Programmable nuclease improvements and compositions and methods for nucleic acid amplification and detection

Abstract

Disclosed herein are compositions, kits, and methods related to improved Cas activity. Through compositions and kits disclosed herein and practice of methods disclosed herein, one attains improved Cas activity such as Cas12 activity relative to Cas proteins in the art such as LbCas12. Further described herein are methods to detect target nucleic acid using a programmable nuclease system. Often, the target nucleic acids are present in at low frequency in the sample. Provided herein are methods for enriching these target nucleic acids for detection. Also described herein are methods to insert a PAM sequence into a target sequence of interest for use in a detection comprising a programmable nuclease.

1 . A composition comprising a programmable nuclease having at least 60% sequence identity to SEQ ID NO:11 and a non-naturally occurring guide nucleic acid. 
     
     
         2 . The composition of  claim 1 , further comprising a detector nucleic acid. 
     
     
         3 . The composition of  claim 1 , wherein the programmable nuclease recognizes a protospacer adjacent motif of 5′-YYN-3′. 
     
     
         4 . The composition of  claim 2 , further comprising a target nucleic acid, wherein when a region of said non-naturally occurring guide nucleic acid hybridizes to a portion of said target nucleic acid adjacent to a protospacer adjacent motif, said programmable nuclease cleaves detector nucleic acids at a rate of at least about 0.1 cleaved detector nucleic acid molecules per minute. 
     
     
         5 . The composition of  claim 4 , wherein the protospacer adjacent motif is 5′-YYN-3′. 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The composition of  claim 1 , wherein the programmable nuclease comprises three partial RuvC domains. 
     
     
         10 . The composition of  claim 1 , wherein the programmable nuclease comprises a RuvC-I subdomain, a RuvC-II subdomain, and a RuvC-III subdomain. 
     
     
         11 . (canceled) 
     
     
         12 . The composition of  claim 1 , wherein the programmable nuclease has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO:11. 
     
     
         13 . The composition of  claim 1 , wherein the programmable nuclease is SEQ ID NO: 11. 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The composition of  claim 1 , wherein the composition further comprises a buffer. 
     
     
         17 . The composition of  claim 16 , wherein the buffer comprises a buffering agent, a salt, a crowding agent, a detergent, or any combination thereof. 
     
     
         18 . The composition of  claim 17 , wherein the buffering agent is at a concentration of from 5 mM to 100 mM. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The composition of  claim 17 , wherein the salt is from 5 mM to 100 mM. 
     
     
         22 . (canceled) 
     
     
         23 . The composition of  claim 17 , wherein the crowding agent is from 0.5% (v/v) to 2% (v/v). 
     
     
         24 . (canceled) 
     
     
         25 . The composition of  claim 17 , wherein the detergent is about 2% (v/v) or less. 
     
     
         26 . (canceled) 
     
     
         27 . The composition of  claim 17 , wherein the buffering agent is HEPES. 
     
     
         28 . The composition of  claim 17 , wherein the salt is potassium acetate, magnesium acetate, sodium chloride, magnesium chloride, or any combination thereof. 
     
     
         29 . The composition of  claim 17 , wherein the crowding agent is glycerol. 
     
     
         30 . The composition of  claim 17 , wherein the detergent is Tween, Triton-X, or any combination thereof. 
     
     
         31 . The composition of  claim 17 , wherein a pH of the composition is from 7 to 8. 
     
     
         32 . The composition of  claim 16 , wherein a pH of the buffer is approximately 7.5. 
     
     
         33 . The composition of  claim 1 , wherein the composition is at a temperature of from 25° C. to 45° C. 
     
     
         34 . The composition of  claim 1 , wherein the programmable nuclease exhibits catalytic activity at a temperature of from 25° C. to 45° C. 
     
     
         35 . The composition of  claim 1 , wherein the programmable nuclease exhibits catalytic activity after heating the composition to a temperature of greater than 45° C. and restoring the temperature to from 25° C. to 45° C. 
     
     
         36 . A method of assaying for a segment of a target nucleic acid in a sample, the method comprising:
 contacting the sample to:
 a reporter comprising a detector nucleic acid; and 
 the composition of  claim 1 , wherein the non-naturally occurring guide nucleic acid hybridizes to a segment of the target nucleic acid; and 
   assaying for a signal produced by cleavage of the detector nucleic acid.   
     
     
         37 .- 183 . (canceled) 
     
     
         184 . The method of  claim 36 , wherein the signal is present prior to cleavage of the detector nucleic acid and changes upon cleavage of the detector nucleic acid or wherein the signal is absent prior to cleavage of the detector nucleic acid and is present upon cleavage of the detector nucleic acid. 
     
     
         185 . The method of  claim 36 , wherein the signal is a calorimetric, potentiometric, amperometric, optical, or piezo-electric signal. 
     
     
         186 . The method of  claim 185 , wherein the optical signal is a fluorescent or colorimetric signal. 
     
     
         187 . The method of  claim 36 , further comprising amplifying the target nucleic acid. 
     
     
         188 . The composition of  claim 1 , wherein the non-naturally occurring guide nucleic acid is a guide RNA. 
     
     
         189 . The composition of  claim 188 , wherein the guide RNA comprises a sequence that is 80% homologous to SEQ ID NO:512. 
     
     
         190 . The composition of  claim 1 , wherein the non-naturally occurring guide nucleic acid has at least 60% sequence identity to SEQ ID NO:512. 
     
     
         191 . The composition of  claim 1 , wherein the non-naturally occurring guide nucleic acid comprises a crRNA. 
     
     
         192 . The composition of  claim 1 , wherein the non-naturally occurring guide nucleic acid comprises RNA having at least 10 nucleotides that hybridize to a region in a target nucleic acid adjacent to a protospacer adjacent motif. 
     
     
         193 . The composition of  claim 1 , further comprising a reporter. 
     
     
         194 . The composition of  claim 193 , wherein the reporter comprises a detector nucleic acid and a detection moiety. 
     
     
         195 . The composition of  claim 194 , wherein the detection moiety comprises a fluorescent moiety, a quenching moiety, a fluorescent dye, an infrared dye, an ultraviolet dye, a fluorescence resonance energy transfer (FRET) pair, a polypeptide, a biotin, an avidin, a streptavidin, a polysaccharide, a polymer, or a nanoparticle. 
     
     
         196 . The composition of  claim 193 , wherein the detector nucleic acid comprises DNA, RNA, or a combination thereof. 
     
     
         197 . The composition of  claim 1 , further comprising an oligonucleotide primer or amplification reagent. 
     
     
         198 . The composition of  claim 197 , wherein the amplification reagent is selected from a recombinase, a single-stranded DNA binding (SSB) protein, a DNA polymerase, an RNA polymerase, a deoxynucleotide triphosphate, a nucleotide triphosphate, a reverse transcriptase, a helicase, a ligase, or any combination thereof.