CAS12A COMPOSITIONS AND METHODS
The present disclosure relates, in some embodiments to compositions comprising a variant Cas12a and one-pot methods of using a variant Cas12a with amplification to detect a sequence of interest.
1 . A composition comprising:
a variant Cas12a having an amino acid sequence at least 90% identical to one or more of SEQ ID NOS: 1-16, wherein the variant Cas12a has thermostable cis recognition activity and thermostable trans nuclease activity;
a crRNA operable with the variant Cas12a; and
optionally, a non-naturally occurring buffer.
2 . A composition according to claim 1 further comprising a trans nuclease substrate.
3 . A composition according to claim 1 , wherein the trans nuclease substrate comprises a fluorophore, a linker polynucleotide, and a quencher, wherein the fluorophore and the quencher are each operably linked to the linker polynucleotide and wherein the quencher is operable to quench the fluorophore.
4 . A composition according to claim 3 , wherein the linker polynucleotide is disposed between the fluorophore and quencher and is susceptible to trans nuclease cleavage.
5 . A composition according to claim 1 , wherein the molar ratio of variant Cas12a: crRNA is 1:1 to 1:20.
6 . A composition according to claim 1 , wherein the amino acid sequence is at least 95% identical to one or more of SEQ ID NOS: 1-16.
7 . A composition according to claim 1 , wherein the composition is cell-free.
8 . A composition according to claim 1 , wherein the composition has a dried, freeze dried, lyophilized, crystalline, or aqueous form.
9 . A composition according to claim 1 , wherein the variant Cas12a is immobilized on a support with or without a linker.
10 . A composition according to claim 1 further comprising a DNA polymerase, a glycosylase, a nicking enzyme, a ligase, a helicase, a recombinase, a crowding agent, a DNA binding protein, a dye, an additive, a ribonucleoprotein, and/or combinations thereof.
11 . A composition according to claim 1 further comprising a Bst DNA polymerase, Bsu DNA polymerase, phi29 DNA polymerase, phi29-XT DNA polymerase, and/or variants thereof.
12 . A composition according to claim 1 further comprising a uracil DNA glycosylase.
13 . A one-pot method comprising contacting at a single temperature and in a single container:
a variant Cas12a having an amino acid sequence at least 90% identical to one or more of SEQ ID NOS: 1-16, wherein the variant Cas12a has thermostable cis recognition activity and thermostable trans nuclease activity;
a crRNA operable with the variant Cas12a, wherein the molar ratio of variant Cas12a:
crRNA is optionally 1:1 to 1:20;
a polynucleotide comprising a nucleic acid sequence of interest;
amplification primers complementary to at least a portion of the sequence of interest and operable to support amplification of the sequence of interest;
optionally, a reverse transcriptase;
a DNA polymerase;
a trans nuclease substrate; and
optionally, a non-naturally occurring buffer.
14 . A method according to claim 13 , wherein the variant Cas12a and the crRNA are contacted in a single container under conditions that permit loop mediated amplification of the sequence of interest and trans nuclease cleavage of the trans nuclease substrate to produce a detectable trans nuclease cleavage marker.
15 . A method according to claim 13 , wherein the method is performed at a single temperature in a range of 50-70° C., 55-65° C., or 50-60° C.
16 . A method according to claim 13 , wherein the amplification primers and the polynucleotide comprising the nucleic acid sequence of interest hybridize to form an amplification substrate for the DNA polymerase.
17 . A method according to claim 14 further comprising amplifying, by the DNA polymerase, the amplification substrate to produce an amplification product.
18 . A method according to claim 17 , wherein the amplifying is selected from genome exponential amplification reaction (GEAR), helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), multiple displacement amplification (MDA), nicking enzyme amplification reaction (NEAR), nucleic acid sequence-based amplification (NASBA), ramification (RAM), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), self-sustained sequence replication (3SR), single primer isothermal amplification (SPIA), strand displacement amplification (SDA), transcription mediated amplification (TMA), or combinations thereof.
19 . A method according to claim 17 , wherein the crRNA hybridizes to the polynucleotide comprising the nucleic acid sequence of interest, the amplification product, or combinations thereof to form a cleavage assembly comprising the crRNA, the polynucleotide, and the variant Cas12a.
20 . A method according to claim 19 , wherein the variant Cas12a cleaves the polynucleotide comprising the nucleic acid sequence of interest.
21 . A method according to claim 20 , wherein the variant Cas12a cleaves the trans nuclease substrate to form a trans nuclease cleavage product.
22 . A method according to claim 21 further comprising detecting the trans nuclease cleavage product.
23 . A method according to claim 22 , wherein detecting the trans nuclease cleavage product comprises optically detecting the trans nuclease cleavage product and/or electrochemically detecting the trans nuclease cleavage product.
24 . A method according to claim 13 , wherein the molar ratio of variant Cas12a: crRNA is 1:1 to 1:20.
25 . A variant Cas12a having an amino acid sequence at least 90% identical to one or more of SEQ ID NOS: 1-16, wherein the variant Cas12a has thermostable cis recognition activity and thermostable trans nuclease activity.