US 11,807,877 B1
CRISPR/Cas activity assays and compositions thereof
Kyle Jeffrey Seamon, Livermore, CA (US); Brooke Nicole Harmon, Livermore, CA (US); Joseph S. Schoeniger, Oakland, CA (US); and Yooli Kim Light, Pleasanton, CA (US)
Assigned to National Technology & Engineering Solutions of Sandia, LLC, Albuquerque, NM (US)
Filed by National Technology & Engineering Solutions of Sandia, LLC, Albuquerque, NM (US)
Filed on Mar. 21, 2019, as Appl. No. 16/360,946.
Claims priority of provisional application 62/646,780, filed on Mar. 22, 2018.
Int. Cl. C07H 21/04 (2006.01); C12N 9/22 (2006.01); G01N 33/542 (2006.01); C12N 15/11 (2006.01); G01N 33/573 (2006.01); C12Q 1/6818 (2018.01)
CPC C12N 9/22 (2013.01) [C12N 15/11 (2013.01); C12N 15/111 (2013.01); C12Q 1/6818 (2013.01); G01N 33/542 (2013.01); G01N 33/573 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 24 Claims
 
1. A method for detecting nuclease activity, the method comprising:
combining a target nuclease with a cleavage substrate and a synthetic guiding component, thereby providing a reaction mixture;
incubating the reaction mixture, thereby providing an incubated reaction mixture;
quenching the incubated reaction mixture under a denaturation condition configured to denature the target nuclease; and
measuring a detectable signal, if any, arising from a reacted cleavage substrate;
wherein the target nuclease is a Cas9 homolog or ortholog;
wherein the cleavage substrate comprises a duplex having a target strand and a non-target strand, and wherein at least one of the target or non-target strand comprises a detectable label;
wherein the target strand comprises a structure having formula (Ia) of 5′-X-Y-T-Z-3′ or formula (Ib) of 5′-Z-T-Y-X-3′ or a salt thereof, wherein:
X is a first binding region comprising a nucleic acid sequence,
Y is a protospacer adjacent motif-binding region comprising a nucleic acid sequence configured to bind to a protospacer adjacent motif in the non-target strand,
T is a target site comprising a nucleic acid sequence configured to bind to a targeting portion of the synthetic guiding component, and
Z is a second binding region comprising a nucleic acid sequence; and
wherein the non-target strand comprises a structure having formula (IIa) of 5′-A-U-B-C-3′ or formula (IIb) of 5′-C-B-U-A-3′ or a salt thereof, wherein:
A is a third binding region comprising a nucleic acid sequence configured to bind to Z or a portion thereof,
U is a complementary target site comprising a nucleic acid sequence configured to bind T or a portion thereof,
B is the protospacer adjacent motif configured to interact with the target nuclease, and
C is a fourth binding region comprising a nucleic acid sequence configured to bind X or a portion thereof,
wherein the synthetic guiding component is configured to interact with the target nuclease, and wherein the cleavage substrate is configured to interact with the synthetic guiding component;
wherein the synthetic guiding component comprises a structure having formula (IIIa) of 5′-D-V-E-L-F-3′ or formula (IIIb) of 5′-F-L-E-V-D-3′ or a salt thereof, wherein:
D is an optional third portion comprising a nucleic acid sequence of from 1 to 20 nucleic acids;
V is a targeting portion comprising a nucleic acid sequence configured to bind to a target site of the cleavage substrate;
E is a first portion comprising a nucleic acid sequence configured to interact with a nuclease configured to bind and/or cleave the cleavage substrate;
L is a linker; and
F is a second portion comprising a nucleic acid sequence configured to interact with the target nuclease and E or a portion thereof;
E comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 150-159, 184-189, 213-220, 249-264, 282-287, 308-311, 331-336, 445-462, 485-496, 520-535, 610-627, 640-645, 664-671, 716-721, 729-734, 740-745, 753-756, 765-770, 779-784, 795-800, 845-852, 861-862, 871-878, 923-931, 940-946, 958-968, 975-981, 1010-1017, 1027-1038, 1078-1083, 1092-1101, 1111-1118, 1135-1150, and 1194-1199;
L comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 160-165, 190-195, 221-227, 265-270, 288-294, 312-317, 337-341, 435-444, 475-484, 508-519, 595-609, 634-639, 658-663, 713-715, 726-728, 737-739, 749-752, 761-764, 775-778, 791-794, 841-844, 857-860, 867-870, 919-922, 937-939, 955-957, 972-974, 1018-1022, 1039-1042, 1074-1077, 1090-1091, 1108-1110, 1127-1134, and 1192-1193; and
F comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 166-183, 196-212, 228-248, 271-281, 295-307, 318-330, 342-349, 425-434, 463-474, 498-507, 580-594, 628-633, 646-657, 710-712, 722-725, 735-736, 746-748, 757-760, 771-774, 785-790, 825-840, 853-856, 863-866, 915-918, 932-936, 947-954, 969-971, 1023-1026, 1043-1046, 1070-1073, 1084-1089, 1102-1107, 1119-1126, and 1180-1191.