REC1 RMSD: 0.261 (225 to 225 atoms)
WED RMSD: 0.289 (198 to 198 atoms)
ΔΔG Flex Value: 0.96

PI = PAM-interacting domain of the combination variant
REC1 = REC1 domain of the combination variant
WED = WED domain of the combination variant

Mutation 9, Combination K538A, Y542A, K595E: The structure of an RNP comprising the combination K538A/Y542A/K595E variant (“combination variant”) nucleic acid-guided nuclease is shown in FIG. 11I. Modeling indicated the following changes to the wildtype LbCas12a structure with the three substitutions: loss of two hydrogen bonds with TS (target strand) at position 595; loss of one hydrogen bond with NTS (non-target); combined loss of three hydrogen bonds at 532/242 positions. Per simulations, the combination variant is structurally stabilizing. The parameters collected from SWISS-MODEL and Rosetta analysis are shown in Table 25.

TABLE 25

Mutation 6: K538A, Y542A, K595E

Global RMSD: 0.976
PI RMSD: 0.351 (89 to 89 atoms)
REC1 RMSD: 0.261 (225 to 225 atoms)
WED RMSD: 0.289 (198 to 198 atoms)
ΔΔG Flex Value: −3.71

PI = PAM-interacting domain of the combination variant
REC1 = REC1 domain of the combination variant
WED = WED domain of the combination variant

In addition to amino acid substitutions, modifications, such as chemical modifications, can be made to amino acids identified by the structural and homology modeling described above. FIG. 6G illustrates an exemplary scheme for acetylating amino acid residue 595 in LbCas12a, a modification which prevents unwinding of dsDNA by blocking entry of a target nucleic acid into the RNP via steric hindrance. LbCas12a is combined with AcrVA5 and the reaction is incubated for 20 minutes at room temperature, resulting in LECas12a that has been acetylated at amino acid residue 595 (K595K^AC). (For a discussion and methods for disabling of Cas12a by ArVA5, see Dong, et al., Nature Structural and Molecular Bio., 26(4):308-14 (2019).) DsDNA is not a substrate for LbCas12a with a K595K^ACmodification; however, ssDNA is a substrate for LbCas12a with a K595K^ACmodification; thus, LbCas12a (K595K^AC) has the desired properties of the variant nucleic acid-guided nucleases described above. In addition to acetylation, phosphorylation and methylation of select amino acid residues may be employed.

Example VIII

Single-Strand Specificity of the Variant Nucleic Acid-Guided Nucleases

In vitro transcription/translation reactions were performed for variant LbaCas12a nucleases as noted in Table 26 using the nucleic acid sequences listed in Table 27:

TABLE 26

Template DNA for IVTT	250 ng

gRNA concentration	100 nM

DNA activator concentration	25 nM

Probe concentration
	500 nM

Reaction volume
	30 pL

Reporter
	5′-FAM-TTATTATT-IABkFQ-3′

Plate	PCR plate 96-well, black

Read temperature
	25° C.

Read duration
	30 minutes

Buffer	NEB r2.1 New England Biolabs ®, Inc.,
	Ipswich, MA)

Na+	50 mM

Mg + 2	10 mM

TABLE 27

Activator

RunX fragment	GCCTTCAGAAGAGGGTGCATTTTCAGGAGGAAGCGAT
(dsDNA + PAM)	GGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 617)

RunX fragment	GCCTTCAGAAGAGGGTGCATGCACAGGAGGAAGCGAT
(dsDNA - PAM)	GGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 618)

Target region in	AGGAGGAAGCGATGGCTTCAGA (SEQ ID NO. 619)
activator

gRNA

LbaCas12a gRNA	gUAAUUUCUACUAAGUGUAGAUAGGAGGAAGCGAUG
	GCUUCAGA (SEQ ID NO. 620)

The results are shown in FIGS. 12A-12G indicating the time for detection of dsDNA and ssDNA both with and without PAM sequences for purified wildtype LbaCas12a and three variants (K538A+K595A, K595A, and K538A+Y542+K595A, and unpurified engineered variants of LbaCas12a:K538D+Y542A+K595D, K595D, K538A+K595D, K538A+K595E, G532A+K538A+Y542A+K595A, K538A+Y542A+K595D, K538D+Y542A+K595A, K538D+Y542D+K595A, and K538E+Y542A+K595A. Note that all variant engineered nucleic acid-guided nucleases slowed down double-strand DNA detection to varying degrees, with the double and triple variants at positions K538, Y542 and K595 of wt LbaCas12a performing best in comparison to wt LbCas12a, while single-strand DNA detection remained high, both in single-strand DNA with a PAM and without a PAM. The following variants were particularly robust: K538D+Y542A+K595D, K538A+K595D, K538A+K595E, G532A+K538A+Y542A+K595A, K538D+Y542A+K595A, and K538D+Y542D+K595D.

FIGS. 13A and 13B show the sequence alignment of many different Cas12a nucleases and orthologs, including in some instances several alignments of the same Cas12a nuclease.

Example IX: Detection of Biomarker Alpha-Synuclein in CSF for Monitoring Progression of Parkinson's Disease

The biomarker α-synuclein, which is found in both aggregated and fibrillar form, has attracted attention as a biomarker of Parkinson's disease. Human α-synuclein is expressed in the brain in the neocortex, hippocampus, substantia nigra, thalamus and cerebellum. It is encoded by the SNCA gene that consists of six exons ranging in size from 42 to 1110 base pairs. The predominant form of α-synuclein is the full-length protein, but other shorter isoforms exist. C-terminal truncation of α-synuclein induces aggregation, suggesting that C-terminal modifications may be involved in Parkinson's pathology. Changes in the levels of α-synuclein have been reported in CSF of Parkinson' patients. The gradual spread of α-synuclein pathology leads to a high concentration of extracellular α-synuclein that can potentially damage healthy neurons. Here, the cascade assay is used to monitor the level of nucleic acids in cerebrospinal fluid (CSF) to monitor the levels of mRNA transcripts that when translated lead to a truncated α-synuclein protein.
A lumbar puncture is performed on an individual, withdrawing approximately 5 mL of cerebrospinal fluid (CSF) for testing. The CSF sample is then treated by phenol-chloroform extraction or oligo dT affinity resins via a commercial kit (see, e.g., the TurboCapture mRNA kit or RNeaxy Pure mRNA Bead Kit from Qiagen®). Briefly, two RNP1s are preassembled as described above in Example II with a first gRNA sequence designed to target the coding sequence of the mRNA transcribed from SNCA gene specific to the C-terminus region of a-synuclein to detect full-length α-synuclein and second gRNA sequence designed to target the coding sequence of the mRNA transcribed from SNCA gene specific to the N-terminus region of α-synuclein to detect all α-synuclein mRNAs. In addition to the gRNA, each RNP1 also comprises an LbCas13a nuclease (i.e., an RNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. A readout is performed by comparing the level of N-terminus coding sequences detected (the level of total α-synuclein mRNA) versus the level of C-terminus coding sequences detected (the level of full-length α-synuclein mRNA).

Example X

Detection of Foot and Mouth Disease Virus from Nasal Swabs

Foot-and-mouth disease (FMD) is a severe and highly contagious viral disease. The FMD virus causes illness in cows, pigs, sheep, goats, deer, and other animals with divided hooves and is a worldwide concern as it can spread quickly and cause significant economic losses. FMD has serious impacts on the livestock trade—a single detection of FMD will stop international trade completely for a period of time. Since the disease can spread widely and rapidly and has grave economic consequences, FMD is one of the animal diseases livestock owners dread most. FMD is caused by a virus, which survives in living tissue and in the breath, saliva, urine, and other excretions of infected animals. FMD can also survive in contaminated materials and the environment for several months under the right conditions.
A nasal swab is performed on a subject, such as a cow or pig, and the nucleic acids extracted using, e.g., the Monarch Total RNA Miniprep Kit (New England Biolabs®, Inc., Ipswich, Mass.). Briefly, an RNP1 is preassembled as described above in Example II with a gRNA sequence designed to a gene from the FMD virus (e.g., to a portion of NCBI Reference Sequence NC 039210.1) and an LbCas12a nuclease (i.e., a DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V, and the readout is positive detection of FMD virus-specific DNA sequences.

Example XI

Detection of Sickle Cell Gene Sequences in Peripheral Blood

Sickle cell disease (SCD) is a group of inherited red blood cell disorders. In someone who has SCD, the hemoglobin is abnormal, which causes the red blood cells to become hard and sticky and look like a C-shaped farm tool called a “sickle.” The sickle cells die early, which causes a constant shortage of red blood cells; in addition, when the sickle-shaped blood cells travel through small blood vessels, they get stuck and clog the blood flow, causing pain and other serious complications such as infection and stroke.
One form of SCD is HbSS. Individuals who have this form of SCD inherit two genes, one from each parent, that code for hemoglobin “S.” Hemoglobin S is an abnormal form of hemoglobin that causes the red cells to become rigid and sickle shaped. This is commonly called sickle cell anemia and is usually the most severe form of the disease. Another form of SCD is HbSC. Individuals who have this form of SCD inherit a hemoglobin “S” gene from one parent and a gene for a different type of abnormal hemoglobin called “C” from the other parent. This is usually a milder form of SCD. A third form of SCD is HbS thalassemia. Individuals who have this form of SCD inherit a hemoglobin “S” gene from one parent and a gene for beta thalassemia, another type of hemoglobin abnormality, from the other parent. There are two types of beta thalassemia: “zero” (HbS beta0) and “plus” (HbS beta+). Those with HbS beta0-thalassemia usually have a severe form of SCD. People with HbS beta+-thalassemia tend to have a milder form of SCD.
A non-invasive prenatal test (NIPT) that uses only maternal cell-free DNA (cfDNA) from peripheral blood permits prenatal detection of sickle cell disease and beta thalassemia by screening without the need for paternal DNA. Such a screening enables patients and healthcare providers to make informed decisions about diagnostic testing and may expand gene therapy treatment options. A 10 mL peripheral blood draw is performed on a pregnant subject into a Streck tube. The blood is treated with lysis-binding buffer and proteinase K under denaturing conditions at 55° C. for 15 minutes in the presence of magnetic beads. Following the heating step, the mixture is incubated for 1 hour at room temperature with mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorf themomixer. The beads are captured on a magnetic stand for 2 minutes, washed three times after which cfDNA is eluted by adding elution buffer and incubating for 5 minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat #WHAWB120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride); Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100 (t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at room temperature. An additional bead purification step is performed using PCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA). Alternatively, there are several kits available commercially that are designed to extract cfDNA including the BioChain® cfPure® Cell free DNA Extraction Kit (BioChain®, Newark, Calif.); the Monarch Genomic DNA Purification Kit and the Monarch HMW DNA Extraction Kit for Blood (New England Biolabs®, Inc., Ipswich, Mass.); and the cfDNA Purification Kit (Active Motif®, Carlsbad, Calif.).
For the cascade assay, three RNP1s are preassembled as described above in Example II with 1) gRNA sequence designed to detect the Hemoglobin S gene variant and an LbCas12a nuclease (i.e., an DNA-specific nuclease); 2) a gRNA sequence designed to detect the Hemoglobin C gene variant and an LbCas 12a nuclease (i.e., an DNA-specific nuclease); and 3) a gRNA sequence designed to detect the gene for beta thalassemia and an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout is detection of the Hemoglobin S gene variant, the detection of the Hemoglobin S variant and the Hemoglobin C variant, and the detection of the Hemoglobin S variant and the β-thalassemia gene.

Example XII

Detection of Donor-Derived Gene Sequences in Peripheral Blood of Transplant Patients

Costly and invasive tissue biopsies to detect allograft rejection after transplantation have numerous limitations; however, assays based on cell-free DNA (cfDNA)—circulating fragments of DNA released from cells, tissues, and organs as they undergo natural cell death—can improve the ability to detect rejection and implement earlier changes in management of the transplanted organ. Rejection, referring to injury of a donated organ caused by the recipient's immune system, often causes allograft dysfunction and even patient death. T-cell mediated acute cellular rejection occurs most often within the first 6 months post-transplant. Acute cellular rejection involves accumulation of CD4+ and CD8+ T-cells in the interstitial space of the allograft as the recipient's immune system recognizes antigens on the donated organ as foreign, initiating an immune cascade that ultimately leads to apoptosis of the targeted cells. As these cells die, genomic DNA is cleaved and fragments of donor derived-cfDNA are released to join the pool of recipient cfDNA in the blood. Using cfDNA as a biomarker for acute cellular rejection is advantageous since it is derived from the injured cells of the donated organ and therefore should represent a direct measure of cell death occurring in the allograft. Further, cfDNA maintains all of the genetic features of the original genomic DNA, allowing the genetic material released from the donated organ to be differentiated from the cfDNA derived from cells of the recipient that are undergoing natural apoptosis.
For organ transplants in which the donor is male and the recipient is female, this “sex mismatch” is leveraged to calculate donor derived-cfDNA levels from within the recipient's total cfDNA pool. Although this approach allows for confident diagnosis of rejection in the allograft, sex-mismatch between the donor and recipient is relatively infrequent and not universally applicable; thus, the presence of other genetic differences between the donor and recipient at a particular locus are leveraged to identify the origin of the circulating cfDNA. Ideally, the recipient would be homozygous for a single base (for example, AA) and at the same locus the donor would be homozygous for a different base (for example, GG). Given the genetic heterogeneity between individuals, hundreds to tens of thousands of potentially informative loci across the genome can be interrogated to distinguish donor derived-cfDNA from recipient cfDNA.
A 10 mL peripheral blood draw is performed on a transplantation subject into a Streck tube. The blood is treated with lysis-binding buffer and proteinase K under denaturing conditions at 55° C. for 15 minutes in the presence of magnetic beads. Following the heating step, the mixture is incubated for 1 hour at room temperature with mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorf themomixer. The beads are captured on a magnetic stand for 2 minutes, washed three times after which cfDNA is eluted by adding elution buffer and incubating for 5 minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat #WHAWB120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride); Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100 (t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at room temperature. An additional bead purification step is performed using PCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA). Also, as stated above, there are several kits available commercially that are designed to extract cfDNA including the BioChain® cfPure® Cell free DNA Extraction Kit (BioChain®, Newark, Calif.); the Monarch Genomic DNA Purification Kit and the Monarch HMW DNA Extraction Kit for Blood (New England Biolabs®, Inc., Ipswich, Mass.); and the cfDNA Purification Kit (Active Motif®, Carlsbad, Calif.).
For the cascade assay, several to many different RNP 1 s are preassembled as described above in Example II with gRNA sequences designed to 1) query Y and/or X chromosome loci in sex mismatch transplantation cases; or 2) gRNA sequences designed to query various loci that are different in the genomic DNA of the recipient and the donor; along with an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout detects the level of donor-specific nucleic acid sequences.

Example XIII

Detection of Microbe Contamination in a Laboratory

DNA that is found in the environment is called “environmental DNA” or eDNA (e-DNA) for short, and it is formally defined as “genetic material obtained directly from environmental samples without any obvious signs of biological source material.” eDNA has been harnessed to detect rare or invasive species and pathogens in a broad range of environments. Samples are typically collected in the form of water, soil, sediment, or surface swabs. The DNA must then be extracted and purified to remove chemicals that may inhibit the cascade reaction. Surface wipe samples are commonly collected to assess microbe contamination in, e.g., a laboratory. The wipe test protocol consists of four distinct stages: removal of DNA from surfaces using absorbent wipes, extraction of DNA from the wipes into a buffer solution, purification of DNA, and analysis of the extract.
For sample collection, sterile 2×2 inch polyester-rayon non-woven wipes are used to wipe down an environmental surface, such as a laboratory bench. Each wipe is placed into a sterile 50 ml conical tube and 10 mL of PBST is transferred to each conical tube using a sterile serological pipette. The tubes are vortexed at the maximum speed for 20 minutes using a Vortex Genie 2. A 200 μL aliquot of the supernatant was processed using a nucleic acid purification kit (QIAmp DNA Blood Mini Kit, QIAGEN, Inc., Valencia, Calif.). The kit lyses the sample, stabilizes and binds DNA to a selective membrane, and elutes the DNA sample.
For the cascade assay, several to many different RNP1s are preassembled as described above in Example II with gRNA sequences designed to detect, e.g., Aspergillus acidus; Parafilaria bovicola; Babesia divergens; Escherichia coli; Pseudomonas aeruginosa; and Dengue virus; along with an LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described in Example II above, an RNP2 is preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a chosen blocked nucleic acid molecule such as U29. The blocked nucleic acid molecule is formed as described above in Example III, and a reporter is formed as described above in Example IV. The reaction mix contains the preassembled RNP1, preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂and 101 mM NaCl. The cascade assay is performed by one of the protocols described above in Example V. The readout is detection of a genomic sequence unique to a pathogen.
While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures.

Claims

We claim:

1. A method for identifying a target nucleic acid of interest in a sample in one minute or less at 16° C. comprising the steps of:

providing a reaction mixture comprising:

first ribonucleoprotein (RNP1) complexes (RNP1s) each comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease;

second ribonucleoprotein complexes (RNP2s) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease optionally comprises a variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved, wherein the variant nuclease comprises at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules, and wherein the variant nuclease exhibits both cis- and trans-cleavage activity;

a plurality of blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein the blocked nucleic acid molecules comprise: a first region recognized by the RNP2 complex; one or more second regions not complementary to the first region forming at least one loop; one or more third regions complementary to and hybridized to the first region forming at least one clamp, wherein optionally the molar ratio of the blocked nucleic acid molecules is at least equal to the molar ratio of the second ribonucleoprotein complexes, and wherein optionally the blocked nucleic acid molecules each comprise at least one bulky modification;

and wherein one of the following conditions is met: 1) providing blocked nucleic acid molecules and ribonucleoprotein complexes where the molar ratio of the blocked nucleic acid molecules is equal to or exceeds the molar ratio of the ribonucleoprotein complexes, 2) the blocked nucleic acid molecules each comprise at least one bulky modification, or 3) the RNP2 comprises at least one variant nuclease engineered such that single stranded DNA is cleaved faster than double stranded DNA is cleaved; and

contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the plurality of blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule, wherein the at least one unblocked nucleic acid molecule binds to RNP2 initiating trans-cleavage of at least one further blocked nucleic acid molecule; and

detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample in one minute or less.

2. The method of claim 1, wherein the reaction mixture further comprises reporter moieties, wherein the reporter moieties produce a detectable signal upon trans-cleavage activity by the RNP2 to identify the presence of one or more nucleic acid targets of interest in the sample.

3. The method of claim 2, wherein the reporter moieties are not coupled to the blocked nucleic acid molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected.

4. The method of claim 2, wherein the reporter moieties are coupled to the blocked nucleic acid molecules, and wherein upon cleavage by RNP2, a signal from the reporter moiety is detected.

5. The method of claim 1, wherein the reaction mixture comprises blocked nucleic acid molecules with bulky modifications and wherein the bulky modifications are about 1 nm in size.

6. The method of claim 6, wherein the reaction mixture comprises blocked nucleic acid molecules with bulky modifications and wherein the bulky modifications are about 0.7 nm in size.

7. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein there are two bulky modifications with one bulky modification located on the 5′ end of the blocked nucleic acid molecule and one bulky modification located on the 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ ends comprising the two bulky modifications are less than 11 nm from one another.

8. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is on a 5′ end of blocked nucleic acid molecules.

9. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is on a 3′ end of the blocked nucleic acid molecules.

10. The method of claim 1, wherein blocked nucleic acid molecules include bulky modifications and wherein the bulky modification is between two internal nucleic acid residues of the blocked nucleic acid molecules.

11. The method of claim 1, wherein the RNP2s comprise a variant nuclease and the variant nuclease comprises at least one mutation to the PAM-acting domain selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs.

12. The method of claim 11, wherein there are at least two mutations to the PAM-acting domain selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs.

13. The method of claim 12, wherein there are at least three mutations to the PAM-acting domain selected from mutations to amino acid residues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acid residues in orthologs.

14. The method of claim 1, wherein the RNP2s comprise a variant nuclease and the variant nuclease comprises at least one mutation to the PAM-acting domain of the variant nucleic acid-guided nuclease and wherein the at least one mutation is selected from mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2; mutations to amino acid residues K534, Y538 and R591 in relation to SEQ ID NO:3; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:4; mutations to amino acid residues K579, N583 and K635 in relation to SEQ ID NO:5; mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:6; from mutations to amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7; mutations to amino acid residues K617, N621 and K678 in relation to SEQ ID NO:8; mutations to amino acid residues K541, N545 and K601 in relation to SEQ ID NO:9; mutations to amino acid residues K569, N573 and K625 in relation to SEQ ID NO:10; mutations to amino acid residues K562, N566 and K619 in relation to SEQ ID NO:11; mutations to amino acid residues K645, N649 and K732 in relation to SEQ ID NO:12; mutations to amino acid residues K548, N552 and K607 in relation to SEQ ID NO:13; mutations to amino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; or mutations to amino acid residues K521, N525 and K577 in relation to SEQ ID NO:15.

15. The method of claim 1, wherein the RNP2s comprise a variant nucleic acid-guided nuclease comprising at least one mutation to the domains that interact with the PAM region or surrounding sequences on the blocked nucleic acid molecules and wherein single stranded DNA is cleaved at least two times faster than double stranded DNA is cleaved.

16. The method of claim 1, wherein the plurality of blocked nucleic acid molecules and the RNP2s are at a molar concentration of at least 2 blocked nucleic acids to 1 RNP2 in the reaction mixture.

17. The method of claim 1, wherein the target nucleic acid molecule of interest is of bacterial or viral origin.

18. The method of claim 1, wherein the target nucleic acid molecule of interest is from a human or other animal.

19. The method of claim 18, wherein the sample is selected from blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, a transudate, an exudate, or fluid obtained from a joint, or a swab of skin or mucosal membrane surface.

20. The method of claim 21, wherein the sample is a blood sample from a transplant patient and the target nucleic acid molecule is a donor-derived genomic sequence.

21. The method of claim 21, wherein the sample is a blood sample from a transplant patient and the target nucleic acid molecules are a hemoglobin S gene and a hemoglobin C gene.

22. The method of claim 20, wherein the target nucleic acid molecule is a pathogen that infects livestock.

23. The method of claim 1, wherein the sample is an environmental sample.

24. The method of claim 23, wherein the sample is selected from the group of a soil sample, an air sample, and a water sample.

25. The method of claim 24, wherein the sample is a sewer sample.

26. The method of claim 1, wherein the target nucleic acid molecule is a pathogen used as a bioweapon.

27. The method of claim 20, wherein the target nucleic acid is a human biomarker.

28. The method of claim 27, wherein the human biomarker is a cancer biomarker.

29. The method of claim 1, wherein there are at least ten target nucleic acid molecules of interest in the sample.

30. The method of claim 29, wherein there are at least twenty target nucleic acid molecules of interest in the sample.