	US 12,410,464 B2
	Detection assays
Ashish Pandey, San Diego, CA (US); Anurup Ganguli, San Diego, CA (US); Ariana Mostafa, San Diego, CA (US); and Jacob Berger, San Diego, CA (US)
Assigned to VedaBio, Inc., San Diego, CA (US)
Filed by VedaBio, Inc., San Diego, CA (US); and The Board of Trustees of the University of Illinois, Urbana, IL (US)
Filed on Feb. 25, 2024, as Appl. No. 18/586,486.
Application 18/586,486 is a continuation of application No. 18/372,098, filed on Sep. 24, 2023, granted, now 11,987,839.
Application 18/372,098 is a continuation of application No. 18/208,272, filed on Jun. 10, 2023, granted, now 11,970,730.
Application 18/208,272 is a continuation of application No. 18/204,337, filed on May 31, 2023, granted, now 11,821,025, issued on Nov. 21, 2023.
Application 18/204,337 is a continuation of application No. 18/106,420, filed on Feb. 6, 2023, granted, now 11,702,686, issued on Jul. 18, 2023.
Application 18/106,420 is a continuation of application No. 17/861,208, filed on Jul. 9, 2022, granted, now 11,639,520, issued on May 2, 2023.
Claims priority of provisional application 63/289,112, filed on Dec. 13, 2021.
Claims priority of provisional application 63/220,987, filed on Jul. 12, 2021.
Prior Publication US 2024/0218428 A1, Jul. 4, 2024
Int. Cl. C12Q 1/6823 (2018.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/682 (2018.01)

CPC C12Q 1/6823 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/682 (2013.01); C12N 2310/20 (2017.05); C12N 2310/315 (2013.01); C12N 2310/321 (2013.01); C12N 2310/3231 (2013.01); C12N 2320/10 (2013.01)]

21 Claims

1. A cascade assay method for detecting a target nucleic acid of interest in a sample comprising the steps of:

(a) providing a reaction mixture comprising:

i. a first ribonucleoprotein (RNP) complex (RNP1) comprising a first Cas12a nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity;

ii. a second ribonucleoprotein complex (RNP2) comprising a second Cas12a nucleic acid-guided nuclease and a second gRNA; wherein the second gRNA is complementary to synthesized activating molecules and the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity;

iii. a plurality of template molecules comprising a sequence having homology to the second gRNA and a primer binding domain;

iv. a plurality of blocked primer molecules comprising a sequence complementary to the primer binding domain of the plurality of template molecules and wherein the blocked primer molecules cannot be extended by a polymerase;

v. a Phi29 or T7 DNA polymerase and dNTPs;

wherein when blocked, the blocked primer molecules cannot bind to the template molecules and be extended by the polymerase and wherein when unblocked, the blocked primer molecules can bind to the primer binding domain of the template molecules and be extended by the polymerase to produce synthesized activating molecules;

(b) contacting the reaction mixture with the sample under conditions that allow target nucleic acids of interest in the sample to bind to the first gRNA;

wherein:

upon binding of the target nucleic acid of interest, the RNP1 becomes active cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase;

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;

at least one synthesized activating molecule binds to the second gRNA, and RNP2 becomes active cleaving at least one further blocked primer molecule;

and