Methods and compositions for processing and amplification of nucleic acids

Abstract

Provided herein are methods and compositions for processing a target nucleic acid sequence. The methods and compositions provided herein comprise a guide complex for recruiting an enzyme which can introduce a cut on the target nucleic acid sequence and a cut on the guide complex. The methods and compositions provided herein comprise adapter regions of the guide complex and / or adapter molecules that modulate a reaction rate. Modified adapters can affect a reaction rate and enhance processing of target nucleic acid sequences. The processed target nucleic acid sequence can be used in further applications such as nucleic acid amplification (e.g., isothermal amplification).

Description

METHODS AND COMPOSITIONS FOR PROCESSING AND AMPLIFICATION OF NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 558,079, filed February 26, 2024, and U.S. Provisional Patent Application No. 63 / 647,925, filed May 15, 2024, the entire contents of which are entirely incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] Nucleic acid amplification techniques such as polymerase chain reaction (PCR) and various isothermal amplification techniques have become an integral part of nucleic acid-based diagnostics and research techniques.SUMMARY OF THE INVENTION

[0003] Recognized herein is a need for improved methods and compositions for processing target nucleic acid molecules with high efficiency and / or simplified optimization process for reaction conditions. The methods and compositions described herein can be used for generating initial products for downstream applications such as isothermal amplifications.

[0004] Provided herein are methods and compositions comprising a non-target binding region (e.g., adapter or adapter region) of a guide polynucleotide that can modulate reaction rate in a targeted manner. The non-target binding region (e.g., adapter or adapter region) can comprise modified nucleotides which may further affect the reaction rate. The adapter can be a region of a guide polynucleotide and / or a separate molecule added into the reaction, and the adapter can act as a rate modulator, while not taking part in the reaction.

[0005] In an aspect, the present disclosure provides a method of processing a single-stranded nucleic acid molecule comprising a target sequence, said method comprising: (a) contacting said single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions sufficient to allow said guide polynucleotide to hybridize to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, wherein said non-target binding region comprises a modified nucleotide, and (ii) a target binding region that hybridizes to said target sequence, and (b) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bind to said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end.

[0006] In some embodiments, said guide polynucleotide further comprises a blocked 3' end non- extendable by a polymerase. In some embodiments, said guide polynucleotide further comprises an unblocked 3’ end. In some embodiments, said non-target binding region comprises at least two modified nucleotides. In some embodiments, said modified nucleotide comprises 2’-O- methoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base. In some embodiments, the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP).

[0007] In some embodiments, the universal base comprises deoxyinosine, nitroindole, 2’- deoxynebularine, or 3 -nitropyrrole. In some embodiments, said modified nucleotide does not comprise adenine, guanine, thymine, or cytosine.

[0008] In some embodiments, a reaction launch rate of said type Ils restriction enzyme is reduced compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non-target binding region comprising said modified nucleotide or (ii) with said non-target binding region that does not comprise said modified nucleotide, and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

[0009] In some embodiments, a reaction launch rate of said type Ils restriction enzyme is increased compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non-target binding region comprising said modified nucleotide or (ii) with said non-target binding region that does not comprise said modified nucleotide, and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

[0010] In an aspect, the present disclosure provides a method of processing a single-stranded nucleic acid molecule comprising a target sequence, said method comprising: (a) contacting said single-stranded nucleic acid molecule with a guide complex and a non-target binding molecule in a reaction, wherein said guide complex comprises a guide polynucleotide under conditions sufficient to allow said guide polynucleotide to hybridize to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, and (ii) a target binding region that hybridizes to said target sequence, and (b) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bindto said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end.

[0011] In some embodiments, said guide polynucleotide further comprises a blocked 3' end non- extendable by a polymerase. In some embodiments, said guide polynucleotide further comprises an unblocked 3' end. In some embodiments, said non-target binding molecule has the same sequence as a sequence of said non-target binding region of said guide polynucleotide. In some embodiments, said non-target binding molecule has a different sequence than a sequence of said non-target binding region of said guide polynucleotide.

[0012] In some embodiments, said non-target binding molecule and / or said non-target binding region of said guide polynucleotide comprises a modified nucleotide. In some embodiments, said modified nucleotide comprises 2’-O-methoxy-ethyl modified nucleotide, aminoethyl- phenoxazine-deoxy cytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base. In some embodiments, the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP). In some embodiments, the universal base comprises deoxyinosine, nitroindole, 2’- deoxynebularine, or 3 -nitropyrrole.

[0013] In some embodiments, the modified nucleotide does not comprise adenine, guanine, thymine, or cytosine. In some embodiments, said non-target binding molecule is soluble. In some embodiments, said non-target binding molecule is not immobilized on a surface. In some embodiments, said non-target binding molecule is immobilized on a surface. In some embodiments, the surface comprises a bead, an antibody, a molecularly imprinted polymer, an aptamer, or a surface of a reaction vial. In some embodiments, said non-target binding molecule is immobilized on said surface via linker.

[0014] In some embodiments, said linker comprises PC Linker Phosphoramidite Spacer Phosphoramidite 9, 5'-Amino-Modifier TEG CE-Phosphoramidite, 5'-Aminooxy-Modifier-l 1- CE Phosphoramidite, Spacer Phosphoramidite 18, Cholesteryl-TEG Phosphoramidite, DNP-TEG Phosphoramidite, 3'-Spacer C3 CPG, 6-FAM-TEG Azide, 5'-DBCO-TEG Phosphoramidite, a- Tocopherol-TEG Phosphoramidite, 5'-Cholesteryl-TEG Phosphoramidite, 3 '-Cholesteryl-TEG CPG, 5'-Biotin II Phosphoramidite, Psoralen C6 Phosphoramidite, dC-CPG 1000, dC-CPG 2000, dG-CPG 2000, dT-CPG 2000, PC Amino-Modifier Phosphoramidite, Azobenzene Phosphoramidite, Thiol-Modifier C6 S-S, 5'-Carboxy -Modifier CIO, 3 '-Protected Biotin Serinol CPG, Protected BiotinLC Serinol Phosphoramidite, 6-Fluorescein Serinol Phosphoramidite, Protected Biotin Serinol Phosphoramidite, Maleimide NHS Ester (SMCC), N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'- dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, N-Methyl-succinimido[3,4-b]-7- oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5- succinoyl long chain alkylamino CPG, Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6- (4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, or any combination thereof.

[0015] In some embodiments, a reaction rate of said reaction is reduced compared to a reaction rate of an otherwise identical reaction without said non-target binding molecule. In some embodiments, a reaction rate of said reaction is increased compared to a reaction rate of an otherwise identical reaction without said non-target binding molecule. In some embodiments, a reaction launch rate of said type Ils restriction enzyme is reduced compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non- target binding region and said non-target binding molecule, (ii) without said non-target binding region, or (iii) with said non-target binding region and without said non-target binding molecule, and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

[0016] In some embodiments, a reaction launch rate of said type Ils restriction enzyme is increased compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non-target binding region and said non-target binding molecule, (ii) without said non-target binding region, or (iii) with said non-target binding region and without said non-target binding molecule, and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

[0017] In some embodiments, said non-target binding molecule has a shorter length than a length of said non-target binding region of said guide polynucleotide. In some embodiments, said non- target binding molecule has a longer length than a length of said non-target binding region of said guide polynucleotide. In some embodiments, said non-target binding molecule has the same length as a length of said non-target binding region of said guide polynucleotide. In some embodiments, said non-target binding region is at least about 12 nucleotides in length.

[0018] In some embodiments, said guide polynucleotide is a first guide polynucleotide, and said guide complex comprises a second guide polynucleotide, wherein said second guide polynucleotide comprises (i) a non-target binding region that is complementary with said non- target binding region of said first guide polynucleotide and (ii) a target binding region configuredto hybridize to said target sequence. In some embodiments, when said first guide polynucleotide of said guide complex is hybridized to said target polynucleotide sequence, said target binding region of said second guide polynucleotide is not hybridized to said target sequence. In some embodiments, said first guide polynucleotide and said second guide polynucleotide hybridize to form a dimer. In some embodiments, said first guide polynucleotide and said second guide polynucleotide hybridize via said non-target binding region of said first guide polynucleotide and said second guide polynucleotide to form said dimer having a double-stranded binding region.

[0019] In some embodiments, said double-stranded binding region comprises said restriction endonuclease recognition sequence. In some embodiments, said type Ils restriction enzyme binds to said double-stranded binding region of said dimer. In some embodiments, the method further comprises amplifying said single-stranded nucleic acid molecule comprising a target sequence, comprising: extending said extendable 3' end using said polymerase.

[0020] In some embodiments, said amplifying occurs at an amplification rate that is reduced compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding region comprising said modified nucleotide. In some embodiments, said amplifying occurs at an amplification rate that is increased compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding region comprising said modified nucleotide. In some embodiments, said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value of an otherwise identical nucleic acid amplification without said non-target binding region comprising said modified nucleotide.

[0021] In some embodiments, said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method. In some embodiments, said existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA). In some embodiments, said cycle threshold value is at most 15 minutes.

[0022] In some embodiments, the method further comprises amplifying said single-stranded nucleic acid molecule comprising a target sequence, comprising: extending said extendable 3' end of said target sequence using said polymerase. In some embodiments, said amplifying occurs at an amplification rate that is reduced compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding molecule. In some embodiments,said amplifying occurs at an amplification rate that is reduced compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding molecule. In some embodiments, said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value of an otherwise identical nucleic acid amplification without said non-target binding molecule.

[0023] In some embodiments, said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method. In some embodiments, said existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA). In some embodiments, said cycle threshold value is at most 15 minutes.

[0024] In some embodiments, the method further comprises amplifying said single-stranded nucleic acid molecule comprising a target sequence, comprising: extending said extendable 3' end with said polymerase to generate an extension product, wherein said extension product displaces said second guide polynucleotide; cutting said first guide polynucleotide within said target binding region to expose an extendable 3' end of said first guide polynucleotide; and extending said extendable 3' end of said first guide polynucleotide using said polymerase to generate a complementary molecule of said target sequence of said single-stranded nucleic acid molecule, thereby amplifying said single-stranded nucleic acid molecule.

[0025] In some embodiments, the method further comprises repeating (d) and (e) to generate a plurality of complementary molecules of said target sequence of said single-stranded nucleic acid molecule. In some embodiments, an additional guide complex binds to said complementary molecule. In some embodiments, the method further comprises using said complementary molecule with said additional guide complex bound thereto as a starting template to generate copies of said target molecule. In some embodiments, said type Ils restriction enzyme comprises N.BstNBI, N.Bst9 I, N.BspD6I, a functional fragment thereof, or a combination thereof. In some embodiments, said blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof. In some embodiments, said ddNTP is ddATP, ddGTP, ddCTP, or ddTTP.

[0026] In some embodiments, said single-stranded nucleic acid molecule with said cut and said guide polynucleotide bound thereto is used as a starting template for an amplification. In some embodiments, said amplification is an isothermal amplification. In some embodiments, saidenzyme exhibits a high-frequency endonuclease activity. In some embodiments, said high- frequency endonuclease activity is from a large subunit of said enzyme. In some embodiments, said enzyme exhibits a low-frequency endonuclease activity. In some embodiments, said low- frequency endonuclease activity is from a small subunit of said enzyme. In some embodiments, said enzyme exhibits at least two differential enzymatic activity rates. In some embodiments, said enzyme comprises at least two or more subunits.

[0027] In some embodiments, each subunit of said at least two or more subunits exhibit a different enzymatic activity rate. In some embodiments, said enzyme is a multimeric enzyme.

[0028] In some embodiments, said at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites. In some embodiments, one of said at least two differential endonuclease activity rates comprises cutting said target sequence of said single-stranded nucleic acid molecule with low frequency. In some embodiments, said reaction launch rate is reduced by reducing said low frequency. In some embodiments, one of said two differential endonuclease activity rates comprises cutting said target binding region of said guide polynucleotide with high frequency. In some embodiments, said two differential endonuclease activity rates are asymmetric or non-equal. In some embodiments, said enzyme comprises BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nucleases, zinc finger nucleases (ZFNs), N.Bst9 I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt. Alwl, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Mval269I, Nb.BpulOI, and Nt.BpulOI, a functional fragment thereof, or a combination thereof.

[0029] In some embodiments, a temperature is changed over a course of said method. In some embodiments, a first activity rate of said at least two differential enzymatic activity rates is favored at a first temperature, and a second activity rate of said at least two differential enzymatic activity rates is favored at a second temperature different from said first temperature. In some embodiments, said enzyme comprises two different active sites or endonuclease domains conferring at least two differential enzymatic activities. In some embodiments, said target binding region is at least about 12 to about 25 nucleotides in length. In some embodiments, a concentration of said guide polynucleotide is at least about 0.1 pM, at least about 1 pM, or about 0.1 pM to about 4 pM.

[0030] In some embodiments, said non-target binding region comprises a palindromic sequence. In some embodiments, said non-target binding region comprises a partially palindromic sequence. In some embodiments, said non-target binding region does not comprise a palindromic sequence. In some embodiments, said non-target binding region is self-complementary. In some embodiments, said single-stranded nucleic acid molecule is a single-stranded deoxyribonucleicacid (ssDNA) or a single- stranded ribonucleic acid (ssRNA). In some embodiments, said polymerase has strand displacement activity. In some embodiments, said single-stranded nucleic acid molecule comprises two or more single-stranded nucleic acid molecules, each singlestranded nucleic acid molecule comprising a different target sequence. In some embodiments, said two or more single-stranded nucleic acid molecules are contained within a single reaction mixture.

[0031] In an aspect, the present disclosure provides a method of processing a plurality of different single-stranded nucleic acid molecules comprising a first target molecule and a second target molecule, said method comprising: (a) contacting said first target molecule with a first guide complex comprising a first guide polynucleotide under conditions sufficient to allow said first guide polynucleotide to hybridize to said first target molecule, wherein said first guide polynucleotide comprises: (i) a first non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and (ii) a first target binding region that hybridizes to said first target molecule; (b) contacting said second target molecule with a second guide complex comprising a second guide polynucleotide under conditions sufficient to allow said second guide polynucleotide to hybridize to said second target molecule, wherein said second guide polynucleotide comprises: (i) a second non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and (ii) a second target binding region that hybridizes to said second target molecule, wherein said first non-target binding region and said second non-target binding region have a different sequence or a different length; and (c) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bind to said restriction endonuclease recognition sequence and cut within said first target molecule or said second target molecule, wherein contacting in (a) and contacting in (b) are in a same mixture.

[0032] In some embodiments, said first guide polynucleotide further comprises (iii) a first blocked 3' end non-extendable by a polymerase. In some embodiments, said first guide polynucleotide further comprises (iii) a first unblocked 3' end. In some embodiments, said second guide polynucleotide further comprises (iii) a second blocked 3' end non-extendable by a polymerase. In some embodiments, said second guide polynucleotide further comprises (iii) a second unblocked 3' end. In some embodiments, said first blocked 3' end and / or said second blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof. In some embodiments, said cut within said first target molecule generates a first extendable 3’ end. In some embodiments, said cut within said second target molecule generates a second extendable 3 ’ end.

[0033] In some embodiments, said first non-target binding region and said second non-target binding region have at most about 98% sequence identity. In some embodiments, said first non- target binding region has a length that is at least two nucleotides longer than a length of said second non-target binding region. In some embodiments, said first non-target binding region has a length that is at least two nucleotides shorter than a length of said second non-target binding region. In some embodiments, said first non-target binding region comprises a first modified nucleotide.

[0034] In some embodiments, said first modified nucleotide comprises 2’-O-methoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base.

[0035] In some embodiments, the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP). In some embodiments, the universal base comprises deoxyinosine, nitroindole, 2’-deoxynebularine, or 3 -nitropyrrole. In some embodiments, said first modified nucleotide does not comprise adenine, guanine, thymine, or cytosine.

[0036] In some embodiments, said second non-target binding region comprises a second modified nucleotide. In some embodiments, said second modified nucleotide comprises 2’-O- methoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base. In some embodiments, the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP).

[0037] In some embodiments, the universal base comprises deoxyinosine, nitroindole, 2’- deoxynebularine, or 3 -nitropyrrole. In some embodiments, said second modified nucleotide does not comprise adenine, guanine, thymine, or cytosine.

[0038] In some embodiments, a reaction launch rate of said type Ils restriction enzyme on said first target molecule is different from a reaction launch rate of said type Ils restriction enzyme on said second target molecule, and wherein said reaction launch rate is a rate for generating copies of extendable products of said first target molecule or said second target molecule with an extendable 3' end per second. In some embodiments, the method further comprises amplifying said plurality of single-stranded nucleic acid molecules comprising said first target molecule and said second target molecule, comprising: extending said first extendable 3' end of said first targetmolecule and said second extendable 3' end of said second target molecule using said polymerase.

[0039] In some embodiments, the method further comprises amplifying said plurality of singlestranded nucleic acid molecules comprising said first target molecule and said second target molecule, comprising: extending said first extendable 3' end of said first target molecule and said second extendable 3' end of said second target molecule using said polymerase to generate a plurality of extension products, wherein said extension products displace said first guide polynucleotide and said second guide polynucleotide; cutting said first guide polynucleotide within said first target binding region to expose a first extendable 3' end of said first guide polynucleotide; cutting said second guide polynucleotide within said second target binding region to expose a second extendable 3' end of said second guide polynucleotide; extending said first extendable 3' end of said first guide polynucleotide using said polymerase to generate a first complementary molecule of said first target molecule of said plurality of single-stranded nucleic acid molecules; and extending said second extendable 3' end of said second guide polynucleotide using said polymerase to generate a second complementary molecule of said second target molecule of said plurality of single-stranded nucleic acid molecules, thereby amplifying said plurality of single-stranded nucleic acid molecules.

[0040] In some embodiments, an amplification rate of said first target molecule is different from an amplification rate of said second target molecule. In some embodiments, said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method. In some embodiments, said existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA). In some embodiments, said cycle threshold value is at most 20 minutes.

[0041] In some embodiments, an additional guide complex binds to said first complementary molecule and / or said second complementary molecule. In some embodiments, the method further comprises using said first complementary molecule with said additional guide complex bound thereto as a starting template to generate copies of said first target molecule. In some embodiments, the method further comprises using said second complementary molecule with said additional guide complex bound thereto as a starting template to generate copies of said second target molecule. In some embodiments, said additional guide complex for said first complementary molecule is different than a secondary guide complex for said secondcomplementary molecule. In some embodiments, said type Ils restriction enzyme comprises N.BstNBI, N.Bst9 I, N.BspD6I, a functional fragment thereof, or a combination thereof. In some embodiments, said first blocked 3' end and / or said second blocked 3’ end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof. In some embodiments, said ddNTP is ddATP, ddGTP, ddCTP, or ddTTP.

[0042] In some embodiments, said plurality of single-stranded nucleic acid molecules with said cut and said first guide polynucleotide and said second guide polynucleotide bound thereto are used as starting templates for an amplification. In some embodiments, said amplification is an isothermal amplification. In some embodiments, said enzyme exhibits a high-frequency endonuclease activity. In some embodiments, said high-frequency endonuclease activity is from a large subunit of said enzyme. In some embodiments, said enzyme exhibits a low-frequency endonuclease activity. In some embodiments, said low-frequency endonuclease activity is from a small subunit of said enzyme. In some embodiments, said enzyme exhibits at least two differential enzymatic activity rates. In some embodiments, said enzyme comprises at least two or more subunits. In some embodiments, each subunit of said at least two or more subunits exhibits a different enzymatic activity rates. In some embodiments, said enzyme is a multimeric enzyme.

[0043] In some embodiments, said at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites. In some embodiments, one of said at least two differential endonuclease activity rates comprises cutting said first target molecule and / or second target molecule of said plurality of single-stranded nucleic acid molecules with low frequency. In some embodiments, one of said two differential endonuclease activity rates comprises cutting said first target binding region of said first guide polynucleotide and / or said second target binding region of said second guide polynucleotide with high frequency. In some embodiments, said two differential endonuclease activity rates are asymmetric or non-equal. In some embodiments, said enzyme comprises BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nucleases, zinc finger nucleases (ZFNs), N.Bst9 I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt. Alwl, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Mval269I, Nb.BpulOI, and Nt.BpulOI, a functional fragment thereof, or a combination thereof.

[0044] In some embodiments, a temperature is changed over a course of said method. In some embodiments, a first activity rate of said at least two differential enzymatic activity rates is favored at a first temperature, and a second activity rate of said at least two differential enzymatic activity rates is favored at a second temperature different from said first temperature. In someembodiments, said enzyme comprises two different active sites or endonuclease domains conferring at least two differential enzymatic activities. In some embodiments, said first target binding region and said second target binding region are each at least about 12 to about 25 nucleotides in length. In some embodiments, a concentration of said first guide polynucleotide and / or said second guide polynucleotide is at least about 0.1 pM, at least about 1 pM, or about 0.1 pM to about 4 pM. In some embodiments, said first non-target binding region and / or said second non-target binding region comprises a palindromic sequence.

[0045] In some embodiments, said first non-target binding region and / or said second non-target binding region is self-complementary. In some embodiments, said plurality of single-stranded nucleic acid molecules are a plurality of single-stranded deoxyribonucleic acid (ssDNA) molecules or a plurality of single-stranded ribonucleic acid (ssRNA) molecules. In some embodiments, said polymerase has strand displacement activity. In some embodiments, said plurality of single-stranded nucleic acid molecules are contained within a single reaction mixture.

[0046] In an aspect, the present disclosure provides a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule having bound thereto a guide complex, wherein said guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of said single-stranded nucleic acid molecule, wherein said non-target binding region comprises a modified nucleotide, and (ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme.

[0047] In an aspect, the present disclosure provides a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule having bound thereto a guide complex and a non-target binding molecule, wherein said guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of said single-stranded nucleic acid molecule, and (ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme.

[0048] In some embodiments, said non-target binding molecule is separated from said guide complex. In some embodiments, said non-target binding molecule is immobilized on a solid surface. In some embodiments, said non-target binding molecule is soluble.

[0049] In an aspect, the present disclosure provides a polynucleotide-polypeptide complex comprising: a plurality of single-stranded nucleic acid molecules having bound thereto a first guide complex and a second guide complex, wherein said first guide complex comprises: (i) a first primary guide polynucleotide comprising, from 5' to 3', a first non-target binding region and a first target binding region that hybridizes with a first target molecule of said plurality of singlestranded nucleic acid molecules; and (ii) a first secondary guide polynucleotide that hybridizes with said first non-target binding region of said first target molecule to form a first doublestranded binding region, wherein said first double-stranded binding region comprises a first restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme; and wherein said second guide complex comprises: (i) second primary guide polynucleotide comprising, from 5' to 3', a second non-target binding region and a second target binding region that hybridizes with a second target molecule of said plurality of single-stranded nucleic acid molecules; and (ii) a second secondary guide polynucleotide that hybridizes with said second non-target binding region of said second target molecule to form a second double-stranded binding region, wherein said second double-stranded binding region comprises a second restriction endonuclease recognition sequence for said enzyme, wherein said first non-target binding region and said second non-target binding region have a different sequence or a different length.

[0050] In some embodiments, said first non-target binding region and said second non-target binding region are configured to be recognized by a same enzyme. In an aspect, the present disclosure provides a kit comprising a guide complex or a guide polynucleotide described herein. In some embodiments, said kit further comprises a probe or a dye for detecting an amplification product generated using said kit. In some embodiments, said kit further comprises an informational material describing an instruction of using said kit.

[0051] In an aspect, the present disclosure provides a method of adjusting a reaction rate of a nucleic acid amplification, said method comprising: (a) contacting a single-stranded nucleic acid molecule comprising a target sequence with a guide complex comprising a guide polynucleotide in a reaction under conditions sufficient to allow said guide polynucleotide to hybridize to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises: (i) a non- target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, and (ii) a target binding region that hybridizes to said target sequence; (b) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bind said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end; (c) changing a sequence or a length of said non-target binding region to provide a changed non-target binding region, introducing a modified nucleotide into said non-target binding region to provide a changed non-target binding region, or adding in a non-target binding molecule in said reaction to adjust said reaction rate; and (d) repeating (a)-(b) with said guide polynucleotide comprising said changed non-target binding region or with said non-target binding molecule in said reaction.

[0052] In some embodiments, said guide polynucleotide further comprises a blocked 3' end non- extendable by a polymerase. In some embodiments, said guide polynucleotide further comprises an unblocked 3' end.

[0053] In an aspect, the present disclosure provides a method of processing a single-stranded nucleic acid molecule comprising a target sequence, said method comprising: (a) contacting said single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where said guide polynucleotide hybridizes to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and (ii) a target binding region configured to hybridize to said target sequence, and (b) introducing said type Ils restriction enzyme under conditions sufficient to cause said type Ils restriction enzyme to bind said restriction endonuclease recognition sequence and cut within said target sequence, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

[0054] In some embodiments, said guide polynucleotide further comprises a blocked 3' end non- extendable by a polymerase. In some embodiments, said guide polynucleotide further comprises an unblocked 3' end.

[0055] In an aspect, the present disclosure provides a method of amplifying a single-stranded nucleic acid molecule comprising a target sequence, said method comprising: (a) contacting said single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where said guide polynucleotide hybridizes to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, and (ii) a target binding region configured to hybridize to said target sequence, and (b) introducing said type Ils restriction enzyme under conditions sufficient to cause said type Ils restriction enzyme to bind said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end; and (c) extending said extendable 3' end using a polymerase, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

[0056] In some embodiments, said guide polynucleotide further comprises a blocked 3' end non- extendable by a polymerase. In some embodiments, said guide polynucleotide further comprises an unblocked 3' end.

[0057] In an aspect, the present disclosure provides a method of amplifying a single-stranded nucleic acid molecule comprising a target sequence, said method comprising: (a) contacting a guide complex with said single-stranded nucleic acid molecule, wherein said guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with said target sequence of said single-stranded nucleic acid molecule, and (ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region binds to an enzyme; (b) cutting said target sequence using said enzyme to expose an extendable 3' end; (c) extending said extendable 3' end with a polymerase to generate an extension product, wherein said extension product displaces said second guide polynucleotide; (d) cutting said first guide polynucleotide within said target binding region to expose an extendable 3' end of said first guide polynucleotide; and (e) extending said extendable 3' end of said first guide polynucleotide using said polymerase to generate a complementary molecule of said target sequence of said single-stranded nucleic acid molecule, thereby amplifying said single-stranded nucleic acid molecule, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

[0058] In some embodiments, said first guide polynucleotide and / or said second guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase. In some embodiments, said first guide polynucleotide and / or said second guide polynucleotide further comprises an unblocked 3' end.

[0059] In an aspect, the present disclosure provides a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule having bound thereto a guide complex, wherein said guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of said single-stranded nucleic acid molecule, and (ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

[0060] In some embodiments, said first guide polynucleotide and / or said second guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase. In someembodiments, said first guide polynucleotide and / or said second guide polynucleotide further comprises an unblocked 3' end.

[0061] In an aspect, the present disclosure provides a system of processing a single-stranded nucleic acid molecule comprising a target sequence, said system comprising: said single-stranded nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide, wherein said guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to said target sequence, and (iii) said enzyme bound to said restriction endonuclease recognition sequence of said non-target binding region, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

[0062] In some embodiments, said guide polynucleotide further comprises a blocked 3' end non- extendable by a polymerase. In some embodiments, said guide polynucleotide further comprises an unblocked 3' end.

[0063] In an aspect, the present disclosure provides a system for processing a plurality of singlestranded nucleic acid molecules, each comprising a different target sequence, said system comprising: a first single-stranded nucleic acid molecule wherein said first single-stranded nucleic acid molecule is bound to a first guide complex comprising a first guide polynucleotide, wherein said first guide polynucleotide comprises: (i) a first non-target binding region comprising a first restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme; (ii) a first target binding region configured to hybridize to a first target sequence; and (iii) a second single-stranded nucleic acid molecule wherein said second singlestranded nucleic acid molecule is bound to a second guide complex comprising a second guide polynucleotide, wherein said second guide polynucleotide comprises: (i) a second non-target binding region comprising a second restriction endonuclease recognition sequence for said enzyme that is a type Ils restriction enzyme; (ii) a second target binding region configured to hybridize to a second target sequence; and wherein said enzyme that is a type Ils restriction enzyme binds to said first restriction endonuclease recognition sequence of said first non-target binding region or said second restriction endonuclease recognition sequence of said second non- target binding region, wherein said first single-stranded nucleic acid molecule or said first target sequence is from Bacillus anthracis, and said second single-stranded nucleic acid molecule or said second target sequence is from Bacillus anthracis.

[0064] In some embodiments, said first guide polynucleotide and / or said second guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase. In someembodiments, said first guide polynucleotide and / or said second guide polynucleotide further comprises an unblocked 3' end.

[0065] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0066] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0068] FIGs. 1A-1O show an example of precursor steps leading to an isothermal amplification cycle according to various embodiments described herein. FIG. 1A depicts the duplexed oligo complex binding to the target nucleic acid strand. FIG. IB depicts endonucleolytic activity on the duplexed oligo / target complex. FIG. 1C depicts a polymerase extending off of the 3' end of the target strand. FIG. ID depicts the polymerase displacing the duplexed guide molecule. FIG. IE depicts endonucleolytic activity on the oligo / extension product complex. FIG. IF depicts a polymerase extending off the 3' end of the cut oligo and displacement of the guide. FIG. 1G depicts endonuclease activity on the newly synthesized portion complementary to the target strand. FIG. 1H depicts a polymerase extending off the 3' end of the cut site and displacement of the synthesized complement to the target strand. FIG. II depicts the displaced complement acting as a new target for the second complementary strand duplexed oligo complex. FIG. 1 Jdepicts the polymerase displacing the second complementary strand duplexed guide molecule. FIG. IK depicts the completed extension on the new guide molecule. FIG. IL depicts endonucleolytic activity on the second complementary strand oligo / extension product complex. FIG. IM depicts a polymerase extending off the 3’ end of the cut site of the second complementary strand of the oligo / extension product complex. FIG. IN depicts endonucleolytic activity on the newly synthesized complementary strand of the second complementary strand guide. FIG. IO depicts the displaced and single stranded synthesized fragments as starting material for a strand displacement amplification reaction.

[0069] FIGs. 2A-2C show a system for the creation of products suitable for amplification by isothermal amplification reactions using a guide molecule with a point mutation relative to the target sequence. FIG. 2A depicts a guide molecule with a point mutation binding to a target DNA and an endonuclease cutting the target. FIG. 2B depicts extension off the target at the 3' end and an endonuclease cutting the guide molecule. FIG. 2C depicts the displacement of the guide complementary to the target after endonucleolytic cutting with subsequent synthesis of a new strand. The opposite strand guide extension will only see the base that originated from the target and not the base that exists in the guide oligo sequence due to the lack of extension off the end of the guide oligo.

[0070] FIGs. 3A-3B show a control experiment where there is no mismatch between the guide and primer. FIG. 3A depicts the guide oligos, probes, and target sequence used in the control experiment with no mismatch between guide oligos and target. FIG. 3B depicts the amplification result of the control reaction without a point mutation.

[0071] FIGs. 4A-4B show an experiment where there is an A to C mismatch between the guide and primer. FIG. 4A depicts the guide oligos, probes, and target sequence used in the mismatch experiment. FIG. 4B depicts the amplification resulting in probe signal that is of target origin; rather than probe signal that is of guide oligo origin indicative of an asymmetric endonuclease activity.

[0072] FIGs. 5A-5B show a control experiment where there is no mismatch between the guide and primer. FIG. 5A depicts the guide oligos, probes, and target sequence used in the control experiment. FIG. 5B depicts the amplification result of the control reaction without a point mutation.

[0073] FIGs. 6A-6B show an experiment where there is an A to C mismatch between the guide and primer. FIG. 6A depicts the guide oligos, probes, and target sequence used in the mismatch experiment. FIG. 6B depicts the amplification resulting in probe signal that is of target origin;rather than probe signal that is of guide oligo origin indicative of an asymmetric endonuclease activity.

[0074] FIGs. 7A-7D illustrate an experiment which uses internal fluorescence to detect the formation of double-stranded nucleic acids and the use of different guides. FIG. 7A depicts a single-stranded DNA (ssDNA) molecule with a 5' quencher and an internal fluorescein-T. Figure discloses SEQ ID NO: 42. FIG. 7B depicts quenched fluorescence when the strand is selfcomplemented. Figure discloses SEQ ID NO: 45. FIG. 7C depicts the binding of a guide molecule to the target ssDNA. Figure discloses SEQ ID NOS 21 and 45, respectively, in order of appearance. FIG. 7D depicts the cut sites which will initiate the formation of extension products and the formation of fluorescent double-stranded nucleic acids. Figure discloses SEQ ID NOS 21 and 45, respectively, in order of appearance.

[0075] FIGs. 8A-8D show the amplification / primer extension results of different primers using Bst polymerase. The 3' extension of the guide molecule is blocked when encountering a 2’0 methyl RNA base or a phosphorylated base. FIG. 8A depicts the amplification / primer extension reaction results using only Bst polymerase. Figure discloses SEQ ID NOS 21-25, respectively in order of appearance. FIG. 8B depicts the amplification / primer extension reaction results using Bst polymerase and endonuclease Nt.BsmAI. Figure discloses SEQ ID NOS 21-25, respectively in order of appearance. FIG. 8C depicts the amplification / primer extension reaction results using Bst polymerase and the endonucleases Nt.BsmAI and N.BstNBI. Figure discloses SEQ ID NOS 21-25, respectively in order of appearance. FIG. 8D depicts the amplification / primer extension reaction results using Bst polymerase and endonuclease N.BstNBI. Figure discloses SEQ ID NOS 21-25, respectively in order of appearance.

[0076] FIGs. 9A-9D show the amplification / primer extension reaction results of different primers using Bst polymerase. The 3' extension of the guide molecule is blocked when encountering a 2’0 methyl RNA base or a phosphorylated base. FIG. 9A depicts the amplification / primer extension reaction results using only Bst polymerase. Figure discloses SEQ ID NOS 26, 23, 28, 22, 30 and 21, respectively in order of appearance. FIG. 9B depicts the amplification / primer extension reaction results using Bst polymerase and endonuclease Nt.BsmAI. Figure discloses SEQ ID NOS 26, 23, 28, 22, 30 and 21, respectively in order of appearance. FIG. 9C depicts the amplification / primer extension reaction results using Bst polymerase and the endonucleases Nt.BsmAI and N.BstNBI. Figure discloses SEQ ID NOS 26, 23, 28, 22, 30 and 21, respectively in order of appearance. FIG. 9D depicts the amplification / primer extension reaction results using Bst polymerase and endonucleaseN.BstNBI. Figure discloses SEQ ID NOS 26, 23, 28, 22, 30 and 21, respectively in order of appearance.

[0077] FIGs. 10A-10B illustrate the cycle threshold results of loop-mediated isothermal amplification (LAMP). FIG. 10A depicts cycle threshold results of LAMP when comparing LAMP to LAMP with differential targeted endonuclease cutting technology (DTECT) priming and DTECT priming on its own. Figure discloses SEQ ID NOS 32-35, respectively in order of appearance. FIG. 10B is a zoomed-in version of FIG. 10A which more clearly shows the difference between the LAMP with DTECT priming and DTECT priming cycle threshold results.

[0078] FIGs. 11A-11D illustrate an experiment which uses internal fluorescence to detect the formation of double-stranded nucleic acids and the use of different guides. FIG. 11A depicts a single-stranded DNA (ssDNA) molecule with a 5' quencher and an internal fluorescein-T. Figure discloses SEQ ID NO: 42. FIG. 11B depicts quenched fluorescence when the strand is selfcomplemented. Figure discloses SEQ ID NO: 45. FIG. 11C depicts a 2’0 methyl bases on the guide molecule. Figure discloses SEQ ID NO: 43. FIG. 11D depicts the cut sites which will initiate the formation of extension products and the formation of fluorescent double-stranded nucleic acids. Figure discloses SEQ ID NOS 44 and 45, respectively, in order of appearance.

[0079] FIGs. 12A-12F show the amplification results of primer guide C (FIG. 12A), primer guide D (FIG. 12B), primer guide E (FIG. 12C), primer guide H (FIG. 12D), primer guide F (FIG. 12E), and primer guide G (FIG. 12F) under different conditions using Bst polymerase, Bst, and Nt.Bsp.

[0080] FIGs. 13A-13B show a comparison between the use of primer guide F, which is unblocked and has a methoxylation block on the guide (FIG. 13A), and primer guide C, which is unblocked and extendable (FIG. 13B).

[0081] FIG. 14 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0082] FIGs. 15A-15B show the results of a triplex isothermal amplification reaction. FIG. 15A shows preliminary performance results of a triplex reaction with three gene targets of a fully virulent Bacillus anthracis strain. The targets included two plasmids, pXOl and pX02, and chromosome. FIG. 15B shows a table with results of each replicate in the experiment across different concentrations. Concentrations ranged from 2x10° c / reaction to 2xl06c / reaction.

[0083] FIG. 16 shows a table with the quantification cycle results and isothermal detection results across pXOl, pX02, and chromosome experimental conditions

[0084] FIG. 17 shows the results of different guide adapters across four reaction repetitions. Guide sets 2 and 4 showed the greatest slowing of reaction rate, as shown by greater cycle threshold values.

[0085] FIGs. 18A-18H show modification of reaction rates following addition of increasing concentration of adapters. FIG. 18A shows cycle threshold values for adapter 1. FIG. 18B shows cycle threshold values for adapter 2. FIG. 18C shows cycle threshold values for adapter 3. FIG. 18D shows cycle threshold values for adapter 4. FIG. 18E shows cycle threshold values for adapter 5. FIG. 18F shows cycle threshold values for adapter 6. FIG. 18G shows cycle threshold values for adapter 7. FIG. 18H shows cycle threshold values for adapter 8.

[0086] FIG. 19 shows results of an amplification reaction with guide polynucleotides comprising blocked or unblocked 3' ends. The concentration of template human RNA was 625 pg per reaction.

[0087] FIG. 20 shows results of an amplification reaction with guide polynucleotides comprising blocked or unblocked 3' ends. The concentration of template human RNA was 62.5 pg per reaction.

[0088] FIG. 21 shows results of a control amplification reaction with guide polynucleotides comprising blocked or unblocked 3' ends. There was no template genetic material added to the reaction.

[0089] FIG. 22 shows a summary of the results from FIGs. 19-21, in Td (minutes).DETAILED DESCRIPTION OF THE INVENTION

[0090] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. It is appreciated that although the vial caps are described in the Figures as having a configuration comprising three void filling caps filling three vials in linear arrangement, that such description is merely illustrative as the inventive concepts described herein contemplate various configurations and numbers of void filling caps.

[0091] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0092] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0093] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.Overview

[0094] The present disclosure provides methods, systems, compositions, and kits for processing target nucleic acid molecules. The methods, systems, compositions, and kits described herein provide for modified regions (e.g., adapter regions) of guide nucleic acid complexes comprising guide polynucleotides. Adapters may also be added into a reaction as non-target binding molecules to modulate a reaction rate. Changes to the adapter region or addition of adapters (e.g., non-target binding molecules) may have an effect on the reaction rate in terms of both launch, as well as logarithmic amplification stage of the reaction.

[0095] The modification of the adapter regions can have an effect of adjusting the rate-limiting step of a low frequency endonuclease cut. This can be achieved through numerous ways. For example, modification of the adapter may comprise the use of modified bases, lengthening or shortening the adapter, changing the base usage of the adapter (such as mismatches, spaces, universal bases, as well as moieties that increase binding strength), changing the interaction of the adapter with other adapters and with itself, or any combination thereof.

[0096] Adapter molecules can also be added into the reaction to affect the reaction rate. By blocking the extension on the 3’ ends, the adapter molecules can act as rate modulators, while not taking part in the reaction. In some embodiments, addition of the adapter molecules to thereaction may increase the reaction rate. In some embodiments, addition of the adapter molecules to the reaction may decrease the reaction rate.

[0097] In some aspects, the present disclosure provides for methods of amplification of nucleic acids (e.g., isothermal amplification). Such a method can involve a cycle of steps such as that depicted in FIGs. 1A through IO. The methods provided herein can offer higher amplification efficiency and easier optimization procedure compared with existing amplifications (e.g. isothermal amplifications). The processed target nucleic acid molecules can be used in various amplification reactions not limited to the amplification or processing methods described herein.

[0098] Such a method can start with the formation of a structure such as that depicted in FIG. 1A, in which a guide nucleic acid complex (or a guide complex) is formed to direct a restriction enzyme to a predetermined site in a nucleic acid. FIG. 1A depicts a nucleic acid strand (e.g., a single-stranded DNA strand or ssDNA strand) (100) comprising a target nucleic acid sequence (101). In some cases, the ssDNA strand can be generated by reverse transcribing a target RNA sequence. In some cases, the ssDNA strand can be generated by denaturing a double-stranded DNA (dsDNA) sequence. In FIG. 1A, a type Ils restriction enzyme (120) is directed to the vicinity of the target site via formation of a guide complex. This guide nucleic acid complex is constituted via self-annealing of single copies of a guide polynucleotide which comprise: a nontarget binding region (e.g., the adapter region) comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme (117), a target binding region configured to hybridize to the target sequence (115), and a blocked 3' end non-extendable by a polymerase (116). In some cases, the 3’ end may not be blocked. Note that in FIG. 1A, self-annealing of the two copies of the guide polynucleotide forms a double-stranded palindromic region that permits binding of the type II restriction enzyme in the vicinity of the target site.

[0099] Such a method can continue in a second stage with the process depicted in FIG. IB and FIG. 1C. After the type Ils restriction enzyme (120) is directed to the vicinity of the target site (101) by the double-stranded palindromic region (two copies of 117) formed by self-annealing of the guide polynucleotides, the type Ils restriction enzyme is able to, characteristic to its activity, cleave single-stranded locations (130, 135) distal to its binding site (FIG. IB). One of these cleavable single-stranded locations (135) is on the nucleic acid strand (101) that comprises the target nucleic acid sequence (101). The other cleavable single-stranded location (130) is located on the guide polynucleotide itself (130). If selective enzymatic conditions, an engineered polymerase, or BspD6I is used, cleavage at one of the sites (e.g. the single-stranded site on the nucleic acid strand (101) that comprises the target nucleic acid sequence (101)) can be favored. Cleavage at the single-stranded site on the nucleic acid strand (101) that comprises the targetnucleic acid sequence (101) generates a free 3' hydroxyl that can then be extended by a stranddisplacing polymerase present in the reaction.

[0100] Such a method can continue in a third stage with the process depicted in FIG. ID through FIG. IF. Extension of the free 3' hydroxyl by the strand-displacing polymerase (140, FIG. 1C) produces a region (160) of the nucleic acid strand (101) that comprises the target nucleic acid sequence (101) that is complementary to the restriction endonuclease recognition sequence for the type Ils restriction enzyme (117) from the guide polynucleotide (FIG. ID). Extension of the nucleic acid (100) displaces the second copy of the guide polynucleotide (116 / 117, lower molecule), that previously formed half of the guide complex. Extension of the nucleic acid (100) with the region complementary to the restriction endonuclease recognition sequence for the type Ils restriction enzyme (160) forms a new double-stranded structure where a type Ils restriction enzyme (120) can bind (FIG. IE). As in the second stage, the type Ils restriction enzyme is able to cleave single-stranded locations (130, 135) distal to its binding site (FIG. IE). While cleavage at the single-stranded site (135) that contains the target nucleic acid site (100) causes the strand (100) to merely be extended again by the polymerase, cleavage at the single-stranded site (130) allows for a new procedure to commence (FIG. IE). Specifically, cleavage at site 130 of FIG. IE on the annealed guide polynucleotide removes the sequence containing the blocked or unblocked 3' end (116) and allows the guide polynucleotide to be extended to comprise a sequence (170) complementary to the strand (100) containing the target nucleic acid site (101) (FIG. IF).

[0101] Such a method can continue in a fourth stage with the process depicted in FIG. 1G and FIG. 1H. As the double-stranded structure of FIG. 1G no longer comprises a blocked 3' end, repeated cleavage at site 130 of FIG. 1G liberates a single strand comprising a sequence (170) complementary to the strand (100) containing the target nucleic acid site (101), and then allows extension of a new strand (171) to replace it. Additionally, the liberated strand (170) can further serve as a new template analogously to the strand 100 of FIG. 1A (FIG. II), which allows for strand 170 to be further cleaved and repeatedly extended as in FIG. 1H (FIG. 1 J). FIG. IK depicts an exemplary completed extension on the new guide molecule.

[0102] In some cases, the method can continue, as seen in FIG. IL, wherein endonucleolytic activity can occur on the second complementary strand oligo / extension product complex (170). FIG. IM depicts a polymerase (140) extending of the 3’ end of the cut site of the second complementary strand of the oligo / extension product complex. Endolytic activity on the newly synthesized strand (130) occurs (FIG. IN) and the displaced, single-stranded synthesizedfragment (42) of FIG. IO can serve as starting material for additional strand displacement amplification reactions.

[0103] In some cases, methods according to the disclosure do not involve amplification and utilize the structure depicted in FIG. 1A to direct cleavage of a single-stranded nucleic acid molecule (100) containing a target site (101) at a specified position (135, FIG. IB).Definitions

[0104] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0105] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)) (which is entirely incorporated by reference herein).

[0106] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0107] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0108] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide may comprise a synthetic nucleotide. A nucleotide may comprise anucleotide analog. A nucleotide may comprise a synthetic nucleotide analog. Nucleotides may be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [aS]dATP, 7- deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. Synthetic nucleotide analogs may include locked nucleic acids (LNAs), bridged nucleic acids (BNAs), fluorinated nucleic acids (also known as fluoromodified nucleic acids), and peptide nucleic acids (PNAs). As used herein, the term “locked nucleic acid” (“LNA”), generally refers to a nucleic acid analog wherein the ribose ring is “locked” with an extra bridge connecting the 2'-oxygen atom with the 4'-carbon atom of the nucleotide such as a methylene bridge (see e.g. WO 99 / 14226, which is incorporated by reference in its entirety herein). As used herein, the term “bridged nucleic acid (BNA),” generally refers to constrained or inaccessible nucleic acid molecules which have a fixed bridge structure at the 2'- or 4'-position. As used herein, “fluorinated nucleic acids” generally refer to nucleic acids which have incorporated a fluorine atom, often at the 2'- or 4'- position. As used herein, the term “peptide nucleic acid (PNA),” generally refers to a nucleotide analog wherein the backbone of the analog, for example a sugar backbone in DNA, is a pseudopeptide. A PNA backbone can comprise, for example, a sequence of repeated N-(2-amino-ethyl)-glycine units. A peptide nucleic acid analog can react as DNA would react in a given environment, and can additionally bind complementary nucleic acid sequences and various proteins. Due to the non-natural backbone, PNAs can be insensitive to endonuclease cleavage in situations where an endonuclease would cleave the equivalent DNA / RNA sequence and in addition, confer specificity and binding to complementary DNA under varying salt conditions. The term “nucleotide,” as used herein, may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fhiorophores). Detectable labels may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.

[0109] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may comprise one or more nucleotide analogs (e.g., including those with an altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5- bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, wyosine, PNAs, and LNAs.

[0110] As used herein, the term “restriction endonuclease,” “restriction enzyme,” or grammatical equivalents thereof generally refers to an enzyme that originates in bacterial host defense and is understood to recognize a specific sequence on an incoming viral DNA and cleave the DNA either at the recognition sequence or at a distinct sequence site. One group of restriction endonucleases are identified as Type IIS. This group can recognize asymmetric DNA sequences and cleaves the DNA at a site outside the cleavage site that is at a defined distance from the recognition site. In some cases, type IIS restriction endonucleases cleave DNA between 1 and 20 nucleotides from the relevant recognition site.

[0111] As used herein, the term “restriction endonuclease recognition sequence” generally refers to a location on a nucleic acid molecule (e.g., DNA molecule) containing specific sequences of nucleotides, which are recognized by various restriction enzymes. These sequences can comprise from 4-8 base pairs to 12-40 base pairs in length. These sites can be palindromic sequences.

[0112] As used herein, the term “polymerase” generally refers to an enzyme that produces a complementary replicate of a nucleic acid molecule using the nucleic acid as a template strand. DNA polymerases bind to the template strand and then move down the template strand adding nucleotides to the free hydroxyl group at the 3' end of a growing chain of nucleic acid. DNA polymerases synthesize complementary DNA molecules from DNA (e.g., DNA-dependent DNA polymerases) or RNA templates (e.g., RNA-dependent DNA polymerases or reverse transcriptases) and RNA polymerases synthesize RNA molecules from DNA templates (e.g., DNA-dependent RNA polymerases which participate in transcription). DNA polymerases generally use a short, preexisting RNA or DNA strand, called a primer, to begin chain growth; and some DNA polymerases can utilize any free 3’ hydroxyl in a DNA duplex for extension. Some DNA polymerases replicate single-stranded templates, while other DNA polymerases displace the strand upstream of the site where they add bases to a chain.

[0113] As used herein, the term “strand displacing,” when used in reference to a polymerase, generally refers to an activity that removes a complementary strand from base-pairing with a template strand being read by the polymerase. Example polymerases having strand displacing activity include the large fragment of Bacillus stearothermophilus polymerase (Bst polymerase), exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, and OmniTaq 2 LA DNA polymerase.

[0114] As used herein, the terms “amplify,” “amplifies,” “amplified,” “amplification,” and “amplicon” generally refer to any method for replicating a nucleic acid. The replication can be conducted with the use of a primer-dependent polymerase. The replication can be enzyme-free amplification. In some cases, amplifying or replicating a target nuclei acid strand also comprises replicating or amplifying a complementary strand of the target nucleic acid strand. Amplified products can be subjected to subsequence analyses, including but not limited to melting curve analysis, nucleotide sequencing, single-strand conformation polymorphism assay, allele-specific oligonucleotide hybridization, Southern blot analysis, and restriction endonuclease digestion.

[0115] The terms “hybridizes,” and “annealing,” as used herein, generally refer to a reaction in which one or more polynucleotides interact to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence sensitive or specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR, or the enzymatic cleavage of a polynucleotide by a ribozyme. A first sequence that can be stabilized via hydrogen bonding with the bases of the nucleotide residues of a second sequence can generally be “hybridizable” to the second sequence. In such a case, the second sequence can also be the to be hybridizable to the first sequence.

[0116] The terms “complement,” “complements,” “complementary,” and “complementarity,” as used herein, generally refer to a sequence that is fully complementary to and hybridizable to the given sequence. In some cases, a first sequence that is hybridizable to a second sequence or set of second sequences is specifically or selectively hybridizable to the second sequence or set of second sequences, such that hybridization to the second sequence or set of second sequences is used. Hybridizable sequences can share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, including at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.

[0117] The isothermal amplification methods described herein can provide advantages over existing nucleic acid amplification methods. Non-limiting examples of isothermal nucleic acid amplification methods can include helicase-dependent amplification, nicking enzyme amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, and nucleic acid sequence based amplification.

[0118] The methods described herein may take advantage of DNA polymerases with high strand-displacement activity and specially designed primer sets to exponentially amplify a target sequence. The methods provided herein may provide a faster time to amplify a target nucleic acid molecule compared to a time with an existing nucleic acid amplification method. The nucleic acid target processed (e.g., nicked or cut mediated by the guide complex or enzyme) by the methods described herein may be used as an initial template to be used with any existing isothermal amplification. Different existing isothermal amplification methods can utilize different DNA polymerases. Loop-mediated isothermal amplification (LAMP) utilizes two sets of specially designed primers, termed inner and outer primers and may be performed under a constant temperature of 50-65°C (122-149°F). A limitation of LAMP can be use of non-specific detection methods, which may result in detection of false positives. Helicase-dependent amplification (HD A) utilizes DNA helicase activity to separate complementary strands of double strand DNA molecules, and thus may avoid temperature cycling to produce single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. The rolling circle amplification (RCA) method utilizes the continuous amplification of a circular DNA template by a strand-displacing DNA polymerase. RCA functions at a constant temperature (e.g., between 37°C-42°C, [98.6-107.6°F]) to produce a long single-stranded DNA molecule with tandem repeats of the circular template. Limitations of RCA may include challenges in mass production of target molecules, purification, and storage. Multiple displacement amplification (MDA) may utilize random exonuclease-resistant primers as well as a q>29 DNA polymerase with strand-displacement activity to produce target DNA strands at a constant temperature, e.g., 30 °C (86°F). MDA may also be used for whole genome amplification. The recombinase polymerase amplification (RPA) method is a low temperature (e.g., 37°C [98.6°F]) isothermal amplification that couples isothermal recombinase-driven primer targeting of a target molecule with stranddisplacement DNA activity. RPA utilizes nucleoprotein complexes formed by oligonucleotide primers and recombinase proteins to guide and facilitate binding to a target DNA strand. Nucleic acid sequence-based amplification (NASBA) is an isothermal, transcription-based amplification method designed for the amplification of single-stranded RNA or DNA sequence and performed at a constant temperature of 41 °C (105.8°F).Compositions and Methods for Processing Nucleic Acid Molecules

[0119] The present disclosure provides methods and compositions for processing nucleic acid molecules comprising target sequences. In some aspects, the present disclosure provides for a method of processing a single- stranded nucleic acid molecule comprising a target sequence. The method can comprise contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a nontarget binding region comprising a restriction endonuclease recognition sequence for an enzyme (e.g., a restriction enzyme). The restriction enzyme can be a type Ils restriction enzyme. The guide polynucleotide can further comprise (ii) a target binding region configured to hybridize to the target sequence. The guide polynucleotide can further comprise (iii) an unblocked 3’ end or a blocked 3' end non-extendable by a polymerase. In some embodiments, the guide polynucleotide further comprises (i), (ii), and (iii) in 5' to 3' order. The non-target binding region can be located at the 5' end of the guide polynucleotide. The target binding region can be located at the 3' end of the guide polynucleotide. In some embodiments, the non-target binding region further comprises a sequence containing a reverse complement of the restriction endonuclease recognition sequence for the type Ils restriction enzyme 3' to the restriction endonuclease recognition sequence for a type Ils restriction enzyme and 5' to the target binding region configured to hybridize to the target sequence. In some embodiments, in (b) the cut exposes an extendable 3' end of the target sequence. In some embodiments, the method further comprises reverse-transcribing the singlestranded nucleic acid molecule from an RNA.

[0120] The guide polynucleotide provided herein can be a forward guide polynucleotide (e.g., Forward Guide Oligo) configured for processing the target nucleic acid molecule in a reaction. The reaction can further comprise a reverse guide polynucleotide (e.g., Reverse Guide Oligo) configured for processing the target nucleic acid molecule or a reverse complement of the target nucleic acid molecule in the reaction.

[0121] Conditions where the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule can be determined empirically or calculated based off of chemical composition of the guide polynucleotide. A variety of tools (e.g., http: / / www.oligoevaluator.com / LoginServlet) are available for calculating annealing / hybridization temperatures and conditions given specific sequences of polynucleotides.

[0122] The target binding region can be of a length sufficient to hybridize to the target site under conditions desirable for the assay (e.g., temperature, pH, ionic strength). In someembodiments, the target binding region is at least about 12 to about 25 nucleotides in length, including 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides. In some embodiments, the target binding region is at least about 12 to about 30 nucleotides in length. In some embodiments, the target binding region is at least about 10 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 15 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 10 to about 30 nucleotides in length. In some embodiments, the target binding region is at least about 15 to about 30 nucleotides in length. In some embodiments, the target binding region is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, the target binding region is at most about 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer nucleotides in length.

[0123] The enzyme described herein can comprise a type Ils restriction enzyme. The type Ils restriction enzyme can comprise one or more enzymes selected from the group consisting of BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nucleases, zinc finger nucleases (ZFNs), N.Bst9 I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt. Alwl, Nt.BbvCI, N.BstNBI, Nt.CviPII, Nb.Mval269I, Nb.BpulOI, Nt.BpulOI, and any combinations thereof. The type Ils restriction enzyme can comprise type Ils nickases such as N.BstNBI, N.BspD6I, , N.Bst9 I and Nt.BstNBI, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutl, or any combination thereof. Alternatively, the type Ils restriction enzyme can comprise BfuAI, BsmAI, BsrDI, or BtsIMutl. Additional examples of Type IIS restriction enzymes can be found at www.neb.com / tools-and-resources / selection-charts / type-iis-restriction-enzymes, which is herein incorporated by reference.

[0124] In some embodiments, the type Ils restriction enzyme comprises an engineered type Ils restriction enzyme that has a nuclease-inactivating mutation in one of its two subunits to create a nickase from an enzyme that is not naturally a nickase. In some embodiments, the type Ils restriction enzyme comprises an engineered type Ils restriction enzyme that has a mutation in one of its two subunits that create different rates of enzymatic activity of cutting one strand over the opposite strand. In some cases, the enzyme comprises two enzymes with different activities or activity rates. In some cases, the enzyme can comprise a subunit of a type Ils restriction enzyme. In some cases, the enzyme can comprise a subunit of a nicking enzyme. In some cases, the enzyme can comprise an activity for introducing a cut on the target nucleic acid sequence. For example, the enzyme can be N.BspD6I. In some cases, the enzyme can comprise an activity for introducing a cut on the complementary strand of the target nucleic acid sequence. In some cases,the enzyme can comprise an activity for introducing a cut on the guide polynucleotide (e.g., the target binding region of the guide polynucleotide). For example, the enzyme can be Nt.BstNBI.

[0125] The blocked 3' end can comprise essentially any 3' chemical structure that prevents extension of the guide polynucleotide by a DNA polymerase. Such structures include, but not limited to, 3' phosphate, 3' thiophosphate, 3'-O-methyl, a PNA, a modified base, a ddNTP, a solid support, or a spacer.

[0126] In some cases, the guide polynucleotide described herein may comprise an unblocked 3' end. The guide polynucleotide may not comprise a blocked 3' end. The unblocked 3’ end may be extended, but it may be non-consequential to the amplification.

[0127] In some cases, the guide polynucleotide can further comprise an additional non-target binding region located at the 3' end of the guide polynucleotide. The additional non-target binding region can comprise an additional site for binding to an enzyme. For example, the additional non-target binding region can comprise an additional restriction endonuclease recognition sequence for binding to a restriction enzyme. The enzyme recruited by the additional non-target binding region can be the same or different from the enzyme that is recruited by the non-target binding region of located at the 5' end of the guide polynucleotide. The additional non-target binding region can function as a blocker to block extension of the 3' end of the guide polynucleotide.

[0128] The method of processing the single-stranded nucleic acid molecule can further comprise introducing the type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence. Optimal temperatures for specific type Ils restriction enzymes can be found in e.g. the Rebase database (accessible at http: / / rebase.neb.com / rebase / rebase.html).

[0129] The method of processing the single-stranded nucleic acid molecule can further comprise extending the extendable 3' end using a polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the polymerase is a DNA-dependent DNA polymerase. In some embodiments, the polymerase comprises a strand-displacing DNA polymerase. In some embodiments, the polymerase comprises a large fragment of Bacillus stearothermophilus polymerase, an exo-Klenow polymerase, a B st 2.0 polymerase, a phi29 DNA polymerase, a T7 exo-polymerase, an OmniTaq 2 LA DNA polymerase, or any combination thereof. In some embodiments, the polymerase may be a IsoFast™ BST polymerase. Such methods can further comprise adding other factors alongside the polymerase sufficient to add nucleotides to the 3' end, including dNTPs, appropriate buffering agents, and cofactors (e.g., divalent cations). The dNTPs may be natural or unnatural dNTPs. The natural dNTPs cancomprise dATP, dCTP, dGTP, dTTP, and / or dUTP. The unnatural dNTPs can be a-thiol dNTPs (e.g., S-dNTPs). S-dNTPS can comprise dATPaS, dCTPaS, dGTPaS, and / or dTTPaS.

[0130] The target sequence processed by the methods provided herein can be used for further downstream applications, e.g., isothermal amplifications. In some cases, the reagents for carrying out the amplification can be in the same mixture as the reagents for target processing. In some aspects, the present disclosure provides for a method of amplifying a single-stranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting the singlestranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) an unblocked 3’ end or a blocked 3' end non-extendable by a polymerase; (b) introducing the type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end; and (c) extending the extendable 3' end of the target sequence using a polymerase. In some embodiments, the guide polynucleotide further comprises (i), (ii), and (iii) in 5' to 3' order. In some embodiments, the non-target binding region further comprises a sequence containing a reverse complement of the restriction endonuclease recognition sequence for the type Ils restriction enzyme 3' to the restriction endonuclease recognition sequence for a type Ils restriction enzyme and 5' to the target binding region configured to hybridize to the target sequence. In some embodiments, the guide polynucleotide is a first guide polynucleotide, and the guide complex comprises a second guide polynucleotide, wherein the second guide polynucleotide comprises (i) a non-target binding region that is complementary with the non- target binding region of the first guide polynucleotide and (ii) a target binding region configured to hybridize to the target sequence. In some cases, when the first guide polynucleotide of the guide complex is hybridized to the target polynucleotide sequence, the target binding region of the second guide polynucleotide of the guide complex is not hybridized to the target sequence. In some embodiments, the first guide polynucleotide and the second guide polynucleotide of the guide complex hybridize to form a dimer. In some embodiments, the first guide polynucleotide and the second guide polynucleotide of the guide complex hybridize at a common 5' region. In some embodiments, the first guide polynucleotide and the second guide polynucleotide hybridize via the non-target binding region of the first guide polynucleotide and the second guide polynucleotide to form the dimer having a double-stranded binding region. In someembodiments, the double-stranded binding region comprises the restriction endonuclease recognition sequence. In some embodiments, the type Ils restriction enzyme binds to the doublestranded binding region of the dimer. A forward guide polynucleotide (or complex) can comprise one or more guide polynucleotides including the first guide polynucleotide and the second guide polynucleotide described herein. The first guide polynucleotide and the second guide polynucleotide can be homodimer or heterodimer. For example, the non-target binding region at the 5’ end of the first guide polynucleotide and the non-target binding region at the 5’ end of the second guide polynucleotide can comprise the same sequence (e.g., a palindromic sequence), and the target binding region at the 3’ end of the first or the second guide polynucleotide can be different. In some embodiments, a target binding region can be configured to hybridize to a target sequence. Alternatively, a target binding region can be configured to hybridize to a different target sequence.

[0131] In some cases, a reverse guide polynucleotide (or complex) can comprise a plurality of guide polynucleotides including the first guide polynucleotide and the second guide polynucleotide. In some cases, a reverse guide polynucleotide and a forward guide polynucleotide can comprise a same sequence (e.g., a palindromic sequence) at the 5’ end such that the reverse guide polynucleotide and the forward guide polynucleotide can hybridize to form a heterodimer. The target binding region of the forward guide polynucleotide and the target binding region of the reverse guide polynucleotide can comprise different sequences.

[0132] In some aspects, the present disclosure provides for a method of amplifying a singlestranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting a guide complex with the single-stranded nucleic acid molecule, wherein the guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with the target sequence of the singlestranded nucleic acid molecule, and (i) a second guide polynucleotide that hybridizes with the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region binds to an enzyme; and (b) cutting the target sequence using the enzyme to expose an extendable 3' end of the target sequence. In some cases, an extendable 3' end is a 3' hydroxyl group. In some embodiments, if a target molecule is an RNA, the method can further comprise reverse-transcribing, prior to contacting the target molecule with the guide complex, the single-stranded nucleic acid molecule from the RNA. For example, the target RNA molecule can be reverse transcribed using a reverse transcriptase to generate a DNA molecule, which can be subject to further processing using the methods described herein. The DNA molecule can be a single-stranded DNA molecule (ssDNA). In somecases, a reverse transcription reaction can be used to make a ssDNA target from an initial RNA target. In some cases, a reverse transcription reaction can comprise a reverse transcriptase and a reverse transcription primer. The reverse transcriptase can comprise avian myeloblastosis virus (AMV) reverse transcriptase (RT), Moloney murine leukemia virus RT (M-MLV RT), telomerase RT, or human immunodeficiency virus type 1 RT (HIV-1 RT).

[0133] For example, a method of amplifying a single-stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) an unblocked 3’ end or a blocked 3' end non-extendable by a polymerase; (b) introducing the type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end; and (c) extending the extendable 3' end of the target sequence using a polymerase, wherein the single-stranded nucleic acid molecule or the target sequence is from Bacillus anthracis.

[0134] As another example, a method of amplifying a single-stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting a guide complex with the singlestranded nucleic acid molecule, wherein the guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with the target sequence of the single-stranded nucleic acid molecule, and (ii) a second guide polynucleotide that hybridizes with the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region binds to an enzyme; (b) cutting the target sequence using the enzyme to expose an extendable 3' end of the target sequence; (c) extending the extendable 3' end of the target sequence with a polymerase to generate an extension product, wherein the extension product displaces the second guide polynucleotide; (d) cutting the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide; and (e) extending the extendable 3' end of the first guide polynucleotide using the polymerase to generate a complementary molecule of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule or the target sequence is from Bacillus anthracis.

[0135] As another example, a method of amplifying a single-stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme and (ii) a target binding region configured to hybridize to the target sequence; (b) introducing the type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end; and (c) extending the extendable 3' end of the target sequence using a polymerase, wherein the singlestranded nucleic acid molecule or the target sequence is from Bacillus anthracis.

[0136] In some cases, the method of amplifying the single-stranded nucleic acid molecule comprising the target sequence further comprises extending the extendable 3' end of the target sequence with a polymerase to generate an extension product, wherein the extension product displaces the second guide polynucleotide. In some cases the polymerase extension creates a double-stranded product displacing the second guide polynucleotide. In some embodiments, the extending comprises incubation in the presence of a DNA polymerase such as strand-displacing DNA polymerase, including any of the strand-displacing polymerases described herein. The extending can also comprise incubation in the presence of factors alongside the polymerase sufficient to add nucleotides to the 3' end, including dNTPs, appropriate buffering agents, and cofactors (e.g. divalent cations). The dNTPs may be natural or unnatural dNTPs. The natural dNTPs can comprise dATP, dCTP, dGTP, dTTP, and / or dUTP. The unnatural dNTPs can be a- thiol dNTPs (e.g., S-dNTPs). S-dNTPS can comprise dATPaS, dCTPaS, dGTPaS, and / or dTTPaS.

[0137] In some cases, the method of amplifying the single-stranded nucleic acid molecule comprising the target sequence further comprises cutting the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide. In some embodiments the cutting can comprise introducing a type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut the first guide polynucleotide within the target binding region. In some embodiments, the extendable 3' end comprises a 3' hydroxyl.

[0138] In some cases, the method of amplifying the single-stranded nucleic acid molecule comprising the target sequence further comprises extending the extendable 3' end of the first guide polynucleotide using a polymerase to generate a complementary molecule of the targetsequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule. The polymerase can be strand-displacing DNA polymerase, including any of the strand-displacing polymerases described herein. The extending can also comprise incubation in the presence of factors alongside the polymerase sufficient to add nucleotides to the 3' end, including dNTPs, appropriate buffering agents, and cofactors (e.g., divalent cations). The dNTPs may be natural or unnatural dNTPs. The natural dNTPs can comprise dATP, dCTP, dGTP, dTTP, and / or dUTP. The unnatural dNTPs can be a-thiol dNTPs (e.g., S-dNTPs). S- dNTPS can comprise dATPaS, dCTPaS, dGTPaS, and / or dTTPaS.

[0139] In some embodiments, the second guide polynucleotide in the method of amplifying a single-stranded nucleic acid molecule comprising a target sequence comprises, from 5' to 3' (i) a non-target binding region that hybridizes with the non-target binding region of the first guide polynucleotide and (ii) a target binding region configured to hybridize with the target sequence. In some embodiments, the method further comprises prior to (b), cutting the first guide polynucleotide within the target binding region using the enzyme, wherein the guide complex dissociates from the single-stranded nucleic acid molecule. In some embodiments, the method further comprises cutting the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide and extending the extendable 3' end of the first guide polynucleotide using a polymerase to generate a complementary molecule of the target sequence of the single-stranded nucleic acid molecule repeatedly to generate a plurality of complementary molecules of the target sequence of the single-stranded nucleic acid molecule. In some embodiments, an additional guide complex binds to the complementary molecule. In some embodiments, the method further comprises using the complementary molecule with the additional guide complex bound thereto as a starting template to generate copies of the target molecule. In some embodiments, the enzyme is a type Ils restriction enzyme. In some embodiments, the type Ils restriction enzyme comprises N.BstNBI, N.Bst9 I and N.BspD6I, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutl, BfuAI, BsmAI, BsrDI, BtsIMutl, a functional fragment thereof, or a combination thereof. In some embodiments, the guide polynucleotide can comprise an unblocked 3’ end. The unblocked 3’ end may be extendable by a polymerase. In some embodiments, the guide polynucleotide comprises a blocked 3' end non-extendable by a polymerase. The blocked 3' end can comprise essentially any 3' chemical structure that prevents extension of the guide polynucleotide by a DNA polymerase, including any structures with such activity described herein. In some embodiments, the blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, or a spacer. In some embodiments, the singlestranded nucleic acid molecule with the cut and the guide polynucleotide bound thereto is used asa starting template for an amplification. In some embodiments, the amplification is an isothermal amplification. In some embodiments, the enzyme comprises asymmetric propensity to cleave one strand of a DNA duplex. In some embodiments, the enzyme exhibits a high-frequency endonuclease activity. In some embodiments, the high-frequency endonuclease activity is from a large subunit of the enzyme. In some embodiments, the enzyme exhibits a low-frequency endonuclease activity. In some embodiments, the low-frequency endonuclease activity is from a small subunit of the enzyme. In some embodiments, the enzyme exhibits at least two differential enzymatic activity rates. In some embodiments, the at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites. In some embodiments, one of the two differential endonuclease activity rates comprises cutting the target sequence of the single-stranded nucleic acid molecule with low frequency. In some embodiments, one of the two differential endonuclease activity rates comprises cutting the target binding region of the guide polynucleotide by with high frequency. In some embodiments, the two differential endonuclease activity rates are asymmetric or not equal. In some embodiments, the enzyme comprises N.BstNBI, N.Bst9 I and N.BspD6I, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutl, BfuAI, BsmAI, BsrDI, BtsIMutl , or a combination thereof.

[0140] In some embodiments, a temperature is changed over the course of the method. In some embodiments, a first activity rate of the at least two differential enzymatic activity rates is favored at a first temperature, and a second activity rate of the at least two differential enzymatic activity rates is favored at a second temperature different from the first temperature. In some embodiments, a first temperature wherein a first enzymatic activity rate is favored can be about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, about 43 °C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C. In some embodiments, a first temperature wherein a first enzymatic activity rate is favored is between about 15°C-50°C, between about 20°C-45°C, between about 30°C-45°C, between about 30°C-40°C, or between about 32°C-39°C. In some embodiments, a second temperature wherein a second enzymatic activity rate is favored can be about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71 °C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, orabout 80°C. In some embodiments, a second temperature wherein a second enzymatic activity rate is favored is between about 45°C-80°C, between about 50°C-80°C, between about 50°C- 70°C, between about 50°C-60°C, between about 52°C-58°C.

[0141] In some embodiments, a temperature may be changed over the course of the method for a period of time. The period of time at which a temperature is changed may benefit the enzymatic activity rate during the reaction. A temperature change can comprise a first temperature or a second temperature. In some embodiments, a first temperature change or a second temperature change may occur over a duration of time of at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, or at least about 15 minutes. In some embodiments, a first temperature change or a second temperature change may occur over a duration of time of at most about 15 minutes, at most about 12 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6.5 minutes, at most about 6 minutes, at most about 5.5 minutes, at most about 5 minutes, at most about 4.5 minutes, at most about 4 minutes, at most about 3.5 minutes, at most about 3 minutes, at most about 2.5 minutes, at most about 2 minutes, at most about 1.5 minutes, at most about 1 minute, at most about 30 seconds, or at most about 15 seconds.

[0142] In some embodiments, a first temperature change or a second temperature change may occur over a duration of time from about 1 minute to about 15 minutes. In some embodiments, the sample may be heated from a range from about 1 minute to about 2 minutes, about 1 minute to about 2.5 minutes, about 1 minute to about 3 minutes, about 1 minute to about 3.5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 1 minute to about 6 minutes, about 1 minute to about 7 minutes, about 1 minute to about 7.5 minutes, about 1 minute to about 10 minutes, about 1 minute to about 15 minutes, about 2 minutes to about 2.5 minutes, about 2 minutes to about 3 minutes, about 2 minutes to about 3.5 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 5 minutes, about 2 minutes to about 6 minutes, about 2 minutes to about 7 minutes, about 2 minutes to about 7.5 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 15 minutes, about 2.5 minutes to about 3 minutes, about 2.5 minutes to about 3.5 minutes, about 2.5 minutes to about 4 minutes, about 2.5 minutes to about 5 minutes, about 2.5 minutes to about 6 minutes, about 2.5 minutes to about 7 minutes, about 2.5 minutes to about 7.5 minutes, about 2.5 minutes to about 10 minutes, about 2.5 minutes to about15 minutes, about 3 minutes to about 3.5 minutes, about 3 minutes to about 4 minutes, about 3 minutes to about 5 minutes, about 3 minutes to about 6 minutes, about 3 minutes to about 7 minutes, about 3 minutes to about 7.5 minutes, about 3 minutes to about 10 minutes, about 3 minutes to about 15 minutes, about 3.5 minutes to about 4 minutes, about 3.5 minutes to about 5 minutes, about 3.5 minutes to about 6 minutes, about 3.5 minutes to about 7 minutes, about 3.5 minutes to about 7.5 minutes, about 3.5 minutes to about 10 minutes, about 3.5 minutes to about 15 minutes, about 4 minutes to about 5 minutes, about 4 minutes to about 6 minutes, about 4 minutes to about 7 minutes, about 4 minutes to about 7.5 minutes, about 4 minutes to about 10 minutes, about 4 minutes to about 15 minutes, about 5 minutes to about 6 minutes, about 5 minutes to about 7 minutes, about 5 minutes to about 7.5 minutes, about 5 minutes to about 10 minutes, about 5 minutes to about 15 minutes, about 6 minutes to about 7 minutes, about 6 minutes to about 7.5 minutes, about 6 minutes to about 10 minutes, about 6 minutes to about 15 minutes, about 7 minutes to about 7.5 minutes, about 7 minutes to about 10 minutes, about 7 minutes to about 15 minutes, about 7.5 minutes to about 10 minutes, about 7.5 minutes to about 15 minutes, or about 10 minutes to about 15 minutes.

[0143] In some embodiments, the enzyme comprises two different active sites or endonuclease domains conferring the at least two differential enzymatic activities. In some embodiments, the target sequence comprises a recognition site specifically recognized by the enzyme or a first activity of the at least two differential enzymatic activities of the enzyme to introduce a cut. In some embodiments, the target binding region of the guide polynucleotide comprises a recognition site specifically recognized by the enzyme or a second activity of the at least two differential enzymatic activities of the enzyme to introduce a cut. The target binding region can be of a length sufficient to hybridize to the target site under conditions desirable for the assay (e.g., temperature, pH, ionic strength). In some embodiments, the target binding region is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, the target binding region is at most about 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or less nucleotides in length. In some embodiments, the target binding region is at least about 15 to about 25 nucleotides in length, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides. In some embodiments, the target binding region is at least about 15 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 10 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 12 to about 25 nucleotides in length.

[0144] In some embodiments, a concentration of the guide polynucleotide is at least about 0.1 pM, at least about 1 pM, or about 0.1 pM to about 4 pM. In some embodiments, a concentration of the guide polynucleotide is at least about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.5 pM, 2.0 pM, 2.5 pM, 3.0 pM, 3.5 pM, 4 pM or more. In some embodiments, the non-target binding region comprises a palindromic sequence. In some embodiments, the non-target binding region is self-complementary or forms a self-annealing dimer under reaction conditions. In some embodiments, the non-target binding region is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. In some embodiments, the non-target binding region is at most about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or less nucleotides in length. In some embodiments, the singlestranded nucleic acid molecule is a single-stranded deoxyribonucleic acid (ssDNA) or a singlestranded ribonucleic acid (ssRNA). In some embodiments, the method further comprises reversetranscribing the single-stranded nucleic acid molecule from an RNA. In some embodiments, the target binding region comprises at least one peptide nucleic acid (PNA) residue. In some embodiments, the polymerase has strand displacement activity.

[0145] In some embodiments, the methods described herein may result in a faster amplification result compared to nucleic acid amplification protocols without the programmed restriction enzyme. A metric of speed of an amplification may be a cycle threshold. A “cycle threshold” can comprise a number of cycles needed for a signal (e.g., fluorescent signal) to exceed a background threshold level. A lower cycle threshold value can indicate a greater amount of target nucleic acid in a sample. In some embodiments, a nucleic acid amplification using the methods described herein can result in a lower cycle threshold compared to loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), rolling circle amplification (RCA), or other amplification methods known in the art. A cycle threshold for a sample processing method described herein may be at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least 18%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% less than a cycle threshold for LAMP. A cycle threshold for a sample processing method described herein may be at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 18%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, or at most about 2% less than a cycle threshold for LAMP. A cycle threshold for a sample processing method described herein may be from about 1% to about 50% less than a cycle threshold for LAMP. A cycle threshold for a sample processing method described herein may be from about 1% to about 2%, about 1% to about 3%,about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% less than a cycle threshold for LAMP.

[0146] In some embodiments, a cycle threshold value for a sample processing method described herein may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40. In some embodiments, a cycle threshold value for a sample processing method described herein may be at most about 40, at most about 35, at most about 30, at most about 25, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1.

[0147] A metric of speed of an amplification may be a quantification cycle value (e.g., a Cq value). A Cq value may refer to the PCR cycle number at which a sample’s reaction curve intersects a threshold line. The value can convey how many cycles it takes to detect a signal from a sample. In some embodiments, quantification cycle value for a sample processing method described herein may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 15, at least about 18, at least about 20, at least about 25, at leastabout 30, at least about 35, or at least about 40. In some embodiments, quantification cycle value for a sample processing method described herein may be at most about 40, at most about 35, at most about 30, at most about 25, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1.

[0148] In some aspects, the present disclosure provides for a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule having bound thereto a guide complex, wherein the guide complex comprises: a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of the single-stranded nucleic acid molecule, and a second guide polynucleotide that hybridizes with the non-target binding region of the first guide molecule to form a doublestranded binding region, wherein the double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme.

[0149] In some aspects, the present disclosure provides for a system of processing a singlestranded nucleic acid molecule comprising a target sequence, the system comprising: the singlestranded nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) an unblocked 3’ end or a blocked 3' end non-extendable by a polymerase; and the enzyme bound to the restriction endonuclease recognition sequence of the non-target binding region.

[0150] For example, a system of processing a single-stranded nucleic acid molecule comprising a target sequence can comprise: the single-stranded nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) an unblocked 3’ end or a blocked 3' end non-extendable by a polymerase; and the enzyme bound to the restriction endonuclease recognition sequence of the non-target binding region, wherein the single-stranded nucleic acid molecule or the target sequence is from Bacillus anthracis.

[0151] Alternatively, a system of processing a single-stranded nucleic acid molecule comprising a target sequence can comprise: the single-stranded nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for anenzyme that is a type Ils restriction enzyme, and (ii) a target binding region configured to hybridize to the target sequence; and the enzyme bound to the restriction endonuclease recognition sequence of the non-target binding region, wherein the single-stranded nucleic acid molecule or the target sequence is from Bacillus anthracis.

[0152] The methods, systems, or kits provided herein can be used to process or analyze one sample or one target nucleic acid molecule or target sequence. Alternatively, the methods, systems or kits provided herein can be used to process or analyze two or more different samples, or two or more different target nucleic acid molecules or target sequences in a same reaction mixture (e.g., a single reaction). For example, the methods, systems or kits provided herein can be used to process or analyze 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different target nucleic acid sequences in a same reaction mixture. In some embodiments, the reaction mixture is lyophilized. In some embodiments, the reaction mixture is not lyophilized.

[0153] In various embodiments, the guide polynucleotide comprises a target binding region. The sequence of the target binding region can be designed according to the target sequence by following similar rules for primer design. For example, primer design can be based on various parameters, including melting temperature of the primers (which may be calculated using the nearest neighbor algorithm shown in John Santa Lucia, Jr., "A unified view of polymers, dumbbell, and oligonucleotide DNA nearest-neighbor thermal dynamics," Proc. Natl. Acad. Sci. USA., Vol.95, 1460-1465(1998) (the contents of which are incorporated herein by reference in their entirety)), primer composition (e.g., nucleotide composition such as GC content may be determined and filtered using software and penalized, as is the composition of the GC content of the hairpin, 3' end of the primer, and the specific parameters that may be evaluated are the homopolymer nucleotides in length, hairpin formation, GC content and amplicon size), predicted dimer-dimer formations, average extension length and the like. In the case of multiplexed reactions with two or more target sequences, the target binding region (or primer) can be designed to minimize cross-reactivity. The non-target binding region of the guide polynucleotide can be designed to be non-hybridizable with the target sequence and contain a sequence that can be recognized by an enzyme (e.g., the restriction enzyme) described herein.

[0154] In some aspects, the present disclosure provides for a method or a system of multiplexing the processing of more than one nucleic acid molecules, each nucleic acid molecule comprising a different target sequence. The method or system can comprise, for each nucleic acid molecule comprising a different target sequence, a nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide. The guide polynucleotide can comprise: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for anenzyme that is a type Ils restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) an unblocked 3’ end or a blocked 3' end non-extendable by a polymerase. The enzyme can bind to the restriction endonuclease recognition sequence of the non-target binding region. In some aspects, a multiplexed processing of one or more nucleic acid molecules comprises using two or more different sets of primers or guide complexes, each targeting a different target. In some aspects, multiplexed processing of one or more nucleic acid molecules comprises a reaction mixture comprising two more different detection probes or fluorophores, each targeting a different target sequence. Each of the two or more different detection probes can be linked to a different fluorophore for multiplexed detection.

[0155] For example, a system for processing a plurality of single-stranded nucleic acid molecules, each comprising a different target sequence can comprise: a first single-stranded nucleic acid molecule wherein the first single-stranded nucleic acid molecule is bound to a first guide complex comprising a first guide polynucleotide, wherein the first guide polynucleotide comprises: (i) a first non-target binding region comprising a first restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme; (ii) a first target binding region configured to hybridize to a first target sequence; and (iii) a first unblocked 3’ end or a first blocked 3' end non-extendable by a polymerase; and a second single-stranded nucleic acid molecule wherein the second single-stranded nucleic acid molecule is bound to a second guide complex comprising a second guide polynucleotide, wherein the second guide polynucleotide comprises: (i) a second non-target binding region comprising a second restriction endonuclease recognition sequence for the enzyme that is a type Ils restriction enzyme; (ii) a second target binding region configured to hybridize to a second target sequence; and (iii) a second unblocked 3’ end or a second blocked 3' end non-extendable by a polymerase; wherein the enzyme that is a type Ils restriction enzyme binds to the first restriction endonuclease recognition sequence of the first non-target binding region or the second restriction endonuclease recognition sequence of the second non-target binding region, wherein the first single-stranded nucleic acid molecule or the first target sequence is from Bacillus antliracis, and the second single-stranded nucleic acid molecule or the second target sequence is from Bacillus anthracis.

[0156] The amplification product can be detected by various methods. The amplification products may be detected by gel electrophoresis, thus detecting reaction products having a specific length. The nucleotides may, for example, be labeled, such as, for example, with biotin. Biotin-labeled amplified sequences may be captured using avidin bound to a signal generating enzyme, for example, peroxidase. Nucleic acid detection methods may employ the use of dyes that specifically stain double-stranded DNA. Intercalating dyes that exhibit enhancedfluorescence upon binding to DNA or RNA can be used. Dyes may be, for example, DNA or RNA intercalating fluorophores and may include but are not limited to the following examples: Acridine orange, ethidium bromide, Hoechst dyes, PicoGreen, propidium iodide, SYBRI (an asymmetrical cyanine dye), SYBRII, TOTO (a thiaxole orange dimer) and YOYO (an oxazole yellow dimer), and the like. Dyes can provide an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with various detection methods and may have varying optimal usage parameters. Nucleic acid detection methods may also employ the use of labeled nucleotides incorporated directly into the target sequence or into probes containing complementary or substantially complementary sequences to the target of interest. Such labels may be radioactive and / or fluorescent in nature. Labeled nucleotides, which can be detected but otherwise function as native nucleotides, can be to be distinguished from modified nucleotides, which do not function as native nucleotides. The production or presence of target nucleic acids and nucleic acid sequences may be detected and monitored by Molecular Beacons. The production or presence of target nucleic acids and nucleic acid sequences may also be detected and monitored by Fluorescence resonance energy transfer (FRET).

[0157] A wide range of fluorophores and / or dyes may be used in the methods described herein according to the present disclosure. Available fluorophores include coumarin; fluorescein; tetrachlorofluorescein; hexachlorofluorescein; Lucifer yellow; rhodamine; BODIPY; tetramethylrhodamine; Cy3; Cy5; Cy7; eosine; Texas red; SYBR Green I; SYBR Gold; 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H), 9'-(9H)xanthene)-5- carboxylic acid, 3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-Hexachloro- Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5- Tetrachloro-Fluorescein ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-Tetrachloro-Fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6- carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium, 9-(2,4- dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2- aminoethyl)amino)naphthalene-l -sulfonic acid); 1,5-IAEDANS (5-((((2- iodoacetyl)amino)ethyl)amino)naphthalene-l -sulfonic acid); DABCYL (4-((4- (dimethylamino)phenyl) azo)benzoic acid) Cy5 (Indodicarbocyanine-5) Cy3 (Indo- dicarbocyanine-3); BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-proprionic acid); Quasar-670 (Bioresearch Technologies); CalOrange (Bioresearch Technologies); and Rox as well as suitable derivatives thereof. Combination fluorophores such asfluorescein-rhodamine dimers may also be suitable. Fluorophores may be chosen to absorb and emit in the visible spectrum or outside the visible spectrum, such as in the ultraviolet or infrared ranges. Suitable quenchers may also include DABCYL and variants thereof, such as DABSYL, DABMI and Methyl Red. Fluorophores may also be used as quenchers, because they tend to quench fluorescence when touching certain other fluorophores. In some cases, quenchers may be chromophores such as DABCYL or malachite green, or fluorophores that may not fluoresce in the detection range when the probe is in the open conformation.

[0158] In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6 at least 7, at least 8, at least 9, at least 10, or more pluralities of single-stranded nucleic acid molecules can be processed in the same reaction. In some embodiments, each plurality of the multiplexed nucleic acid molecules is derived from a different sample.

[0159] A sample described herein can comprise a biological sample. A sample can comprise a single-stranded nucleic acid molecule. Alternatively, a sample can comprise a double-stranded nucleic acid molecule.

[0160] A sample can comprise a fluid sample. Non-limiting examples of fluid samples can include blood, plasma, urine, feces saliva, sweat, tears, pericardial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, gastric juice, respiratory secretion, semen, synovial fluid, or amniotic fluid.

[0161] In some embodiment, the sample comprises a blood sample, a swab sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, a pleural fluid sample, a rectal sample, a vaginal sample, a stool sample, a sputum sample, and / or a lymph sample for nucleic acid amplification. In some embodiments, the swab sample comprises a vaginal swab, an oral swab, a nasopharyngeal swab, a nasal swab, and / or a rectal swab. In some embodiments, the sample is selected from the group consisting of peripheral blood, sputum, nasopharyngeal swab, nasopharyngeal wash, bronchoalveolar lavage, endotracheal aspirate, and combinations thereof. In some embodiments, the sample is a solid sample. In some embodiments, the sample is a liquid sample. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject has a disease, a condition, or an infection. In some embodiments, the sample comprises a purified sample. In some embodiments, the sample is a combination of two, three, four, five, or more types of samples. In some embodiments, the sample comprises one, two, three, four, five, six, seven, eight, nine, ten, or more target nucleic acid molecules.

[0162] A sample may be obtained invasively (e.g., tissue biopsy) or non-invasively (e.g., venipuncture). The sample may be an environmental sample. The sample may be a water sample (e.g., a water sample obtained from a lake, stream, river, estuary, bay, or ocean). The sample maybe a soil sample. The sample may be a tissue or fluid sample from a subject, such as saliva, semen, blood (e.g., whole blood), serum, synovial fluid, tear, urine, or plasma. The sample may be a tissue sample, such as a skin sample or tumor sample. The sample may be obtained from a portion of an organ of a subject. The sample may be a cellular sample. The sample may be a cell-free sample (e.g., a plasma sample comprising cell-free analytes or nucleic acids). A sample may be a solid sample or a liquid sample. A sample may be a biological sample or a non- biological sample. A sample may comprise an in-vitro sample or an ex -vivo sample. Nonlimiting examples of a sample include an amniotic fluid, bile, bacterial sample, breast milk, buffy coat, cells, cerebrospinal fluid, chromatin DNA, ejaculate, nucleic acids, plant-derived materials, RNA, saliva, semen, blood, serum, soil, synovial fluid, tears, tissue, urine, water, whole blood or plasma, and / or any combination and / or any fraction thereof. In one example, the sample may be a plasma sample that may comprise DNA. In another example, the sample may comprise a cell sample that may comprise cell-free DNA.

[0163] A sample may be a mammalian sample. For example, a sample may be a human sample. Alternatively, a sample may be a non-human animal sample. Non-limiting examples of a nonhuman sample include a cat sample, a dog sample, a goat sample, a guinea pig sample, a hamster sample, a mouse sample, a pig sample, a non-human primate sample (e.g., a gorilla sample, an ape sample, an orangutan sample, a lemur sample, or a baboon sample), a rat sample, a sheep sample, a cow sample, and a zebrafish sample.

[0164] The sample may comprise nucleic acids (e.g., circulating and / or cell-free DNA fragments). Nucleic acids may be derived from eukaryotic cells, prokaryotic cells, or non-cellular sources (e.g., viral particles). A nucleic acid may refer to a substance whose molecules consist of many nucleotides linked in a long chain. Non-limiting examples of the nucleic acid include an artificial nucleic acid analog (e.g., a peptide nucleic acid, a morpholino oligomer, a locked nucleic acid, a glycol nucleic acid, or a threose nucleic acid), chromatin, niRNA, cDNA, DNA, single stranded DNA, double stranded DNA, genomic DNA, plasmid DNA, or RNA. A nucleic acid may be double stranded or single stranded. A sample may comprise a nucleic acid that may be intracellular. Alternatively, a sample may comprise a nucleic acid that may be extracellular (e.g., cell-free). A sample may comprise a nucleic acid (e.g., chromatin) that may be fragmented.

[0165] A sample can be obtained from a virus, a bacterium, an archaea, or a eukarya. In some embodiments, a sample is obtained from a bacterium. A bacterium can be a spherical-shaped bacterium, a rod-shaped bacterium, a spiral-shaped bacterium, a comma-shaped bacterium, or a corkscrew-shaped bacterium. Non-limiting examples of bacteria are Streptococcus pneumoniae, Streptococcus pyogenes, Legionella pneumonia, Bordetella bronchiseptica, Enterobacteraerogenes, Pasteurella multocida, Proteus mirabilis, Staphylococcus aureus, Haemophilus influenzae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia pneumoniae and Chlamydia trachomatis.

[0166] In some embodiments, the bacterium is a gram-positive bacterium. In some embodiments, the bacterium is aerobic. In some embodiments, the bacterium is a spore-bearing bacillus. In some embodiments, the bacterium is Bacillus anthracis. In some embodiments, the bacterium is anaerobic. In some embodiments, the bacterium is of the Clostridium species.

[0167] In some embodiments, the methods and / or systems described herein may be used to process a single-stranded nucleic acid molecule. In some embodiments, the methods and / or systems described herein may be used to process a plurality of single-stranded nucleic acid molecules. In some embodiments, the plurality of single-stranded nucleic acid molecules comprises a first single-stranded nucleic acid molecule, a second single-stranded nucleic acid molecule, a third single-stranded nucleic acid molecule, a fourth single-stranded nucleic acid molecule, a fifth single-stranded nucleic acid molecule, a sixth single-stranded nucleic acid molecule, a seventh single-stranded nucleic acid molecule, an eighth single-stranded nucleic acid molecule, or more. In some embodiments, the single-stranded nucleic acid molecule comprises a target sequence.

[0168] For example, a method of processing a single- stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and (ii) a target binding region configured to hybridize to the target sequence; and (b) introducing the type Ils restriction enzyme under conditions sufficient to cause the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence, wherein the singlestranded nucleic acid molecule or the target sequence is from Bacillus anthracis.

[0169] The guide polynucleotide can comprise a blocked 3' end. In some embodiments, the guide polynucleotide may not comprise a blocked 3' end (e.g., the guide polynucleotide may comprise an unblocked 3' end). The unblocked 3' end can be extendable by a polymerase.

[0170] In some embodiments, a single-stranded nucleic acid molecule or a target sequence is from Bacillus anthracis. In some embodiments, the methods and / or systems provided herein comprises two or more single-stranded nucleic acid molecules and each single-stranded nucleicacid molecule is from Bacillus anthracis. In some embodiments, in a plurality of single-stranded nucleic acid molecules, at least one single-stranded nucleic acid molecule is from Bacillus anthracis.

[0171] In some embodiments, a first single-stranded nucleic acid molecule, a single-stranded nucleic acid molecule, and a third single- stranded nucleic acid molecule are from a virulence plasmid o Bacillus anthracis chromosome. In some embodiments, a first single-stranded nucleic acid molecule is from a virulence plasmid. In some embodiments, a first single-stranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a second singlestranded nucleic acid molecule is from a virulence plasmid. In some embodiments, a second single-stranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a third single-stranded nucleic acid molecule is from a virulence plasmid. In some embodiments, a third single-stranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule are from a virulence plasmid. In some embodiments, a first single-stranded nucleic acid molecule and a third single-stranded nucleic acid molecule are from a virulence plasmid. In some embodiments, a second single-stranded nucleic acid molecule and a third single-stranded nucleic acid molecule are from a virulence plasmid.

[0172] In some embodiments, a first single-stranded nucleic acid molecule is from a virulence plasmid and a second single- stranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a first single-stranded nucleic acid molecule is from a virulence plasmid and a third single-stranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a second single-stranded nucleic acid molecule is from a virulence plasmid and a third single-stranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a first single-stranded nucleic acid molecule is from a virulence plasmid, a second single-stranded nucleic acid molecule is from a virulence plasmid, and a third single-stranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a first single-stranded nucleic acid molecule is from a virulence plasmid, a third single-stranded nucleic acid molecule is from a virulence plasmid, and a second singlestranded nucleic acid molecule is from a Bacillus anthracis chromosome. In some embodiments, a second single-stranded nucleic acid molecule is from a virulence plasmid, a third singlestranded nucleic acid molecule is from a virulence plasmid, and a first single-stranded nucleic acid molecule is from a Bacillus anthracis chromosome.

[0173] In some embodiments, a first target sequence, a target sequence, and a third target sequence are from a virulence plasmid ox Bacillus anthracis chromosome. In some embodiments,a first target sequence is from a virulence plasmid. In some embodiments, a first target sequence is from a Bacillus anthracis chromosome. In some embodiments, a second target sequence is from a virulence plasmid. In some embodiments, a second target sequence is from a Bacillus anthracis chromosome. In some embodiments, a third target sequence is from a virulence plasmid. In some embodiments, a third target sequence is from a Bacillus anthracis chromosome. In some embodiments, a first target sequence and a second target sequence are from a virulence plasmid. In some embodiments, a first target sequence and a third target sequence are from a virulence plasmid. In some embodiments, a second target sequence and a third target sequence are from a virulence plasmid.

[0174] In some embodiments, a first target sequence is from a virulence plasmid and a second target sequence is from a Bacillus anthracis chromosome. In some embodiments, a first target sequence is from a virulence plasmid and a third target sequence is from a Bacillus anthracis chromosome. In some embodiments, a second target sequence is from a virulence plasmid and a third target sequence is from a Bacillus anthracis chromosome. In some embodiments, a first target sequence is from a virulence plasmid, a second target sequence is from a virulence plasmid, and a third target sequence is from a Bacillus anthracis chromosome. In some embodiments, a first target sequence is from a virulence plasmid, a third target sequence is from a virulence plasmid, and a second target sequence is from a Bacillus anthracis chromosome. In some embodiments, a second target sequence is from a virulence plasmid, a third target sequence is from a virulence plasmid, and a first target sequence is from a Bacillus anthracis chromosome.

[0175] In some embodiments, the virulence plasmid can be pXOl or pX02. In some embodiments a first virulence plasmid can be pXOl and a second virulence plasmid can be pX02. In some embodiments a first virulence plasmid can be pX02 and a second virulence plasmid can be pXOl.

[0176] In some embodiments, a sample is obtained from a virus. A virus can be a doublestranded DNA virus, a single-stranded DNA virus, a double-stranded RNA virus, a singlestranded RNA virus, a positive sense single-stranded reverse transcriptase virus, or a doublestranded DNA reverse transcriptase virus. In some cases, the sample comprises a human gene such as RPP30. In some embodiments, sample preparation can comprise extracting nucleic acids from a sample. In some embodiments, sample preparation can comprise extracting nucleic acids from a sample by heating the sample. For example, a target nucleic acid (e.g., target RNA, target DNA) may be extracted or released from a biological sample during heating phases of nucleic acid amplification. Alternatively or in addition to the heating, a target nucleic acid (e.g., target RNA, target DNA) may be extracted or released from a biological sample using a cartridgesystem wherein a sample can be mixed with a lysis buffer and then drawn through a filter thereby capturing the target nucleic acid in the filter. In some cases, a cartridge system can also comprise washing steps to remove contaminants. An elution buffer can be added to the cartridge to remove the target nucleic acid from the filter for further processing or analysis. The cartridge system can be an automated cartridge system. In some cases, the cartridge system can be the Ml Sample Prep® Cartridge Kit (SKU:3000536, Biomeme, Inc.). In some cases, the sample preparation method described herein can use the cartridge system for automated sample processing. Details of the sample preparation cartridge and related methods is described in the U.S. Application No. 16 / 817,733, the entire content of which is incorporated herein by reference. It is to be understood that the sample described herein can be processed by various other methods or any commercially available nucleic acid extraction kits or methods.

[0177] In some aspects, the present disclosure provides for a kit comprising any of the guide complexes or any of the guide polynucleotides described herein. In some embodiments, the kit further comprises a probe or a dye for detecting an amplification product generated using the kit. In some embodiments, the kit further comprises an informational material describing an instruction of using the kit. In some embodiments, the information comprises optimal reaction temperatures for amplification using the guide complexes or the guide polynucleotides, or optimal buffer conditions for the same. In some embodiments, the kit further comprises a type II restriction enzyme compatible with the guide polynucleotides or guide complexes as described herein. In some embodiments, the kit further comprises a strand-displacing polymerase. The kits can be compartmentalized for ease of use and can include one or more containers with reagents. In some embodiments, all of the kit components are packaged together. Alternatively, one or more individual components of the kit can be provided in a separate package from the other kits components.Methods of Processing Nucleic Acid Molecules using Guide Polynucleotides with Variations of Non-Target Binding Region

[0178] The present invention can comprise guide molecules comprising a non-target binding region. The non-target binding region can be an adapter, an adapter region, and / or an adapter sequence described herein. Adapter regions of guide polynucleotides and / or adapter molecules (e.g., non-target binding molecules) may be modified to modulate a reaction rate of reactions described herein. An adapter molecule, adapter region, adapter sequence, or any combination thereof may be adjusted to increase guanine-cytosine content (e.g., make more GC rich), increase adenine-thymine content (e.g., make more AT rich), increase a length of the adapter, decrease alength of the adapter, or any combination thereof. In some embodiments, the adapters may comprise modified bases, which may be varied (e.g., inserted and / or substituted) into the adapter to affect the reaction rate. The adapter may comprise one or more substitutions in an endonuclease complimentary recognition site to affect a reaction rate. Variations of the nontarget binding region described herein may be used to increase or decrease a reaction rate of a reaction (e.g., a nucleic acid amplification reaction) described herein.

[0179] A reaction may be a multiplexed reaction in which two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) target sequences can be amplified simultaneously. Addition of the non-target binding region, modification of the non-target binding regions and / or non-target binding molecules, or any combination thereof may enhance a multiplexed reaction by preventing or minimizing “cross-talk” between the target sequences that may not share adapters. Without wishing to be bound by theory, addition of multiple adapters may inhibit the different reactions of the multiplexed reaction from interacting with one another, reducing a complexity of the reaction. The non-target binding molecules and / or non-target binding regions described herein can promote the interaction of a forward guide molecule with a reverse. The use of different guide polynucleotides comprising different adapter (e.g., non-target binding region) sequences may provide for greater precision and control of reaction launch time.

[0180] In some aspects, the present disclosure provides a method of processing a nucleic acid molecule. The nucleic acid molecule can be a single-stranded nucleic acid molecule. The singlestranded nucleic acid molecule can comprise a target sequence. In some embodiments, the method can comprise contacting a single-stranded nucleic acid molecule with a guide complex. A guide complex may comprise a guide polynucleotide described herein. The guide polynucleotide can comprise a non-target binding region. The non-target binding region may comprise a sequence that can be recognized by a restriction endonuclease (e.g., a restriction endonuclease recognition sequence). The non-target binding region may comprise a sequence that can be recognized by an enzyme. The non-target binding region may comprise a sequence that can be recognized by a restriction enzyme (e.g., a Type I, Type II, Type III, or Type IV restriction enzyme). The non-target binding region may comprise a sequence that can be recognized by a type Ils restriction enzyme. The non-target binding region may comprise at least one modified nucleotide. The guide polynucleotide may hybridize to the single-stranded nucleic acid molecule. In some embodiments, the method can comprise introducing the type Ils restriction enzyme. The enzyme may be introduced under conditions that allow it to bind to the restriction endonuclease recognition sequence of the guide polynucleotide. In some embodiments, the enzyme may bind to the restriction endonuclease recognition sequence. In some embodiments, the enzyme may notbind to the restriction endonuclease recognition sequence. Once bound to the restriction endonuclease recognition sequence, the enzyme may cut within the target sequence of the singlestranded nucleic acid molecule. The cut can generate an extendable 3’ end of the target sequence. The target sequence may be from Bacillus anthracis.

[0181] For example, the method of processing a single-stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions sufficient to allow the guide polynucleotide to hybridize to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, wherein the non-target binding region comprises a modified nucleotide, (ii) a target binding region that hybridizes to the target sequence, and (iii) a blocked 3' end non-extendable by a polymerase; and (b) introducing the type Ils restriction enzyme under conditions sufficient to allow the type Ils restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3 ' end of the target sequence.

[0182] As another example, a method of processing a single-stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions sufficient to allow the guide polynucleotide to hybridize to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, wherein the non-target binding region comprises a modified nucleotide, and (ii) a target binding region that hybridizes to the target sequence; and (b) introducing the type Ils restriction enzyme under conditions sufficient to allow the type Ils restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end of the target sequence.

[0183] In some embodiments, a guide polynucleotide may comprise from 5’ end to 3’ end: a non-target binding region, a target binding region, and a non-extendable 3’ end (e.g., a guide molecule PNA sequence). Modifications to the guide polynucleotide can modulate the rate of a reaction in processing of the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule). The guide polynucleotide may be modified at any point in the sequence of the polynucleotide. For example, the guide polynucleotide may be modified at a non-target binding region. As another example, the guide polynucleotide may be modified at a non-extendable 3’ end. In some embodiments, a guide polynucleotide described herein may comprise modifiednucleotides at the non-target binding region and the non-extendable 3’ end. Without wishing to be bound by theory, the modification at the 3’ end of the guide polynucleotide may help prevent the extension by a polymerase. By blocking the extension on the 3’ ends, the guide polynucleotides may act as rate modulators and not take part in the reaction.

[0184] The non-target binding region may comprise one or more modified nucleotides. In some embodiments, the non-target binding region can comprise at least about 1 modified nucleotide, at least about 2 modified nucleotides, at least about 3 modified nucleotides, at least about 4 modified nucleotides, at least about 5 modified nucleotides, at least about 6 modified nucleotides, at least about 7 modified nucleotides, at least about 8 modified nucleotides, at least about 9 modified nucleotides, at least about 10 modified nucleotides, or greater than about 10 modified nucleotides. In some embodiments, the non-target binding region can comprise at most about 10 modified nucleotides, at most about 9 modified nucleotides, at most about 8 modified nucleotides, at most about 7 modified nucleotides, at most about 6 modified nucleotides, at most about 5 modified nucleotides, at most about 4 modified nucleotides, at most about 3 modified nucleotides, at most about 2 modified nucleotides, at most about 1 modified nucleotides, or less than about 1 modified nucleotide.

[0185] In some embodiments, the non-target binding region can comprise from about 1 modified nucleotide to about 12 modified nucleotides. In some embodiments, the non-target binding region can comprise from about 1 modified nucleotide to about 2 modified nucleotides, about 1 modified nucleotide to about 3 modified nucleotides, about 1 modified nucleotide to about 4 modified nucleotides, about 1 modified nucleotide to about 5 modified nucleotides, about 1 modified nucleotide to about 6 modified nucleotides, about 1 modified nucleotide to about 7 modified nucleotides, about 1 modified nucleotide to about 8 modified nucleotides, about 1 modified nucleotide to about 9 modified nucleotides, about 1 modified nucleotide to about 10 modified nucleotides, about 1 modified nucleotide to about 11 modified nucleotides, about 1 modified nucleotide to about 12 modified nucleotides, about 2 modified nucleotides to about 3 modified nucleotides, about 2 modified nucleotides to about 4 modified nucleotides, about 2 modified nucleotides to about 5 modified nucleotides, about 2 modified nucleotides to about 6 modified nucleotides, about 2 modified nucleotides to about 7 modified nucleotides, about 2 modified nucleotides to about 8 modified nucleotides, about 2 modified nucleotides to about 9 modified nucleotides, about 2 modified nucleotides to about 10 modified nucleotides, about 2 modified nucleotides to about 11 modified nucleotides, about 2 modified nucleotides to about 12 modified nucleotides, about 3 modified nucleotides to about 4 modified nucleotides, about 3 modified nucleotides to about 5 modified nucleotides, about 3 modified nucleotides to about 6modified nucleotides, about 3 modified nucleotides to about 7 modified nucleotides, about 3 modified nucleotides to about 8 modified nucleotides, about 3 modified nucleotides to about 9 modified nucleotides, about 3 modified nucleotides to about 10 modified nucleotides, about 3 modified nucleotides to about 11 modified nucleotides, about 3 modified nucleotides to about 12 modified nucleotides, about 4 modified nucleotides to about 5 modified nucleotides, about 4 modified nucleotides to about 6 modified nucleotides, about 4 modified nucleotides to about 7 modified nucleotides, about 4 modified nucleotides to about 8 modified nucleotides, about 4 modified nucleotides to about 9 modified nucleotides, about 4 modified nucleotides to about 10 modified nucleotides, about 4 modified nucleotides to about 11 modified nucleotides, about 4 modified nucleotides to about 12 modified nucleotides, about 5 modified nucleotides to about 6 modified nucleotides, about 5 modified nucleotides to about 7 modified nucleotides, about 5 modified nucleotides to about 8 modified nucleotides, about 5 modified nucleotides to about 9 modified nucleotides, about 5 modified nucleotides to about 10 modified nucleotides, about 5 modified nucleotides to about 11 modified nucleotides, about 5 modified nucleotides to about 12 modified nucleotides, about 6 modified nucleotides to about 7 modified nucleotides, about 6 modified nucleotides to about 8 modified nucleotides, about 6 modified nucleotides to about 9 modified nucleotides, about 6 modified nucleotides to about 10 modified nucleotides, about 6 modified nucleotides to about 11 modified nucleotides, about 6 modified nucleotides to about 12 modified nucleotides, about 7 modified nucleotides to about 8 modified nucleotides, about 7 modified nucleotides to about 9 modified nucleotides, about 7 modified nucleotides to about 10 modified nucleotides, about 7 modified nucleotides to about 11 modified nucleotides, about 7 modified nucleotides to about 12 modified nucleotides, about 8 modified nucleotides to about 9 modified nucleotides, about 8 modified nucleotides to about 10 modified nucleotides, about 8 modified nucleotides to about 11 modified nucleotides, about 8 modified nucleotides to about 12 modified nucleotides, about 9 modified nucleotides to about 10 modified nucleotides, about 9 modified nucleotides to about 11 modified nucleotides, about 9 modified nucleotides to about 12 modified nucleotides, about 10 modified nucleotides to about 11 modified nucleotides, about 10 modified nucleotides to about 12 modified nucleotides, or about 11 modified nucleotides to about 12 modified nucleotides.

[0186] The modified nucleotide of the guide polynucleotide may comprise 2’ -O-m ethoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base. The unnatural base can comprise a a-thiol deoxynucleotide triphosphate (dNTP) and / or a dideoxyribonucleotidetriphosphate (ddNTP). A deoxynucleotide triphosphate may be dATP, dGTP, dCTP, dTTP, or any combination thereof. A dideoxyribonucleotide triphosphate can be ddATP, ddGTP, ddCTP, ddTTP, or any combination thereof. In some embodiments, the universal base can comprise deoxy Inosine, nitroindole, 2’-deoxynebularine, 3 -nitropyrrole, or any combination thereof.

[0187] In some embodiments, a modified nucleotide may not comprise adenine, guanine, thymine, or cytosine.

[0188] In some embodiments, the guide polynucleotide may comprise modifications to increase guanine and cytosine content (e.g., make the guide polynucleotide more GC rich). In some embodiments, the guide polynucleotide may comprise modifications to increase adenine and thymine content (e.g., make the guide polynucleotide more AT rich).

[0189] In some embodiments, modifications to the guide polynucleotide may increase a length of the guide polynucleotide. In some embodiments, a length of a guide polynucleotide may be increased by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or greater than about 100 nucleotides. In some embodiments, a length of a guide polynucleotide may be increased by at most about 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, or less than about 1 nucleotide. In some embodiments, a length of a guide polynucleotide may be increased from about 1 nucleotide to about 100 nucleotides. In some embodiments, a length of a guide polynucleotide may be increased from about 1 nucleotide to about 2 nucleotides, about 1 nucleotide to about 3 nucleotides, about 1 nucleotide to about 4 nucleotides, about 1 nucleotide to about 5 nucleotides, about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 1 nucleotide to about 25 nucleotides, about 1 nucleotide to about 50 nucleotides, about 1 nucleotide to about 75 nucleotides, about 1 nucleotide to about 100 nucleotides, about 2 nucleotides to about 3 nucleotides, about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 5 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 20 nucleotides, about 2 nucleotides to about 25 nucleotides, about 2 nucleotides to about 50 nucleotides, about 2 nucleotides to about 75 nucleotides, about 2 nucleotides to about 100 nucleotides, about 3 nucleotides to about 4 nucleotides, about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 15 nucleotides, about 3 nucleotides to about 20 nucleotides, about 3 nucleotides to about 25 nucleotides, about 3 nucleotides to about 50 nucleotides, about 3 nucleotides to about 75 nucleotides, about 3 nucleotides to about 100 nucleotides, about 4 nucleotides to about 5 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 20nucleotides, about 4 nucleotides to about 25 nucleotides, about 4 nucleotides to about 50 nucleotides, about 4 nucleotides to about 75 nucleotides, about 4 nucleotides to about 100 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 75 nucleotides, about 5 nucleotides to about 100 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 75 nucleotides, about 10 nucleotides to about 100 nucleotides, about 15 nucleotides to about 20 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 75 nucleotides, about 15 nucleotides to about 100 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 75 nucleotides, about 20 nucleotides to about 100 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 75 nucleotides, about 25 nucleotides to about 100 nucleotides, about 50 nucleotides to about 75 nucleotides, about 50 nucleotides to about 100 nucleotides, or about 75 nucleotides to about 100 nucleotides.

[0190] In some embodiments, modifications to the guide polynucleotide may shorten a length of the guide polynucleotide. In some embodiments, a length of a guide polynucleotide may be shortened by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or greater than about 100 nucleotides. In some embodiments, a length of a guide polynucleotide may be shortened by at most about 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, or less than about 1 nucleotide. In some embodiments, a length of a guide polynucleotide may be shortened from about 1 nucleotide to about 100 nucleotides. In some embodiments, a length of a guide polynucleotide may be shortened from about 1 nucleotide to about 2 nucleotides, about 1 nucleotide to about 3 nucleotides, about 1 nucleotide to about 4 nucleotides, about 1 nucleotide to about 5 nucleotides, about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 1 nucleotide to about 25 nucleotides, about 1 nucleotide to about 50 nucleotides, about 1 nucleotide to about 75 nucleotides, about 1 nucleotide to about 100 nucleotides, about 2 nucleotides to about 3 nucleotides, about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 5 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 20 nucleotides, about 2 nucleotides to about 25 nucleotides, about 2 nucleotides to about 50 nucleotides, about 2 nucleotides to about 75nucleotides, about 2 nucleotides to about 100 nucleotides, about 3 nucleotides to about 4 nucleotides, about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 15 nucleotides, about 3 nucleotides to about 20 nucleotides, about 3 nucleotides to about 25 nucleotides, about 3 nucleotides to about 50 nucleotides, about 3 nucleotides to about 75 nucleotides, about 3 nucleotides to about 100 nucleotides, about 4 nucleotides to about 5 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 20 nucleotides, about 4 nucleotides to about 25 nucleotides, about 4 nucleotides to about 50 nucleotides, about 4 nucleotides to about 75 nucleotides, about 4 nucleotides to about 100 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 50 nucleotides, about 5 nucleotides to about 75 nucleotides, about 5 nucleotides to about 100 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 75 nucleotides, about 10 nucleotides to about 100 nucleotides, about 15 nucleotides to about 20 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 75 nucleotides, about 15 nucleotides to about 100 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 75 nucleotides, about 20 nucleotides to about 100 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 75 nucleotides, about 25 nucleotides to about 100 nucleotides, about 50 nucleotides to about 75 nucleotides, about 50 nucleotides to about 100 nucleotides, or about 75 nucleotides to about 100 nucleotides.

[0191] In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced. The reaction launch rate can be a rate for generating copies of extendable products of the target sequence with an extendable 3' end per second. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the guide polynucleotide comprising a modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the non-target binding region of the guide polynucleotide comprising a modified nucleotide. In some embodiments, a reaction launchrate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the 3’ end (e.g., the non-extendable 3’ end) of the guide polynucleotide comprising a modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction with the non-target binding region that does not comprise the modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction with the 3’ end region that does not comprise the modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction with an alternative guide polynucleotide that comprises less modified nucleotides than the guide polynucleotide.

[0192] In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non-target binding region that does not comprise the modified nucleotide. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced by at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non-target binding region that does not comprise the modified nucleotide. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced from about 1% to about 50% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non- target binding region that does not comprise the modified nucleotide. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1%to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non-target binding region that does not comprise the modified nucleotide.

[0193] In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the guide polynucleotide comprising a modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the non-target binding region of the guide polynucleotide comprising a modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the 3’ end (e.g., the non-extendable 3’ end) of the guide polynucleotide comprising a modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction with the non-target binding region that does not comprise themodified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction with the 3’ end region that does not comprise the modified nucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction with an alternative guide polynucleotide that comprises less modified nucleotides than the guide polynucleotide.

[0194] In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non-target binding region that does not comprise the modified nucleotide. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased by at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non-target binding region that does not comprise the modified nucleotide. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased from about 1% to about 50% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non- target binding region that does not comprise the modified nucleotide. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% as compared to an otherwise identical reaction (i) without the non-target binding region of the guide polynucleotide comprising a modified nucleotide or (ii) with the non-target binding region that does not comprise the modified nucleotide

[0195] In some aspects, the present disclosure provides a method of processing a nucleic acid molecule (e.g., a single- stranded nucleic acid molecule comprising a target sequence) in which the single-stranded nucleic acid molecule can be contacted with a guide complex and a non-target binding molecule. The non-target binding molecule may not have a complementary binding region to a sequence of the single-stranded nucleic acid molecule. In some embodiments, the non-target binding molecule may be added to the reaction directly. The guide complex can comprise a guide polynucleotide comprising a non-target binding region. The non-target binding region of the may comprise a sequence that can be recognized by a type Ils restriction enzyme. The non-target binding region may comprise at least one modified nucleotide. The guide polynucleotide may hybridize to the single-stranded nucleic acid molecule. In some embodiments, the method can comprise introducing the type Ils restriction enzyme. The enzyme may be introduced under conditions that allow it to bind to the restriction endonuclease recognition sequence of the guide polynucleotide. The enzyme (e.g., the type Ils restriction enzyme) can bind to a restriction endonuclease recognition sequence of the non-target binding region and initiate a cut within the target sequence of the single-stranded nucleic acid molecule. The cut may generate an extendable 3' end of the target sequence and comprise processing of the single-stranded nucleic acid molecule. The target sequence may be from Bacillus anthracis.

[0196] For example, the method of processing a single-stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting the single-stranded nucleic acid molecule with a guide complex and a non-target binding molecule in a reaction, wherein the guide complex comprises a guide polynucleotide under conditions sufficient to allow the guide polynucleotide to hybridize to the single-stranded nucleic acid molecule, wherein the guidepolynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, (ii) a target binding region that hybridizes to the target sequence, and (iii) a blocked 3' end non-extendable by a polymerase; and (b) introducing the type Ils restriction enzyme under conditions sufficient to allow the type Ils restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end of the target sequence.

[0197] As another example, the method of processing a single-stranded nucleic acid molecule comprising a target sequence can comprise: (a) contacting the single-stranded nucleic acid molecule with a guide complex and a non-target binding molecule in a reaction, wherein the guide complex comprises a guide polynucleotide under conditions sufficient to allow the guide polynucleotide to hybridize to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, (ii) a target binding region that hybridizes to the target sequence, and (iii) an unblocked 3' end extendable by a polymerase; and (b) introducing the type Ils restriction enzyme under conditions sufficient to allow the type Ils restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end of the target sequence

[0198] In some embodiments, the non-target binding molecule described herein may have the same sequence as a sequence of the non-target binding region of the guide polynucleotide. In some embodiments, the non-target binding molecule described herein may have a different sequence than a sequence of the non-target binding region of the guide polynucleotide.

[0199] In some embodiments, the non-target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof may comprise a modified nucleotide. The non-target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof may comprise one or more modified nucleotides. In some embodiments, the target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof can comprise at least about 1 modified nucleotide, at least about 2 modified nucleotides, at least about 3 modified nucleotides, at least about 4 modified nucleotides, at least about 5 modified nucleotides, at least about 6 modified nucleotides, at least about 7 modified nucleotides, at least about 8 modified nucleotides, at least about 9 modified nucleotides, at least about 10 modified nucleotides, or greater than about 10 modified nucleotides. In some embodiments, the target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof can comprise at most about 10 modified nucleotides, at most about 9 modified nucleotides, at most about 8 modified nucleotides, at most about 7modified nucleotides, at most about 6 modified nucleotides, at most about 5 modified nucleotides, at most about 4 modified nucleotides, at most about 3 modified nucleotides, at most about 2 modified nucleotides, at most about 1 modified nucleotides, or less than about 1 modified nucleotide.

[0200] The non-target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof may comprise 2’-O-methoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, an unnatural base, or any combination thereof. The unnatural base can comprise a a-thiol deoxynucleotide triphosphate (dNTP) and / or a dideoxyribonucleotide triphosphate (ddNTP). A deoxynucleotide triphosphate may be dATP, dGTP, dCTP, dTTP, or any combination thereof. A dideoxyribonucleotide triphosphate can be ddATP, ddGTP, ddCTP, ddTTP, or any combination thereof. In some embodiments, the universal base can comprise deoxy Inosine, nitroindole, 2’-deoxynebularine, 3 -nitropyrrole, or any combination thereof. In some embodiments, a modified nucleotide of the non-target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof may not comprise adenine, guanine, thymine, or cytosine.

[0201] In some embodiments, the non-target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof may comprise modifications to increase guanine and cytosine content (e.g., make the guide polynucleotide more GC rich). In some embodiments, the non-target binding molecule, the non-target binding region of the guide polynucleotide, or any combination thereof may comprise modifications to increase adenine and thymine content (e.g., make the guide polynucleotide more AT rich).

[0202] The non-target binding molecule may be soluble. The non-target binding molecule may be immobilized on a surface. The non-target binding molecule may not be immobilized on a surface. In some embodiments, the surface can comprise a bead. In some embodiments, the surface may comprise a reaction vial. In some embodiments, the surface can comprise an antibody (e.g., the non-target binding molecule can be immobilized on an antibody). In some embodiments, the surface may comprise a synthetic antibody (e.g., a chemical antibody). A surface can be a molecularly imprinted polymer (MIP). The MIP can be generated by molding the shape and chemical functionalities of a target into a synthetic polymer. A MIP can be generated by polymerizing monomers in the presence of a template molecule. The monomers may be extracted, leaving complementary cavities in the polymeric matrix that can have affinity for the original monomers. In some embodiments, the surface may comprise an aptamer. Theaptamer can comprise a short sequence (e.g., 20-100 bases and / or 3-20 kDa) that binds a nontarget binding molecule. In some embodiments, the aptamer can be a short sequence of artificial DNA, RNA, XNA, peptide, or any combination thereof, that binds a non-target binding molecule.

[0203] In some embodiments, the non-target binding molecule may be immobilized on a surface via a linker. The linker can comprise PC Linker Phosphoramidite (e.g., 3 -(4,4'- Dimethoxytrityl)-l-(2-nitrophenyl)-propan-l-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite), Spacer Phosphoramidite 9 (e.g., 9-O-Dimethoxytrityl-triethylene glycol, l-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), 5'-Amino-Modifier TEG CE-Phosphoramidite (e.g., 10-(O-trifluoroacetamido-N-ethyl)-tri ethyleneglycol- 1 -[(2-cyanoethyl)-(N, N-diisopropyl)]- phosphoramidite), 5'-Aminooxy-Modifier-l 1-CE Phosphoramidite (e.g., 10-[N-Dimethoxytrityl- aminooxyethyl)]-triethyleneglycol-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), Spacer Phosphoramidite 18 (e.g., 18-O-Dimethoxytritylhexaethyleneglycol,l-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite), Cholesteryl-TEG Phosphoramidite (e.g., 1- Dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2- cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite), DNP-TEG Phosphoramidite (e.g., 1- Dimethoxytrityloxy-3-O-[N-(2,4-dinitrophenyl)-3-N-aminopropyl-(triethyleneglycol)]-glyceryl- 2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), 3'-Spacer C3 CPG (e.g., (1- Dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPG), 6-F AM- TEG Azide (e.g., 6-fluoresceinyl-2-aminoethyl-diethyleneglycolyl-ethyl azide), 5'-DBCO-TEG Phosphoramidite (e.g., 10-(6-oxo-6-(dibenzo[b,f]azacyclooct-4-yn-l-yl)-capramido-N-ethyl)-O- triethyleneglycol-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), a-Tocopherol-TEG Phosphoramidite (e.g., l-Dimethoxytrityloxy-3-O-[(9-DL-a-tocopheryl)-triethyleneglycol-l-yl]- glyceryl-2-O-[(2-cyanoethyl)-(N,N,-diisopropyl)]-phosphoramidite), 5'-Cholesteryl-TEG Phosphoramidite (e.g., 10-O-[l-propyl-3-N-carbamoylcholesteryl]-triethyleneglycol-l-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), 3 '-Cholesteryl-TEG CPG (e.g., 1- Dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O- succinoyl-long chain alkylamino-CPG), 5'-Biotin II Phosphoramidite (e.g., [l-N-(4,4'- Dimethoxytrityl)-biotinyl-6-aminoethoxyethyl]-2-cyanoethyl-(N,N-diisopropyl)- phosphoramidite), Psoralen C6 Phosphoramidite (e.g., 6-[4'-(Hydroxymethyl)-4,5',8- trimethylpsoralen]-hexyl-l-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), dC-CPG 1000 (e.g., 5'-Dimethoxytrityl-N-benzoyl-2'-deoxyCytidine, 3'-succinoyl-long chain alkylamino- CPG 1000), dC-CPG 2000 (e.g., 5'-Dimethoxytrityl-N-benzoyl-2'-deoxyCytidine, 3'-succinoyl- long chain alkylamino-CPG 2000), dG-CPG 2000 (e.g., 5'-Dimethoxytrityl-N-isobutyryl-2'-deoxyGuanosine, 3'-succinoyl-long chain alkylamino-CPG 2000), dT-CPG 2000 (e.g., 5'- Dimethoxytrityl-2'-deoxy Thymidine, 3'-succinoyl-long chain alkylamino-CPG 2000), PC Amino-Modifier Phosphoramidite (e.g., [(6-Trifluoroacetylamidocaproamidomethyl)-l-(2- nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite), Azobenzene Phosphoramidite (e.g., 3-O-(Dimethoxytrityl)-2-N-(4-carboxyazobenzene)-D-threonin-l-yl-O- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), Thiol-Modifier C6 S-S (e.g., l-O- Dimethoxytrityl-hexyl-disulfide,l'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), 5'- Carboxy -Modifier CIO (e.g., 10-Carboxy-decyl-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite, N-hydroxysuccinimide ester), 3'-Protected Biotin Serinol CPG (e.g., 3- Dimethoxytrityloxy-2-(3-((4-t-butylbenzoyl)-biotinyl)propanamido)propyl-l-O-succinyl-long chain alkylamino-CPG), Protected BiotinLC Serinol Phosphoramidite (e.g., 3- Dimethoxytrityloxy-2-(3-((4-t-butylbenzoyl)-biotinyl-3-aminopropyl)-diethyleneglycolyl- propylamido-glycanoylamido)propyl-l-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), 6-Fluorescein Serinol Phosphoramidite (e.g., 3 -Dimethoxytrityloxy -2-(3-(6-carboxy-(di-O- pivaloyl-fluorescein)propanamido)propyl)-l-O-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite), Protected Biotin Serinol Phosphoramidite (e.g., 3-Dimethoxytrityloxy-2-(3- ((4-t-butylbenzoyl)-biotinyl)propanamido)propyl-l-O-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite), Maleimide NHS Ester (SMCC) (e.g., 4-(N-maleimidomethyl) cyclohexanoic acid-N-hydroxysuccinimidyl ester), Glen UnySupport™ 500 (e.g., N-Methyl-succinimido[3,4- b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), Glen UnySupport™ 2000 (e.g., N-Methyl-succinimido[3,4-b]-7- oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), Glen UnySupport™ 1000 (e.g., N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6- (4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), High Load Glen UnySupport™ (e.g., N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2. l]heptane-6-(4,4'- dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), Glen UnySupport™ 1400 (e.g., Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), or any combination thereof.

[0204] In some embodiments, a reaction rate of a reaction may be reduced as compared to a reaction rate of an otherwise identical reaction without the non-target binding molecule. In some embodiments, the reaction rate may be reduced by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to a reaction rate of anotherwise identical reaction without the non-target binding molecule. In some embodiments, the reaction rate may be reduced by at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to a reaction rate of an otherwise identical reaction without the non-target binding molecule. In some embodiments, the reaction rate may be reduced from about 1% to about 50% as compared to a reaction rate of an otherwise identical reaction without the non-target binding molecule. In some embodiments, the reaction rate may be reduced from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% as compared to a reaction rate of an otherwise identical reaction without the non- target binding molecule.

[0205] In some embodiments, a reaction rate of a reaction may be increased as compared to a reaction rate of an otherwise identical reaction without the non-target binding molecule. In some embodiments, the reaction rate may be increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to a reaction rate of an otherwise identical reaction without the non-target binding molecule. In some embodiments, thereaction rate may be increased by at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to a reaction rate of an otherwise identical reaction without the non-target binding molecule. In some embodiments, the reaction rate may be increased from about 1% to about 50% as compared to a reaction rate of an otherwise identical reaction without the non-target binding molecule. In some embodiments, the reaction rate may be increased from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% as compared to a reaction rate of an otherwise identical reaction without the non- target binding molecule

[0206] In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the non-target binding region and the non-target binding molecule. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the non- target binding region of the guide polynucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced as compared to a reaction launchrate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without with the non-target binding region and without the non-target binding molecule.

[0207] In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to an otherwise identical reaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced by at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to an otherwise identical reaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced from about 1% to about 50% as compared to an otherwise identical reaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be reduced from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% as compared to an otherwise identical reaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule.

[0208] In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the non-target binding region and the non-target binding molecule. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without the non- target binding region of the guide polynucleotide. In some embodiments, a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased as compared to a reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) in an otherwise identical reaction without with the non-target binding region and without the non-target binding molecule.

[0209] In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to an otherwise identical reaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased by at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to an otherwise identical reaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased from about 1% to about 50% as compared to an otherwise identicalreaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule. In some embodiments, the reaction launch rate of the enzyme (e.g., type Ils restriction enzyme) may be increased from about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, about 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50%, about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% as compared to an otherwise identical reaction (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule.

[0210] In some embodiments, the non-target binding molecule can have a same length as the length of the non-target binding region of the guide polynucleotide. In some embodiments, the non-target binding molecule can have a different length than the non-target binding region of the guide polynucleotide. In some embodiments, the non-target binding molecule may have a shorter length than a length of the non-target binding region of the guide polynucleotide. In some embodiments, a length of the non-target binding molecule may be at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or greater than about 50 nucleotides shorter than a length of the non-target binding region of the guide polynucleotide. In some embodiments, a length of the non-target binding molecule may be at most about 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, or less than 1 nucleotide shorter than a length of the non-target binding region of the guide polynucleotide. Insome embodiments, the non-target binding molecule may have a longer length than a length of the non-target binding region of the guide polynucleotide. In some embodiments, a length of the non-target binding molecule may be at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or greater than about 50 nucleotides longer than a length of the non-target binding region of the guide polynucleotide. In some embodiments, a length of the non-target binding molecule may be at most about 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, or less than 1 nucleotide longer than a length of the non-target binding region of the guide polynucleotide.

[0211] In some embodiments, the non-target binding region of the guide polynucleotide may be at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, or greater than about 25 nucleotides in length. In some embodiments, the non-target binding region of the guide polynucleotide may be at most about 25 nucleotides, 20 nucleotides, at most about 18 nucleotides, at most about 15 nucleotides, at most about 12 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, at most about 4 nucleotides, at most about 3 nucleotides, at most about 2 nucleotides, or less than about 2 nucleotides in length.

[0212] In some embodiments, the non-target binding region of the guide polynucleotide may be from about 2 nucleotides to about 20 nucleotides in length. In some embodiments, the non-target binding region of the guide polynucleotide may be from about 2 nucleotides to about 3 nucleotides, about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 5 nucleotides, about 2 nucleotides to about 6 nucleotides, about 2 nucleotides to about 7 nucleotides, about 2 nucleotides to about 8 nucleotides, about 2 nucleotides to about 9 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 12 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 20 nucleotides, about 3 nucleotides to about 4 nucleotides, about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 6 nucleotides, about 3 nucleotides to about 7 nucleotides, about 3 nucleotides to about 8 nucleotides, about 3 nucleotides to about 9 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 12 nucleotides, about 3 nucleotides to about 15 nucleotides, about 3 nucleotides to about 20 nucleotides, about 4 nucleotides to about 5 nucleotides, about 4 nucleotides to about 6 nucleotides, about 4 nucleotides to about 7 nucleotides, about 4 nucleotides to about 8nucleotides, about 4 nucleotides to about 9 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 12 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 20 nucleotides, about 5 nucleotides to about 6 nucleotides, about 5 nucleotides to about 7 nucleotides, about 5 nucleotides to about 8 nucleotides, about 5 nucleotides to about 9 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 12 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 6 nucleotides to about 7 nucleotides, about 6 nucleotides to about 8 nucleotides, about 6 nucleotides to about 9 nucleotides, about 6 nucleotides to about 10 nucleotides, about 6 nucleotides to about 12 nucleotides, about 6 nucleotides to about 15 nucleotides, about 6 nucleotides to about 20 nucleotides, about 7 nucleotides to about 8 nucleotides, about 7 nucleotides to about 9 nucleotides, about 7 nucleotides to about 10 nucleotides, about 7 nucleotides to about 12 nucleotides, about 7 nucleotides to about 15 nucleotides, about 7 nucleotides to about 20 nucleotides, about 8 nucleotides to about 9 nucleotides, about 8 nucleotides to about 10 nucleotides, about 8 nucleotides to about 12 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotides to about 20 nucleotides, about 9 nucleotides to about 10 nucleotides, about 9 nucleotides to about 12 nucleotides, about 9 nucleotides to about 15 nucleotides, about 9 nucleotides to about 20 nucleotides, about 10 nucleotides to about 12 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 12 nucleotides to about 15 nucleotides, about 12 nucleotides to about 20 nucleotides, or about 15 nucleotides to about 20 nucleotides in length.

[0213] The guide polynucleotide described herein may be a first guide polynucleotide. The guide complex may comprise a second guide polynucleotide. The second guide polynucleotide may comprise a non-target binding region. The non-target binding region of the second guide polynucleotide may comprise a sequence that can be complementary with a non-target binding region of the first guide polynucleotide. The second guide polynucleotide may comprise a target binding region. The target binding region can be configured to hybridize to the target sequence of the single-stranded nucleic acid molecule.

[0214] For example, when the guide complex described herein comprises a second guide polynucleotide, the second guide polynucleotide may comprise (i) a non-target binding region that is complementary with the non-target binding region of the first guide polynucleotide and (ii) a target binding region configured to hybridize to the target sequence.

[0215] In some cases, when the first guide polynucleotide of the guide complex hybridizes to the target sequence of the single-stranded nucleic acid molecule, the target binding region of thesecond guide polynucleotide may not hybridize to the target sequence. In some cases, when the second guide polynucleotide of the guide complex hybridizes to the target sequence of the singlestranded nucleic acid molecule, the target binding region of the first guide polynucleotide may not hybridize to the target sequence. In some embodiments, the first guide polynucleotide and the second guide polynucleotide can hybridize to form a dimer. The first guide polynucleotide and the second guide polynucleotide can form a dimer prior to hybridizing to a target sequence of the single-stranded nucleic acid molecule. The first guide polynucleotide and the second guide polynucleotide can form a dimer after hybridizing to a target sequence of the single-stranded nucleic acid molecule.

[0216] In some embodiments, the first guide polynucleotide and the second guide polynucleotide can hybridize via a non-target binding region. The non-target binding region can be of the first guide polynucleotide and second guide polynucleotide to form the dimer. The dimer may comprise a double-stranded binding region. In some embodiments the doublestranded binding region can have a restriction endonuclease recognition sequence. In some embodiments the double-stranded binding region can have one or more restriction endonuclease recognition sequences. The enzyme (e.g., type Ils restriction enzyme) of a reaction described herein may bind to the double-stranded binding region of the dimer.

[0217] In some embodiments, the methods described herein may further comprise amplifying the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence). The amplifying may occur when the 3 ’end of the target sequence is extended by a polymerase. For example, the methods described herein may further comprise amplifying the single-stranded nucleic acid molecule comprising a target sequence by extending the extendable 3' end of the target sequence using a polymerase. The amplification of the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence) may occur at an amplification rate.

[0218] The amplification rate of a reaction described herein may be reduced as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding region comprising a modified nucleotide. In some embodiments, the amplification rate may be reduced by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding region comprising a modified nucleotide. In some embodiments, the amplification rate may be reduced by at most about 50%, at most about40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding region comprising a modified nucleotide. In some embodiments, the amplification rate may be reduced from about 1% to about 50% as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding region comprising a modified nucleotide.

[0219] The amplification rate of a reaction described herein may be increased as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding region comprising a modified nucleotide. In some embodiments, the amplification rate may be increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50% as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding region and / or non-extendable 3’ end comprising a modified nucleotide. In some embodiments, the amplification rate may be increased by at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding and / or non-extendable 3’ end region comprising a modified nucleotide. In some embodiments, the amplification rate may be increased from about 1% to about 50% as compared to an amplification rate of an otherwise identical amplification reaction without the non-target binding region and / or non-extendable 3’ end comprising a modified nucleotide.

[0220] An amplification reaction with a guide polynucleotide comprising a modified nucleotide may shorten a cycle threshold (cycle threshold value) in the nucleic acid amplification reaction. The term “cycle threshold” refers to the number of cycles used to amplify a target nucleic acid molecule to a detectable level. In some cases, when isothermal amplifications are used, time to result value can be used and it refers to the time used to amplify a target nucleic acid molecule to a detectable level.

[0221] In some embodiments, amplifying the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence) with a guide polynucleotide comprising a modified nucleotide may shorten a cycle threshold or time to result value as compared to a cyclethreshold or a time to result value of an otherwise identical nucleic acid amplification without the non-target binding region comprising a modified nucleotide. In some embodiments, amplifying the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence) with a guide polynucleotide comprising a modified nucleotide may shorten a cycle threshold or time to result value as compared to a cycle threshold or a time to result value of an otherwise identical nucleic acid amplification without the non-extendable 3’ region comprising a modified nucleotide. In some embodiments, amplifying the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence) with the guide polynucleotide and the non-target binding molecule may shorten a cycle threshold or time to result value as compared to a cycle threshold or a time to result value of an otherwise identical nucleic acid amplification (i) without the non-target binding region and the non-target binding molecule, (ii) without the non-target binding region, or (iii) with the non-target binding region and without the non-target binding molecule.

[0222] In some embodiments, amplifying the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence) with a guide polynucleotide comprising a modified nucleotide may shorten a cycle threshold or time to result value as compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method. In some embodiments, amplifying the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence) with the guide polynucleotide and the non-target binding molecule may shorten a cycle threshold or time to result value as compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method. An existing nucleic acid amplification method may be a method as described herein. In some embodiments, the existing nucleic acid amplification method comprises loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA).

[0223] In some embodiments, a cycle threshold of the amplification reaction using the methods described herein may be at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 18 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, or greater than about 30 minutes. In some embodiments, a cycle threshold of the amplification reaction using the methods described herein may be at most about 30 minutes, at most about 25 minutes, at most about 20 minutes, at most-n-about 18 minutes, at most about 15 minutes, at most about 12 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, at most about 5 minutes, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, or less than about 1 minute.

[0224] In some embodiments, a cycle threshold of the amplification reaction using the methods described herein may be from about 1 minute to about 30 minutes. In some embodiments, a cycle threshold of the amplification reaction using the methods described herein may be from about 1 minute to about 2 minutes, about 1 minute to about 3 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 1 minute to about 8 minutes, about 1 minute to about 10 minutes, about 1 minute to about 12 minutes, about 1 minute to about 15 minutes, about 1 minute to about 20 minutes, about 1 minute to about 25 minutes, about 1 minute to about 30 minutes, about 2 minutes to about 3 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 5 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 12 minutes, about 2 minutes to about 15 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 25 minutes, about 2 minutes to about 30 minutes, about 3 minutes to about 4 minutes, about 3 minutes to about 5 minutes, about 3 minutes to about 8 minutes, about 3 minutes to about 10 minutes, about 3 minutes to about 12 minutes, about 3 minutes to about 15 minutes, about 3 minutes to about 20 minutes, about 3 minutes to about 25 minutes, about 3 minutes to about 30 minutes, about 4 minutes to about 5 minutes, about 4 minutes to about 8 minutes, about 4 minutes to about 10 minutes, about 4 minutes to about 12 minutes, about 4 minutes to about 15 minutes, about 4 minutes to about 20 minutes, about 4 minutes to about 25 minutes, about 4 minutes to about 30 minutes, about 5 minutes to about 8 minutes, about 5 minutes to about 10 minutes, about 5 minutes to about 12 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 25 minutes, about 5 minutes to about 30 minutes, about 8 minutes to about 10 minutes, about 8 minutes to about 12 minutes, about 8 minutes to about 15 minutes, about 8 minutes to about 20 minutes, about 8 minutes to about 25 minutes, about 8 minutes to about 30 minutes, about 10 minutes to about 12 minutes, about 10 minutes to about 15 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 30 minutes, about 12 minutes to about 15 minutes, about 12 minutes to about 20 minutes, about 12 minutes to about 25 minutes, about 12 minutes to about 30 minutes, about 15 minutes to about 20 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 30 minutes, about 20 minutes to about 25 minutes, about 20 minutes to about 30 minutes, or about 25 minutes to about 30 minutes.

[0225] In some embodiments, the methods described herein may further comprise amplifying the nucleic acid molecule (e.g., the single-stranded nucleic acid molecule comprising the target sequence) to generate an extension product. The extension product may displace a guide polynucleotide (e.g., a second guide polynucleotide). An enzyme (e.g., type Ils restriction enzyme) can cut a first guide polynucleotide within the target binding region. Cutting the first guide polynucleotide within the target binding region can expose an extendable 3’ end of the first guide polynucleotide. In some embodiments, the 3’ end of the first guide polynucleotide can be extended using a polymerase. The polymerase may be a polymerase described herein. Extension of the 3’ end of the first guide polynucleotide can generate a complementary molecule of the target sequence of the single-stranded nucleic acid molecule. For example, the methods described herein may further comprise amplifying the single-stranded nucleic acid molecule comprising a target sequence, comprising: (a) extending the extendable 3' end of the target sequence with the polymerase to generate an extension product, wherein the extension product displaces the second guide polynucleotide; (b) cutting the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide; and (c) extending the extendable 3' end of the first guide polynucleotide using the polymerase to generate a complementary molecule of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule.

[0226] The amplifying may be repeated a number of times (e.g., at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 250, 500, or greater than about 500 times) to generate a plurality of complementary molecule of the target sequence. In some embodiments, an additional guide complex (e.g., comprising an additional guide polynucleotide) may bind to a complementary molecule of the target sequence. Once bound to the complementary molecule of the target sequence, the additional guide complex can use the complementary molecule as a starting template to generate copies of a target molecule (e.g., target sequence). An amplification described herein may be an isothermal nucleic acid amplification.

[0227] In some embodiments, an enzyme (e.g., a type Ils restriction enzyme) can be N.BstNBI, N.Bst9 I, N.BspD6I, a functional fragment thereof, or any combination thereof.

[0228] The blocked 3' end of the guide polynucleotide may comprise a peptide nucleic acid (PNA), a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof. In some embodiments, the ddNTP can be ddNTP is ddATP, ddGTP, ddCTP, or ddTTP. In some embodiments, the blocked 3' end of the guide polynucleotide can comprise one or more PNA, one or more modified bases, one or more phosphate groups, one ormore ddNTPs, one or more solid supports, one or more spacer (e.g., C3 spacer), or any combination thereof.

[0229] In some embodiments, the enzyme (e.g., a type Ils restriction enzyme) can exhibit a rate of activity. In some embodiments, the enzyme (e.g., a type Ils restriction enzyme) can exhibit high-frequency endonuclease activity. In some embodiments, the high-frequency endonuclease activity can be from a large subunit of the enzyme. In some embodiments, the high-frequency endonuclease activity can be from a small subunit of the enzyme. In some embodiments, the enzyme (e.g., a type Ils restriction enzyme) can exhibit low-frequency endonuclease activity. In some embodiments, the low-frequency endonuclease activity can be from a large subunit of the enzyme. In some embodiments, the low-frequency endonuclease activity can be from a small subunit of the enzyme.

[0230] In some embodiments, the enzyme may be a multimeric enzyme. Multimeric enzymes can refer to enzymes with multiple subunits (e.g., 2, 3, 4, 5 or more subunits) and may exhibit cooperative behavior among subunits in binding of substrates. In some embodiments, binding of a substrate to one subunit of the enzyme may influence the subsequent binding of substrate to another subunit of the enzyme. In some embodiments, binding of a substrate to one subunit of the enzyme may not influence the subsequent binding of substrate to another subunit of the enzyme. In some embodiments, an enzyme (e.g., a type Ils restriction enzyme) can exhibit at least two differential enzymatic activity rates. The enzyme may comprise at least two or more subunits. In some embodiments, an enzyme may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more subunits. In some embodiments, at least two of the subunits of the enzyme (e.g., a type Ils restriction enzyme) can exhibit different enzymatic activity rates. In some embodiments, the at least two differential enzymatic activity rates may comprise two differential endonuclease activity rates. For example, one or more subunits of the enzyme may comprise high-frequency endonuclease activity and one or more subunits may comprise low-frequency endonuclease activity. In some embodiments, the at least two differential enzymatic activity rates of the enzyme can comprise two differential endonuclease activity rates when cutting two different cutting sites.

[0231] In some embodiments, one of the differential endonuclease activity rates can comprise cutting the target sequence of the single-stranded nucleic acid molecule with low frequency. In some embodiments, reducing a reaction launch rate of a reaction described herein may be achieved by reducing the low frequency. In some embodiments, one of the differential endonuclease activity rates can comprise cutting the target sequence of the single-stranded nucleic acid molecule with high frequency. In some embodiments, reducing a reaction launchrate of a reaction described herein may be achieved by reducing the high frequency. In some embodiments, increasing a reaction launch rate of a reaction described herein may be achieved by increasing the low frequency. In some embodiments, increasing a reaction launch rate of a reaction described herein may be achieved by increasing the high frequency.

[0232] In some embodiments, the two differential endonuclease activity rates can be asymmetric. In some embodiments, the two differential endonuclease activity rates can be nonequal. In some embodiments, an enzyme described herein may comprise BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nucleases, zinc finger nucleases (ZFNs), N.Bst9 I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt. Alwl, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Mval269I, Nb.BpulOI, and Nt.BpulOI, a functional fragment thereof, or any combination thereof. In some embodiments, an enzyme can comprise two or more different active sites or endonuclease domains (e.g., at least about 2, 3, 4, 5, or greater than about 5 different active sites or endonuclease domains). The two or more different active sites or endonuclease domains can confer at least two differential enzymatic activities (e.g., at least about 2, 3, 4, 5, or greater than about 5 different enzymatic activities).

[0233] In some embodiments, the target binding region may be a length described herein. In some embodiments, the target binding region can be from about 2 nucleotides to about 50 nucleotides in length. In some embodiments, the target binding region can be from about 2 nucleotides to about 3 nucleotides, about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 5 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 12 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 18 nucleotides, about 2 nucleotides to about 20 nucleotides, about 2 nucleotides to about 25 nucleotides, about 2 nucleotides to about 30 nucleotides, about 2 nucleotides to about 50 nucleotides, about 3 nucleotides to about 4 nucleotides, about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 12 nucleotides, about 3 nucleotides to about 15 nucleotides, about 3 nucleotides to about 18 nucleotides, about 3 nucleotides to about 20 nucleotides, about 3 nucleotides to about 25 nucleotides, about 3 nucleotides to about 30 nucleotides, about 3 nucleotides to about 50 nucleotides, about 4 nucleotides to about 5 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 12 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 18 nucleotides, about 4 nucleotides to about 20 nucleotides, about 4 nucleotides to about 25 nucleotides, about 4 nucleotides to about 30 nucleotides, about 4 nucleotides to about 50 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 12 nucleotides, about 5nucleotides to about 15 nucleotides, about 5 nucleotides to about 18 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 50 nucleotides, about 10 nucleotides to about 12 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 50 nucleotides, about 12 nucleotides to about 15 nucleotides, about 12 nucleotides to about 18 nucleotides, about 12 nucleotides to about 20 nucleotides, about 12 nucleotides to about 25 nucleotides, about 12 nucleotides to about 30 nucleotides, about 12 nucleotides to about 50 nucleotides, about 15 nucleotides to about 18 nucleotides, about 15 nucleotides to about 20 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 50 nucleotides, about 18 nucleotides to about 20 nucleotides, about 18 nucleotides to about 25 nucleotides, about 18 nucleotides to about 30 nucleotides, about 18 nucleotides to about 50 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 50 nucleotides, about 25 nucleotides to about 30 nucleotides, about 25 nucleotides to about 50 nucleotides, or about 30 nucleotides to about 50 nucleotides in length. For example, the target binding region may be at least 12 nucleotides to about 25 nucleotides in length.

[0234] A concentration of the guide polynucleotide may be at least about 0.001 pM, at least about 0.005 pM, at least about 0.01 pM, at least about 0.05 pM, at least about 0.1 pM, at least about 0.5 pM, at least about 1.0 pM, at least about 1.5 pM, at least about 2.0 pM, at least about 2.5 pM, at least about 3.0 pM, at least about 3.5 pM, at least about 4.0 pM, at least about 4.5 pM, at least about 5.0 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, at least about 10 pM, or greater than about 10 pM. A concentration of the guide polynucleotide may be at most about 10 pM, at most about 9 pM, at most about 8 pM, at most about 7 pM, at most about 6 pM, at most about 5 pM, at most about 4.5 pM, at most about 4 pM, at most about 3.5 pM, at most about 3.0 pM, at most about 2.5 pM, at most about 2.0 pM, at most about 1.5 pM, at most about 1.0 pM, at most about 0.5 pM, at most about 0.1 pM, at most about 0.05 pM, at most about 0.01 pM, at most about 0.005 pM, at most about 0.001 pM, or less than about 0.001 pM.

[0235] A concentration of the guide polynucleotide may be from about 0.01 pM to about 5 pM. A concentration of the guide polynucleotide may be from about 0.01 pM to about 0.1 pM, about 0.01 pM to about 0.5 pM, about 0.01 pM to about 1 pM, about 0.01 pM to about 1.5 pM, about0.01 pM to about 2 pM, about 0.01 pM to about 2.5 pM, about 0.01 pM to about 3 pM, about 0.01 pM to about 3.5 pM, about 0.01 pM to about 4 pM, about 0.01 pM to about 4.5 pM, about 0.01 pM to about 5 pM, about 0.1 pM to about 0.5 pM, about 0.1 pM to about 1 pM, about 0.1 pM to about 1.5 pM, about 0.1 pM to about 2 pM, about 0.1 pM to about 2.5 pM, about 0.1 pM to about 3 pM, about 0.1 pM to about 3.5 pM, about 0.1 pM to about 4 pM, about 0.1 pM to about 4.5 pM, about 0.1 pM to about 5 pM, about 0.5 pM to about 1 pM, about 0.5 pM to about 1.5 pM, about 0.5 pM to about 2 pM, about 0.5 pM to about 2.5 pM, about 0.5 pM to about 3 pM, about 0.5 pM to about 3.5 pM, about 0.5 pM to about 4 pM, about 0.5 pM to about 4.5 pM, about 0.5 pM to about 5 pM, about 1 pM to about 1.5 pM, about 1 pM to about 2 pM, about 1 pM to about 2.5 pM, about 1 pM to about 3 pM, about 1 pM to about 3.5 pM, about 1 pM to about 4 pM, about 1 pM to about 4.5 pM, about 1 pM to about 5 pM, about 1.5 pM to about 2 pM, about 1.5 pM to about 2.5 pM, about 1.5 pM to about 3 pM, about 1.5 pM to about 3.5 pM, about 1.5 pM to about 4 pM, about 1.5 pM to about 4.5 pM, about 1.5 pM to about 5 pM, about 2 pM to about 2.5 pM, about 2 pM to about 3 pM, about 2 pM to about 3.5 pM, about 2 pM to about 4 pM, about 2 pM to about 4.5 pM, about 2 pM to about 5 pM, about 2.5 pM to about 3 pM, about 2.5 pM to about 3.5 pM, about 2.5 pM to about 4 pM, about 2.5 pM to about 4.5 pM, about 2.5 pM to about 5 pM, about 3 pM to about 3.5 pM, about 3 pM to about 4 pM, about 3 pM to about 4.5 pM, about 3 pM to about 5 pM, about 3.5 pM to about 4 pM, about 3.5 pM to about 4.5 pM, about 3.5 pM to about 5 pM, about 4 pM to about 4.5 pM, about 4 pM to about 5 pM, or about 4.5 pM to about 5 pM.

[0236] In some embodiments, the non-target binding region of the guide polynucleotide may comprise a palindromic sequence. A palindromic sequence can refer to a nucleic acid sequence in a double-stranded DNA or RNA molecule whereby reading in a direction (e.g. in a 5’ to 3’ direction) on one strand can be identical to the sequence in the same direction (e.g. in a 5’ to 3’ direction) on a complementary strand. For example, the DNA sequence 5’-ATCCGAT-3’ can be palindromic with its nucleotide-by-nucleotide complement 3’-TAGGCTA-5’. In some embodiments, the non-target binding region of the guide polynucleotide may not comprise a palindromic sequence. In some embodiments, a non-target binding region of the guide polynucleotide may comprise a partially palindromic sequence. For example, the non-target binding region of the guide polynucleotide may comprise a sequence wherein about one or more nucleotides of the sequence are not palindromic with a complementary strand. For example, a partial palindromic sequence may comprise the sequence 5’-ATCCGAT-3’ on one strand and the sequence 5’-ATCCGCT-3’ on another strand. For another example, the non-target binding region of the guide polynucleotide may comprise a first subsequence that is palindromic, and a secondsubsequence that is not palindromic. In some embodiments, the non-target binding region can be self-complementary.

[0237] The single-stranded nucleic acid molecule described herein can be a single- stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), or any combination thereof. In some embodiments, the single-stranded nucleic acid molecule can comprise two or more single-stranded nucleic acid molecules. In some embodiments, the two or more singlestranded nucleic acid molecules can comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 250, 500 or greater than about 500 single-stranded nucleic acid molecules. In some embodiments, each single- stranded nucleic acid molecule can comprise a different target sequence. In some embodiments, the two or more single-stranded nucleic acid molecules can be contained within a single reaction mixture. A reaction mixture described herein may comprise a Tris, potassium phosphate, sodium chloride, ethylenediaminetetraacetic acid (EDTA), potassium chloride, nonoxynol-9, at least one saccharide, dextran, a cyclodextrin, or any combination thereof. A reaction mixture may comprise at least one single-stranded nucleic acid molecule comprising a target sequence, at least one guide complex, one or more target probes, at least one polymerase, at least one restriction enzyme, or any combination thereof.

[0238] In some aspects, the present disclosure provides a method of processing different singlestranded nucleic acid molecules. The different single-stranded nucleic acid molecules can comprise a first target molecule and a second target molecule. In some embodiments, the different single-stranded nucleic acid molecules can comprise additional target molecules (e.g., third, fourth, fifth, sixth, seventh, eighth, or more target molecules). In some embodiments, the first target molecule may be contacted with a first guide complex. The first guide complex can comprise a guide polynucleotide (e.g., a first guide polynucleotide) as described herein. A first non-target binding region of the first guide polynucleotide can hybridize to the first target molecule. In some embodiments, the second target molecule may be contacted with a second guide complex. The second guide complex can comprise a guide polynucleotide (e.g., a second guide polynucleotide) as described herein. A second non-target binding region of the second guide polynucleotide can hybridize to the second target molecule. In some embodiments, a first non-target binding region and a second non-target binding region can comprise a different sequence, a different length, or any combination thereof. The method can comprise introducing an enzyme (e.g., a type Ils restriction enzyme) and the type Ils restriction enzyme may cut within a first target molecule and / or a second target molecule.

[0239] For example, a method of processing a plurality of different single-stranded nucleic acid molecules comprising a first target molecule and a second target molecule can comprise: (a)contacting the first target molecule with a first guide complex comprising a first guide polynucleotide under conditions sufficient to allow the first guide polynucleotide to hybridize to the first target molecule, wherein the first guide polynucleotide comprises: (i) a first non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and (ii) a first target binding region that hybridizes to the first target molecule; (b) contacting the second target molecule with a second guide complex comprising a second guide polynucleotide under conditions sufficient to allow the second guide polynucleotide to hybridize to the second target molecule, wherein the second guide polynucleotide comprises: (i) a second non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and (ii) a second target binding region that hybridizes to the second target molecule, wherein the first non-target binding region and the second non-target binding region have a different sequence or a different length; and (c) introducing the type Ils restriction enzyme under conditions sufficient to allow the type Ils restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the first target molecule or the second target molecule, wherein contacting in (a) and contacting in (b) are in a same mixture.

[0240] In some embodiments, the first guide polynucleotide may comprise a first blocked 3' end wherein the 3’ end may not be extended by a polymerase. In some embodiments, the second guide polynucleotide may comprise a second blocked 3' end wherein the 3’ end may not be extended by a polymerase. In some embodiments, both the first guide polynucleotide and the second guide polynucleotide may comprise blocked 3' ends non-extendable by a polymerase. The blocked 3’ end of the first guide polynucleotide, second guide polynucleotide, or any combination thereof may comprise a modification as described herein. In some embodiments, the first guide polynucleotide may not comprise a first blocked 3' end. For example, the first guide polynucleotide may comprise a first unblocked 3' end. In some embodiments, the second guide polynucleotide may not comprise a second blocked 3' end. For example, the second guide polynucleotide may comprise a second unblocked 3' end. In some embodiments, the systems, methods, and / or complexes described herein may comprise a first guide polynucleotide with a first blocked 3' end and a second guide polynucleotide with a second blocked 3' end. In some embodiments, the systems, methods, and / or complexes described herein may comprise a first guide polynucleotide with a blocked 3' end (e.g., a first blocked 3' end) and a second guide polynucleotide with an unblocked 3' end (e.g., a second unblocked 3' end). In some embodiments, the systems, methods, and / or complexes described herein may comprise a first guide polynucleotide with an unblocked 3' end (e.g., a first unblocked 3' end) and a second guidepolynucleotide with a blocked 3' end (e.g., a second blocked 3' end). In some embodiments, the systems, methods, and / or complexes described herein may comprise a first guide polynucleotide with a first unblocked 3' end and a second guide polynucleotide with a second unblocked 3' end.

[0241] In some embodiments, a cut within the first target molecule may generate a first extendable 3’ end. In some embodiments, a cut within the second target molecule may generate a second extendable 3’ end. In some embodiments, the first non-target binding region and the second non-target binding region can comprise at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity. In some embodiments, the first non-target binding region and the second non-target binding region can comprise at most about 99.9%, at most about 99.5%, at most about 99%, at most about 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 94%, at most about 93%, at most about 92%, at most about 91%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, or less than about 60% sequence identity.

[0242] In some embodiments, the first non-target binding region and the second non-target binding region can comprise from about 50% to about 99.5% sequence identity. In some embodiments, the first non-target binding region and the second non-target binding region can comprise from about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 92%, about 50% to about 94%, about 50% to about 96%, about 50% to about 98%, about 50% to about 99%, about 50% to about 99.5%, about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 92%, about 60% to about 94%, about 60% to about 96%, about 60% to about 98%, about 60% to about 99%, about 60% to about 99.5%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 92%, about 70% to about 94%, about 70% to about 96%, about 70% to about 98%, about 70% to about 99%, about 70% to about 99.5%, about 80% to about 85%, about 80% to about 90%, about 80% to about 92%, about 80% to about 94%, about 80% to about 96%, about 80% to about 98%, about 80% to about 99%, about 80% to about 99.5%, about 85% to about 90%, about 85% to about 92%, about 85% to about 94%, about 85% to about 96%, about 85% to about 98%, about 85% to about 99%, about 85% to about 99.5%, about 90% to about 92%, about 90% to about 94%, about 90% to about 96%, about 90% to about 98%, about 90% to about 99%, about 90% to about 99.5%, about 92% to about 94%, about 92% to about 96%, about 92% to about 98%, about 92%to about 99%, about 92% to about 99.5%, about 94% to about 96%, about 94% to about 98%, about 94% to about 99%, about 94% to about 99.5%, about 96% to about 98%, about 96% to about 99%, about 96% to about 99.5%, about 98% to about 99%, about 98% to about 99.5%, or about 99% to about 99.5% sequence identity.

[0243] In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, or greater than about 20 nucleotides longer than a length of a second non-target binding region of the second guide polynucleotide. In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at most about 20 nucleotides, at most about 18 nucleotides, at most about 15 nucleotides, at most about 12 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, at most about 4 nucleotides, at most about 3 nucleotides, at most about 2 nucleotides, at most about 1 nucleotide, or less than about 1 nucleotide longer than a length of a second non-target binding region of the second guide polynucleotide.

[0244] In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is from about 1 nucleotide to about 20 nucleotides longer than a length of a second non-target binding region of the second guide polynucleotide. In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is from about 1 nucleotide to about 2 nucleotides, about 1 nucleotide to about 3 nucleotides, about 1 nucleotide to about 4 nucleotides, about 1 nucleotide to about 5 nucleotides, about 1 nucleotide to about 6 nucleotides, about 1 nucleotide to about 7 nucleotides, about 1 nucleotide to about 8 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 2 nucleotides to about 3 nucleotides, about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 5 nucleotides, about 2 nucleotides to about 6 nucleotides, about 2 nucleotides to about 7 nucleotides, about 2 nucleotides to about 8 nucleotides, about 2 nucleotides to about 9 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 20 nucleotides, about 3 nucleotides to about 4 nucleotides, about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 6 nucleotides, about 3 nucleotides to about 7nucleotides, about 3 nucleotides to about 8 nucleotides, about 3 nucleotides to about 9 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 15 nucleotides, about 3 nucleotides to about 20 nucleotides, about 4 nucleotides to about 5 nucleotides, about 4 nucleotides to about 6 nucleotides, about 4 nucleotides to about 7 nucleotides, about 4 nucleotides to about 8 nucleotides, about 4 nucleotides to about 9 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 20 nucleotides, about 5 nucleotides to about 6 nucleotides, about 5 nucleotides to about 7 nucleotides, about 5 nucleotides to about 8 nucleotides, about 5 nucleotides to about 9 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 6 nucleotides to about 7 nucleotides, about 6 nucleotides to about 8 nucleotides, about 6 nucleotides to about 9 nucleotides, about 6 nucleotides to about 10 nucleotides, about 6 nucleotides to about 15 nucleotides, about 6 nucleotides to about 20 nucleotides, about 7 nucleotides to about 8 nucleotides, about 7 nucleotides to about 9 nucleotides, about 7 nucleotides to about 10 nucleotides, about 7 nucleotides to about 15 nucleotides, about 7 nucleotides to about 20 nucleotides, about 8 nucleotides to about 9 nucleotides, about 8 nucleotides to about 10 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotides to about 20 nucleotides, about 9 nucleotides to about 10 nucleotides, about 9 nucleotides to about 15 nucleotides, about 9 nucleotides to about 20 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, or about 15 nucleotides to about 20 nucleotides longer than a length of a second nontarget binding region of the second guide polynucleotide.

[0245] In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, or greater than about 20 nucleotides shorter than a length of a second non-target binding region of the second guide polynucleotide. In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at most about 20 nucleotides, at most about 18 nucleotides, at most about 15 nucleotides, at most about 12 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, at most about 4 nucleotides, at most about 3 nucleotides, at most about 2nucleotides, at most about 1 nucleotide, or less than about 1 nucleotide shorter than a length of a second non-target binding region of the second guide polynucleotide.

[0246] In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is from about 1 nucleotide to about 20 nucleotides shorter than a length of a second non-target binding region of the second guide polynucleotide. In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is from about 1 nucleotide to about 2 nucleotides, about 1 nucleotide to about 3 nucleotides, about 1 nucleotide to about 4 nucleotides, about 1 nucleotide to about 5 nucleotides, about 1 nucleotide to about 6 nucleotides, about 1 nucleotide to about 7 nucleotides, about 1 nucleotide to about 8 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 2 nucleotides to about 3 nucleotides, about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 5 nucleotides, about 2 nucleotides to about 6 nucleotides, about 2 nucleotides to about 7 nucleotides, about 2 nucleotides to about 8 nucleotides, about 2 nucleotides to about 9 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 20 nucleotides, about 3 nucleotides to about 4 nucleotides, about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 6 nucleotides, about 3 nucleotides to about 7 nucleotides, about 3 nucleotides to about 8 nucleotides, about 3 nucleotides to about 9 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 15 nucleotides, about 3 nucleotides to about 20 nucleotides, about 4 nucleotides to about 5 nucleotides, about 4 nucleotides to about 6 nucleotides, about 4 nucleotides to about 7 nucleotides, about 4 nucleotides to about 8 nucleotides, about 4 nucleotides to about 9 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 20 nucleotides, about 5 nucleotides to about 6 nucleotides, about 5 nucleotides to about 7 nucleotides, about 5 nucleotides to about 8 nucleotides, about 5 nucleotides to about 9 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 6 nucleotides to about 7 nucleotides, about 6 nucleotides to about 8 nucleotides, about 6 nucleotides to about 9 nucleotides, about 6 nucleotides to about 10 nucleotides, about 6 nucleotides to about 15 nucleotides, about 6 nucleotides to about 20 nucleotides, about 7 nucleotides to about 8 nucleotides, about 7 nucleotides to about 9 nucleotides, about 7 nucleotides to about 10 nucleotides, about 7 nucleotides to about 15 nucleotides, about 7 nucleotides to about 20 nucleotides, about 8 nucleotides to about 9nucleotides, about 8 nucleotides to about 10 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotides to about 20 nucleotides, about 9 nucleotides to about 10 nucleotides, about 9 nucleotides to about 15 nucleotides, about 9 nucleotides to about 20 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, or about 15 nucleotides to about 20 nucleotides shorter than a length of a second non-target binding region of the second guide polynucleotide.

[0247] In some embodiments, the first non-target binding region, second non-target binding region, or any combination thereof may comprise one or more modified nucleotides. The modified nucleotide may comprise any modified nucleotide described herein.

[0248] In some embodiments, a reaction launch rate of a type Ils restriction enzyme on the first target molecule can be different from a reaction launch rate of the type Ils restriction enzyme on the second target molecule. In some embodiments, a reaction launch rate of a type Ils restriction enzyme on the first target molecule can be faster than a reaction launch rate of the type Ils restriction enzyme on the second target molecule. In some embodiments, a reaction launch rate of a type Ils restriction enzyme on the first target molecule can be at least about, at most about, or about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or lOx faster than a reaction launch rate of the type Ils restriction enzyme on the second target molecule. In some embodiments, a reaction launch rate of a type Ils restriction enzyme on the first target molecule can be slower than a reaction launch rate of the type Ils restriction enzyme on the second target molecule. In some embodiments, a reaction launch rate of a type Ils restriction enzyme on the first target molecule can be at least about, at most about, or about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or lOx slower than a reaction launch rate of the type Ils restriction enzyme on the second target molecule. In some embodiments, the enzyme may be a restriction enzyme, a polymerase, a reverse transcriptase, or any combination thereof. In some embodiments, a reaction described herein may comprise an avian myeloblastosis virus (AMV) reverse transcriptase.

[0249] In some embodiments, the plurality of single-stranded nucleic acid molecules comprising a first target molecule and a second target molecule may be amplified. Amplifying can comprise extending a 3' end of the first target molecule, a 3' end of the second target molecule, or any combination thereof. The 3’ end of the first target molecule and / or the second target molecule may be extended using a polymerase as described herein. The amplifying may comprise extending a 3' end of the first target molecule, a 3' end of the second target molecule, or any combination thereof to generate a plurality of extension products. An extension product of the plurality of extension products may comprise an extension product as described herein. Insome embodiments, the plurality of single-stranded nucleic acid molecules can be in a same reaction mixture.

[0250] For example, a method described herein may comprise amplifying the plurality of single-stranded nucleic acid molecules comprising the first target molecule and the second target molecule, comprising: (a) extending the first extendable 3' end of the first target molecule and the second extendable 3' end of the second target molecule using the polymerase. As another example, a method described herein may comprise amplifying the plurality of single-stranded nucleic acid molecules comprising the first target molecule and the second target molecule, comprising: (a) extending the first extendable 3' end of the first target molecule and the second extendable 3' end of the second target molecule using the polymerase to generate a plurality of extension products, wherein the extension products displace the first guide polynucleotide and the second guide polynucleotide; (b) cutting the first guide polynucleotide within the first target binding region to expose a first extendable 3' end of the first guide polynucleotide; (c) cutting the second guide polynucleotide within the second target binding region to expose a second extendable 3' end of the second guide polynucleotide; and (d) extending the first extendable 3' end of the first guide polynucleotide using the polymerase to generate a first complementary molecule of the first target molecule of the plurality of single-stranded nucleic acid molecules; and (e) extending the second extendable 3' end of the second guide polynucleotide using the polymerase to generate a second complementary molecule of the second target molecule of the plurality of single-stranded nucleic acid molecules, thereby amplifying the plurality of singlestranded nucleic acid molecules.

[0251] An amplification rate of the first target molecule may be different than an amplification rate of the second target molecule. In some embodiments, an amplification rate of the first target molecule may be faster than an amplification rate of the second target molecule. In some embodiments, an amplification rate of the first target molecule may be at least about, at most about, or about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or lOx faster than an amplification rate of the second target molecule. In some embodiments, an amplification rate of the first target molecule may be slower than an amplification rate of the second target molecule. In some embodiments, an amplification rate of the first target molecule may be at least about, at most about, or about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or lOx slower than an amplification rate of the second target molecule.

[0252] In some embodiments, an additional guide complex may bind to a first complementary molecule, a second complementary molecule, or any combination thereof. The additional guide complex bound to the first complementary molecule may serve as a starting template to generate additional copies of a first target molecule. The additional guide complex bound to the secondcomplementary molecule may serve as a starting template to generate additional copies of a second target molecule. In some embodiments, the additional guide complex bound to a first complementary molecule can be different than an additional guide complex bound to a second complementary molecule. In some embodiments, a reaction can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10 guide complexes. In some embodiments, a reaction can comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than about 1 guide complex.

[0253] In some embodiments, methods comprising the plurality of single-stranded nucleic acid molecules comprising the first target molecule and the second target molecule may comprise a type Ils restriction enzyme described herein. The enzyme (e.g., type Ils restriction enzyme) may be a multimeric enzyme described herein. The multimeric enzyme can comprise differential activity, wherein different subunits of the same enzyme (e.g., N.BstNBI, N.Bst9 I, N.BspD6I, a functional fragment thereof, or a combination thereof) may comprise different endonuclease activity.

[0254] The one or more enzymes (e.g., type Ils restriction enzyme) described herein may cut the first target molecule and / or the second target molecule of the plurality of single-stranded nucleic acid molecules with low frequency, high frequency, or any combination thereof. The differential enzyme activity rates of the one or more enzymes (e.g., type Ils restriction enzymes) may be asymmetric or non-equal.

[0255] The length of the first target binding region of the first guide polynucleotide, the second target binding region of the second guide polynucleotide, or any combination thereof may comprise a length of a non-target binding region as described herein. A concentration of the first guide polynucleotide, the second guide polynucleotide, or any combination thereof may comprise a concentration of a guide polynucleotide as described herein. The first target binding region of the first guide polynucleotide, the second target binding region of the second guide polynucleotide, or any combination thereof may comprise a palindromic sequence. The first target binding region of the first guide polynucleotide, the second target binding region of the second guide polynucleotide, or any combination thereof may not comprise a palindromic sequence. In some embodiments, the first target binding region of the first guide polynucleotide, the second target binding region of the second guide polynucleotide, or any combination thereof may comprise a partial palindromic sequence as described herein.Polynucleotide-Polypeptide Complexes

[0256] In some aspects, the present disclosure provides a polynucleotide-polypeptide complex. The polynucleotide-polypeptide complex can comprise a single-stranded nucleic acid molecule.The single-stranded nucleic acid molecule may be bound to a guide complex, wherein the guide complex comprises a guide complex described herein. For example, the guide complex may comprise a first guide polynucleotide comprising a non-target binding region and a target binding region. The target binding region may hybridize with a target sequence of the single-stranded nucleic acid molecule. The non-target binding region of the first guide polynucleotide may comprise a modified nucleotide. The guide complex may further comprise a second guide polynucleotide. The second guide polynucleotide may hybridize with the non-target binding region of the first guide polynucleotide. Hybridization of the second guide polynucleotide with the non-target binding region of the first guide polynucleotide can form a double-stranded binding region. The double-stranded binding region can comprise a restriction endonuclease recognition sequence. The restriction endonuclease recognition sequence may be recognized by an enzyme (e.g., a type Ils restriction enzyme).

[0257] For example, a polynucleotide-polypeptide complex can comprise: a single-stranded nucleic acid molecule having bound thereto a guide complex, wherein the guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of the single-stranded nucleic acid molecule, wherein the non-target binding region comprises a modified nucleotide, and (ii) a second guide polynucleotide that hybridizes with the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme.

[0258] In some aspects, the present disclosure provides a polynucleotide-polypeptide complex. The polynucleotide-polypeptide complex can comprise a single-stranded nucleic acid molecule bound to a guide complex and a non-target binding molecule. The guide complex can comprise a guide complex as described herein. For example, the polynucleotide-polypeptide complex can comprise: a single-stranded nucleic acid molecule having bound thereto a guide complex and a non-target binding molecule, wherein the guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of the single-stranded nucleic acid molecule, and (ii) a second guide polynucleotide that hybridizes with the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme. In some embodiments, the non-target binding molecule can be separated from the guide complex. In some embodiments, the non-target binding molecule may be soluble. Thenon-target binding molecule may be immobilized on a surface. The non-target binding molecule may not be immobilized on a surface. In some embodiments, the surface can comprise a bead. In some embodiments, the surface may comprise a reaction vial. In some embodiments, the surface can comprise an antibody (e.g., the non-target binding molecule can be immobilized on an antibody). In some embodiments, the surface may comprise a synthetic antibody (e.g., a chemical antibody). A surface can be a molecularly imprinted polymer (MIP). The MIP can be generated by molding the shape and chemical functionalities of a target into a synthetic polymer. A MIP can be generated by polymerizing monomers in the presence of a template molecule. The monomers may be extracted, leaving complementary cavities in the polymeric matrix that can have affinity for the original monomers. In some embodiments, the surface may comprise an aptamer. The aptamer can comprise a short sequence (e.g., 20-100 bases and / or 3-20 kDa) that binds a non-target binding molecule. In some embodiments, the aptamer can be a short sequence of artificial DNA, RNA, XNA, peptide, or any combination thereof, that binds a non-target binding molecule.

[0259] In some embodiments, the non-target binding molecule may be immobilized on a surface via a linker. A linker can comprise PC Linker Phosphoramidite (e.g., 3 -(4,4'- Dimethoxytrityl)-l-(2-nitrophenyl)-propan-l-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite), Spacer Phosphoramidite 9 (e.g., 9-O-Dimethoxytrityl-triethylene glycol, l-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), 5'-Amino-Modifier TEG CE-Phosphoramidite (e.g., 10-(O-trifluoroacetamido-N-ethyl)-tri ethyleneglycol- 1 -[(2-cyanoethyl)-(N, N-diisopropyl)]- phosphoramidite), 5'-Aminooxy-Modifier-l 1-CE Phosphoramidite (e.g., 10-[N-Dimethoxytrityl- aminooxyethyl)]-triethyleneglycol-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), Spacer Phosphoramidite 18 (e.g., 18-O-Dimethoxytritylhexaethyleneglycol,l-[(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite), Cholesteryl-TEG Phosphoramidite (e.g., 1- Dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O-(2- cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite), DNP-TEG Phosphoramidite (e.g., 1- Dimethoxytrityloxy-3-O-[N-(2,4-dinitrophenyl)-3-N-aminopropyl-(triethyleneglycol)]-glyceryl- 2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), 3'-Spacer C3 CPG (e.g., (1- Dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPG), 6-F AM- TEG Azide (e.g., 6-fluoresceinyl-2-aminoethyl-diethyleneglycolyl-ethyl azide), 5'-DBCO-TEG Phosphoramidite (e.g., 10-(6-oxo-6-(dibenzo[b,f]azacyclooct-4-yn-l-yl)-capramido-N-ethyl)-O- triethyleneglycol-l-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), a-Tocopherol-TEG Phosphoramidite (e.g., l-Dimethoxytrityloxy-3-O-[(9-DL-a-tocopheryl)-triethyleneglycol-l-yl]- glyceryl-2-O-[(2-cyanoethyl)-(N,N,-diisopropyl)]-phosphoramidite), 5'-Cholesteryl-TEGPhosphoramidite (e.g., 10-O-[l-propyl-3-N-carbamoylcholesteryl]-triethyleneglycol-l-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), 3'-Cholesteryl-TEG CPG (e.g., 1- Dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-glyceryl-2-O- succinoyl-long chain alkylamino-CPG), 5'-Biotin II Phosphoramidite (e.g., [l-N-(4,4'- Dimethoxytrityl)-biotinyl-6-aminoethoxyethyl]-2-cyanoethyl-(N,N-diisopropyl)- phosphoramidite), Psoralen C6 Phosphoramidite (e.g., 6-[4'-(Hydroxymethyl)-4,5',8- trimethylpsoralen]-hexyl-l-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), dC-CPG 1000 (e.g., 5'-Dimethoxytrityl-N-benzoyl-2'-deoxyCytidine, 3'-succinoyl-long chain alkylamino- CPG 1000), dC-CPG 2000 (e.g., 5'-Dimethoxytrityl-N-benzoyl-2'-deoxyCytidine, 3'-succinoyl- long chain alkylamino-CPG 2000), dG-CPG 2000 (e.g., 5'-Dimethoxytrityl-N-isobutyryl-2'- deoxyGuanosine, 3'-succinoyl-long chain alkylamino-CPG 2000), dT-CPG 2000 (e.g., 5'- Dimethoxytrityl-2'-deoxy Thymidine, 3'-succinoyl-long chain alkylamino-CPG 2000), PC Amino-Modifier Phosphoramidite (e.g., [(6-Trifluoroacetylamidocaproamidomethyl)-l-(2- nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite), Azobenzene Phosphoramidite (e.g., 3-O-(Dimethoxytrityl)-2-N-(4-carboxyazobenzene)-D-threonin-l-yl-O- [(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), Thiol-Modifier C6 S-S (e.g., l-O- Dimethoxytrityl-hexyl-disulfide,l'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), 5'- Carboxy -Modifier CIO (e.g., 10-Carboxy-decyl-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite, N-hydroxysuccinimide ester), 3'-Protected Biotin Serinol CPG (e.g., 3- Dimethoxytrityloxy-2-(3-((4-t-butylbenzoyl)-biotinyl)propanamido)propyl-l-O-succinyl-long chain alkylamino-CPG), Protected BiotinLC Serinol Phosphoramidite (e.g., 3- Dimethoxytrityloxy-2-(3-((4-t-butylbenzoyl)-biotinyl-3-aminopropyl)-diethyleneglycolyl- propylamido-glycanoylamido)propyl-l-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), 6-Fluorescein Serinol Phosphoramidite (e.g., 3 -Dimethoxytrityloxy -2-(3 -(6-carboxy-(di-O- pivaloyl-fluorescein)propanamido)propyl)-l-O-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite), Protected Biotin Serinol Phosphoramidite (e.g., 3-Dimethoxytrityloxy-2-(3- ((4-t-butylbenzoyl)-biotinyl)propanamido)propyl-l-O-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite), Maleimide NHS Ester (SMCC) (e.g., 4-(N-maleimidomethyl) cyclohexanoic acid-N-hydroxysuccinimidyl ester), Glen UnySupport™ 500 (e.g., N-Methyl-succinimido[3,4- b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), Glen UnySupport™ 2000 (e.g., N-Methyl-succinimido[3,4-b]-7- oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), Glen UnySupport™ 1000 (e.g., N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6- (4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), High Load GlenUnySupport™ (e.g., N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2. l]heptane-6-(4,4'- dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), Glen UnySupport™ 1400 (e.g., Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG), or any combination thereof.

[0260] In some aspects, the present disclosure provides a polynucleotide-polypeptide complex comprising a plurality of single-stranded nucleic acid molecules. In some embodiments, the plurality of single-stranded nucleic acid molecules may comprise at least a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule. The plurality of single-stranded nucleic acid molecules may have a first guide complex and a second guide complex bound to the single-stranded nucleic acid molecules. The first guide complex can comprise a guide complex (e.g., a first guide complex) described herein, wherein the first guide complex comprises a first primary guide polynucleotide. The first guide complex can comprise a first secondary guide polynucleotide. The first secondary guide polynucleotide may hybridize with a non-target binding region of the first primary guide polynucleotide (e.g., first non-target binding region). Hybridization of the first secondary guide polynucleotide to the first non-target binding region can form a first double-stranded binding region. The first double-stranded binding region can comprise a sequence recognized by an enzyme (e.g., a type Ils restriction enzyme). The second guide complex can comprise a guide complex (e.g., a second guide complex) described herein, wherein the second guide complex comprises a second primary guide polynucleotide. The second guide complex can comprise a second secondary guide polynucleotide. The second secondary guide polynucleotide may hybridize with a non-target binding region of the second primary guide polynucleotide (e.g., second non-target binding region). Hybridization of the second secondary guide polynucleotide to the second non-target binding region can form a second double-stranded binding region. The second double-stranded binding region can comprise a sequence recognized by an enzyme (e.g., a type Ils restriction enzyme).

[0261] As another example, the polynucleotide-polypeptide complex can comprise: a singlestranded nucleic acid molecule having bound thereto a guide complex, wherein the guide complex comprises: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of the singlestranded nucleic acid molecule, and (ii) a second guide polynucleotide that hybridizes with the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region comprises a restriction endonuclease recognitionsequence for an enzyme that is a type Ils restriction enzyme, wherein the single-stranded nucleic acid molecule or the target sequence is from Bacillus anthracis.

[0262] For example, a polynucleotide-polypeptide complex can comprise: a plurality of singlestranded nucleic acid molecules having bound thereto a first guide complex and a second guide complex, wherein the first guide complex comprises: (i) a first primary guide polynucleotide comprising, from 5' to 3', a first non-target binding region and a first target binding region that hybridizes with a first target molecule of the plurality of single-stranded nucleic acid molecules; and (ii) a first secondary guide polynucleotide that hybridizes with the first non-target binding region of the first target molecule to form a first double-stranded binding region, wherein the first double-stranded binding region comprises a first restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme; and wherein the second guide complex comprises: (i) a second primary guide polynucleotide comprising, from 5' to 3', a second non- target binding region and a second target binding region that hybridizes with a second target molecule of the plurality of single-stranded nucleic acid molecules; and (ii) a second secondary guide polynucleotide that hybridizes with the second non-target binding region of the second target molecule to form a second double-stranded binding region, wherein the second doublestranded binding region comprises a second restriction endonuclease recognition sequence for the enzyme, wherein the first non-target binding region and the second non-target binding region have a different sequence or a different length.

[0263] In some embodiments, the first non-target binding region and the second non-target binding region can comprise from about 50% to about 99.5% sequence identity. In some embodiments, the first non-target binding region and the second non-target binding region can comprise from about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 92%, about 50% to about 94%, about 50% to about 96%, about 50% to about 98%, about 50% to about 99%, about 50% to about 99.5%, about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 92%, about 60% to about 94%, about 60% to about 96%, about 60% to about 98%, about 60% to about 99%, about 60% to about 99.5%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 92%, about 70% to about 94%, about 70% to about 96%, about 70% to about 98%, about 70% to about 99%, about 70% to about 99.5%, about 80% to about 85%, about 80% to about 90%, about 80% to about 92%, about 80% to about 94%, about 80% to about 96%, about 80% to about 98%, about 80% to about 99%, about 80% to about 99.5%, about 85% to about 90%, about 85% to about 92%, about 85% to about 94%, about 85% to about 96%, about 85% to about 98%, about 85% toabout 99%, about 85% to about 99.5%, about 90% to about 92%, about 90% to about 94%, about 90% to about 96%, about 90% to about 98%, about 90% to about 99%, about 90% to about 99.5%, about 92% to about 94%, about 92% to about 96%, about 92% to about 98%, about 92% to about 99%, about 92% to about 99.5%, about 94% to about 96%, about 94% to about 98%, about 94% to about 99%, about 94% to about 99.5%, about 96% to about 98%, about 96% to about 99%, about 96% to about 99.5%, about 98% to about 99%, about 98% to about 99.5%, or about 99% to about 99.5% sequence identity.

[0264] In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, or greater than about 20 nucleotides longer than a length of a second non-target binding region of the second guide polynucleotide. In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at most about 20 nucleotides, at most about 18 nucleotides, at most about 15 nucleotides, at most about 12 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, at most about 4 nucleotides, at most about 3 nucleotides, at most about 2 nucleotides, at most about 1 nucleotide, or less than about 1 nucleotide longer than a length of a second non-target binding region of the second guide polynucleotide.

[0265] In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at least about 1 nucleotide, at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 20 nucleotides, or greater than about 20 nucleotides shorter than a length of a second non-target binding region of the second guide polynucleotide. In some embodiments, a first non-target binding region of the first guide polynucleotide can have a length that is at most about 20 nucleotides, at most about 18 nucleotides, at most about 15 nucleotides, at most about 12 nucleotides, at most about 10 nucleotides, at most about 9 nucleotides, at most about 8 nucleotides, at most about 7 nucleotides, at most about 6 nucleotides, at most about 5 nucleotides, at most about 4 nucleotides, at most about 3 nucleotides, at most about 2nucleotides, at most about 1 nucleotide, or less than about 1 nucleotide shorter than a length of a second non-target binding region of the second guide polynucleotide.

[0266] In some embodiments, the first non-target binding region of the first primary guide polynucleotide and the second non-target binding region of the second primary guide polynucleotide can be configured to be recognized by a same enzyme (e.g., the same type Ils restriction enzyme).Methods of Adjusting Reaction Rate

[0267] In some aspects, the present disclosure provides a method of adjusting a reaction rate. The reaction rate may be of a nucleic acid amplification. The nucleic acid amplification may be an isothermal nucleic acid amplification. The method may comprise contacting a nucleic acid molecule (e.g., a single- stranded nucleic acid molecule comprising a target sequence) with a guide complex described herein. The guide complex can comprise a guide polynucleotide. The guide polynucleotide can comprise a non-target binding region. In some embodiments, the method can comprise changing a sequence, a length, or any combination thereof of the non-target binding region of the guide polynucleotide to provide a changed non-target binding region. In some embodiments, the method can comprise introducing a modified nucleotide to the non-target binding region of the guide polynucleotide to provide a changed non-target binding region. In some embodiments, the method can comprise (i) changing a sequence of the non-target binding region, (ii) changing a length of the non-target binding region, (iii) introducing a modified nucleotide to the non-target binding region, or (iv) any combination thereof, to provide a changed non-target binding region. In some embodiments, the method can comprise adding in a non- target binding molecule to the reaction to adjust a reaction rate. In some embodiments, the method may comprise providing a changed non-target binding region described herein and adding in a non-target binding molecule to the reaction to adjust a reaction rate.

[0268] For example, a method of adjusting a reaction rate can comprise: (a) contacting a singlestranded nucleic acid molecule comprising a target sequence with a guide complex comprising a guide polynucleotide in a reaction under conditions sufficient to allow the guide polynucleotide to hybridize to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, and (ii) a target binding region that hybridizes to the target sequence; (b) introducing the type Ils restriction enzyme under conditions sufficient to allow the type Ils restriction enzyme to bind the restriction endonuclease recognition sequence and cut within the target sequence to generate an extendable 3' end; (c) changing a sequence or alength of the non-target binding region to provide a changed non-target binding region, introducing a modified nucleotide into the non-target binding region to provide a changed non- target binding region, or adding in a non-target binding molecule in the reaction to adjust the reaction rate; and (d) repeating (a)-(b) with the guide polynucleotide comprising the changed non-target binding region or with the non-target binding molecule in the reaction.Computer Systems

[0269] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 14 shows a computer system 1401 that can be programmed or otherwise configured to analyze polynucleotide-polypeptide complexes. Alternatively or in addition to, the computer system 1401 can be programmed or otherwise configured to analyze single-stranded nucleic acid molecule processing data. The computer system 1401 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0270] The computer system 1401 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1405, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1401 also includes memory or memory location 1410 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1415 (e.g., hard disk), communication interface 1420 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1425, such as cache, other memory, data storage and / or electronic display adapters. The memory 1410, storage unit 1415, interface 1420 and peripheral devices 1425 are in communication with the CPU 1405 through a communication bus (solid lines), such as a motherboard. The storage unit 1415 can be a data storage unit (or data repository) for storing data. The computer system 1401 can be operatively coupled to a computer network (“network”) 1430 with the aid of the communication interface 1420. The network 1430 can be the Internet, an internet and / or extranet, or a...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of processing a single-stranded nucleic acid molecule comprising a target sequence, said method comprising:(a) contacting said single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions sufficient to allow said guide polynucleotide to hybridize to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises:(i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, wherein said nontarget binding region comprises a modified nucleotide, and(ii) a target binding region that hybridizes to said target sequence, and(b) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bind to said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end.

2. The method of claim 1, wherein said guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

3. The method of claim 1, wherein said guide polynucleotide further comprises an unblocked3’ end.

4. The method of any one of claims 1-3, wherein said non-target binding region comprises at least two modified nucleotides.

5. The method of any one of claims 1-4, wherein said modified nucleotide comprises 2’-O- methoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base.

6. The method of claim 5, wherein the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP).

7. The method of claim 5, wherein the universal base comprises deoxyinosine, nitroindole,2’-deoxynebularine, or 3 -nitropyrrole.

8. The method of any one of claims 1-4, wherein said modified nucleotide does not comprise adenine, guanine, thymine, or cytosine.

9. The method of any one of claims 1-8, wherein a reaction launch rate of said type Ils restriction enzyme is reduced compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non-target binding region comprising said modified nucleotide or (ii) with said non-target binding region that does not comprise said modified nucleotide, and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

10. The method of any one of claims 1-8, wherein a reaction launch rate of said type Ils restriction enzyme is increased compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non-target binding region comprising said modified nucleotide or (ii) with said non-target binding region that does not comprise said modified nucleotide, and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

11. A method of processing a single-stranded nucleic acid molecule comprising a target sequence, said method comprising:(a) contacting said single-stranded nucleic acid molecule with a guide complex and a non-target binding molecule in a reaction, wherein said guide complex comprises a guide polynucleotide under conditions sufficient to allow said guide polynucleotide to hybridize to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises:(i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, and(ii) a target binding region that hybridizes to said target sequence, and(b) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bind to said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end.

12. The method of claim 11, wherein said guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

13. The method of claim 11, wherein said guide polynucleotide further comprises an unblocked 3' end.

14. The method of any one of claims 11-13, wherein said non-target binding molecule has the same sequence as a sequence of said non-target binding region of said guide polynucleotide.

15. The method of any one of claims 11-13, wherein said non-target binding molecule has a different sequence than a sequence of said non-target binding region of said guide polynucleotide.

16. The method of any one of claims 11-15, wherein said non-target binding molecule and / or said non-target binding region of said guide polynucleotide comprises a modified nucleotide.

17. The method of claim 16, wherein said modified nucleotide comprises 2’-O-methoxy- ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base.

18. The method of claim 17, wherein the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP).

19. The method of claim 17, wherein the universal base comprises deoxyinosine, nitroindole, 2’-deoxynebularine, or 3 -nitropyrrole.

20. The method of claim 16, wherein the modified nucleotide does not comprise adenine, guanine, thymine, or cytosine.

21. The method of any one of claims 11-20, wherein said non-target binding molecule is soluble.

22. The method of any one of claims 11-21, wherein said non-target binding molecule is not immobilized on a surface.

23. The method of any one of claims 11-21, wherein said non-target binding molecule is immobilized on a surface.

24. The method of claim 23, wherein the surface comprises a bead, an antibody, a molecularly imprinted polymer, an aptamer, or a surface of a reaction vial.

25. The method of claim 23 or 24, wherein said non-target binding molecule is immobilized on said surface via linker.

26. The method of claim 25, wherein said linker comprises PC Linker Phosphoramidite Spacer Phosphoramidite 9, 5'-Amino-Modifier TEG CE-Phosphoramidite, 5'-Aminooxy- Modifier-ll-CE Phosphoramidite, Spacer Phosphoramidite 18, Cholesteryl-TEG Phosphoramidite, DNP-TEG Phosphoramidite, 3'-Spacer C3 CPG, 6-FAM- TEG Azide,5'-DBC0-TEG Phosphoramidite, a-Tocopherol-TEG Phosphoramidite, 5'-Cholesteryl- TEG Phosphoramidite, 3'-Cholesteryl-TEG CPG, 5'-Biotin II Phosphoramidite, Psoralen C6 Phosphoramidite, dC-CPG 1000, dC-CPG 2000, dG-CPG 2000, dT-CPG 2000, PC Amino-Modifier Phosphoramidite, Azobenzene Phosphoramidite, Thiol-Modifier C6 S-S, 5'-Carboxy-Modifier CIO, 3'-Protected Biotin Serinol CPG, Protected BiotinLC Serinol Phosphoramidite, 6-Fluorescein Serinol Phosphoramidite, Protected Biotin Serinol Phosphoramidite, Maleimide NHS Ester (SMCC), N-Methyl-succinimido[3,4-b]-7- oxabicyclo[2.2.1]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'- dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, N-Methyl-succinimido[3,4- b]-7-oxabicyclo[2.

2. l]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, N-Methyl-succinimido[3,4-b]-7-oxabicyclo[2.2.1]heptane-6-(4,4'- dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, Methyl-succinimido[3,4- b]-7-oxabicyclo[2.

2. l]heptane-6-(4,4'-dimethoxytrityloxy)-5-succinoyl long chain alkylamino CPG, or any combination thereof.

27. The method of any one of claims 11-26, wherein a reaction rate of said reaction is reduced compared to a reaction rate of an otherwise identical reaction without said nontarget binding molecule.

28. The method of any one of claims 11-26, wherein a reaction rate of said reaction is increased compared to a reaction rate of an otherwise identical reaction without said nontarget binding molecule.

29. The method of any one of claims 11-28, wherein a reaction launch rate of said type Ils restriction enzyme is reduced compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non-target binding region and said non-target binding molecule, (ii) without said non-target binding region, or (iii) with said non-target binding region and without said non-target binding molecule, and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

30. The method of any one of claims 11-28, wherein a reaction launch rate of said type Ils restriction enzyme is increased compared to a reaction launch rate of said type Ils restriction enzyme in an otherwise identical reaction (i) without said non-target binding region and said non-target binding molecule, (ii) without said non-target binding region, or (iii) with said non-target binding region and without said non-target binding molecule,and wherein said reaction launch rate is a rate for generating copies of extendable products of said target sequence with an extendable 3' end per second.

31. The method of any one of claims 11-30, wherein said non-target binding molecule has a shorter length than a length of said non-target binding region of said guide polynucleotide.

32. The method of any one of claims 11-30, wherein said non-target binding molecule has a longer length than a length of said non-target binding region of said guide polynucleotide.

33. The method of any one of claims 11-30, wherein said non-target binding molecule has the same length as a length of said non-target binding region of said guide polynucleotide.

34. The method of any one of claims 1-33, wherein said non-target binding region is at least about 12 nucleotides in length.

35. The method of any one of claims 1-34, wherein said guide polynucleotide is a first guide polynucleotide, and said guide complex comprises a second guide polynucleotide, wherein said second guide polynucleotide comprises (i) a non-target binding region that is complementary with said non-target binding region of said first guide polynucleotide and (ii) a target binding region configured to hybridize to said target sequence.

36. The method of claim 35, wherein when said first guide polynucleotide of said guide complex is hybridized to said target polynucleotide sequence, said target binding region of said second guide polynucleotide is not hybridized to said target sequence.

37. The method of claim 35 or 36, wherein said first guide polynucleotide and said second guide polynucleotide hybridize to form a dimer.

38. The method of claim 37, wherein said first guide polynucleotide and said second guide polynucleotide hybridize via said non-target binding region of said first guide polynucleotide and said second guide polynucleotide to form said dimer having a doublestranded binding region.

39. The method of claim 38, wherein said double-stranded binding region comprises said restriction endonuclease recognition sequence.

40. The method of claim 38, wherein said type Ils restriction enzyme binds to said doublestranded binding region of said dimer.

41. The method of any one of claims 1-10, further comprising amplifying said singlestranded nucleic acid molecule comprising a target sequence, comprising:(c) extending said extendable 3' end using said polymerase.

42. The method of claim 41, wherein said amplifying occurs at an amplification rate that is reduced compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding region comprising said modified nucleotide.

43. The method of claim 41, wherein said amplifying occurs at an amplification rate that is increased compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding region comprising said modified nucleotide.

44. The method of claim 41, wherein said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value of an otherwise identical nucleic acid amplification without said non-target binding region comprising said modified nucleotide.

45. The method of claim 41, wherein said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method.

46. The method of claim 45, wherein said existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA).

47. The method of any one of claims 44-46, wherein said cycle threshold value is at most 15 minutes.

48. The method of any one of claims 11-40, further comprising amplifying said singlestranded nucleic acid molecule comprising a target sequence, comprising:(c) extending said extendable 3' end of said target sequence using said polymerase.

49. The method of claim 48, wherein said amplifying occurs at an amplification rate that is reduced compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding molecule.

50. The method of claim 48, wherein said amplifying occurs at an amplification rate that is reduced compared to an amplification rate of an otherwise identical amplification reaction without said non-target binding molecule.

51. The method of claim 48, wherein said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value of an otherwise identical nucleic acid amplification without said non-target binding molecule.

52. The method of claim 48, wherein said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method.

53. The method of claim 48, wherein said existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA).

54. The method of any one of claims 51-53, wherein said cycle threshold value is at most 15 minutes.

55. The method of any one of claims 1-40, further comprising amplifying said singlestranded nucleic acid molecule comprising a target sequence, comprising:(c) extending said extendable 3' end with said polymerase to generate an extension product, wherein said extension product displaces said second guide polynucleotide;(d) cutting said first guide polynucleotide within said target binding region to expose an extendable 3' end of said first guide polynucleotide; and(e) extending said extendable 3' end of said first guide polynucleotide using said polymerase to generate a complementary molecule of said target sequence of said single-stranded nucleic acid molecule, thereby amplifying said single-stranded nucleic acid molecule.

56. The method of claim 55, further comprising repeating (d) and (e) to generate a plurality of complementary molecules of said target sequence of said single-stranded nucleic acid molecule.

57. The method of claim 55 or 56, wherein an additional guide complex binds to said complementary molecule.

58. The method of claim 57, further comprising using said complementary molecule with said additional guide complex bound thereto as a starting template to generate copies of said target molecule.

59. The method of any one of claims 1-58, wherein said type Ils restriction enzyme comprises N.BstNBI, N.Bst9 I, N.BspD6I, a functional fragment thereof, or a combination thereof.

60. The method of any one of claims 2, 4-10, 12, and 14-59, wherein said blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof.

61. The method of claim 60, wherein said ddNTP is ddATP, ddGTP, ddCTP, or ddTTP.

62. The method of any one of claims 1-61, wherein said single-stranded nucleic acid molecule with said cut and said guide polynucleotide bound thereto is used as a starting template for an amplification.

63. The method of claim 62, wherein said amplification is an isothermal amplification.

64. The method of any one of claims 1-63, wherein said enzyme exhibits a high-frequency endonuclease activity.

65. The method of claim 64, wherein said high-frequency endonuclease activity is from a large subunit of said enzyme.

66. The method of any one of claims 1-65, wherein said enzyme exhibits a low-frequency endonuclease activity.

67. The method of claim 66, wherein said low-frequency endonuclease activity is from a small subunit of said enzyme.

68. The method of any one of claims 1-67, wherein said enzyme exhibits at least two differential enzymatic activity rates.

69. The method of any one of claims 1-68, wherein said enzyme comprises at least two or more subunits.

70. The method of claim 69, wherein each subunit of said at least two or more subunits exhibit a different enzymatic activity rate.

71. The method of any one of claims 1-69, wherein said enzyme is a multimeric enzyme.

72. The method of claim 68, wherein said at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites.

73. The method of claim 68, wherein one of said at least two differential endonuclease activity rates comprises cutting said target sequence of said single-stranded nucleic acid molecule with low frequency.

74. The method of claim 73, wherein said reaction launch rate is reduced by reducing said low frequency.

75. The method of claim 68, wherein one of said two differential endonuclease activity rates comprises cutting said target binding region of said guide polynucleotide with high frequency.

76. The method of claim 68, wherein said two differential endonuclease activity rates are asymmetric or non-equal.

77. The method of claim 68, wherein said enzyme comprises BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nucleases, zinc finger nucleases (ZFNs), N.Bst9 I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt. Alwl, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Mval269I, Nb.BpulOI, and Nt.BpulOI, a functional fragment thereof, or a combination thereof.

78. The method of any one of claims 68-77, wherein a temperature is changed over a course of said method.

79. The method of claim 78, wherein a first activity rate of said at least two differential enzymatic activity rates is favored at a first temperature, and a second activity rate of said at least two differential enzymatic activity rates is favored at a second temperature different from said first temperature.

80. The method of any one of claims 68-79, wherein said enzyme comprises two different active sites or endonuclease domains conferring at least two differential enzymatic activities.

81. The method of any one of claims 1-80, wherein said target binding region is at least about 12 to about 25 nucleotides in length.

82. The method of any one of claims 1-81, wherein a concentration of said guide polynucleotide is at least about 0.1 pM, at least about 1 pM, or about 0.1 pM to about 4 pM.

83. The method of any one of claims 1-82, wherein said non-target binding region comprises a palindromic sequence.

84. The method of any one of claims 1-82, wherein said non-target binding region comprises a partially palindromic sequence.

85. The method of any one of claims 1-82, wherein said non-target binding region does not comprise a palindromic sequence.

86. The method of any one of claims 1-84, wherein said non-target binding region is self- complementary.

87. The method of any one of claims 1-86, wherein said single-stranded nucleic acid molecule is a single-stranded deoxyribonucleic acid (ssDNA) or a single-stranded ribonucleic acid (ssRNA).

88. The method of any one of claims 1-87, wherein said polymerase has strand displacement activity.

89. The method of any one of claims 1-88, wherein said single-stranded nucleic acid molecule comprises two or more single-stranded nucleic acid molecules, each singlestranded nucleic acid molecule comprising a different target sequence.

90. The method of claim 89, wherein said two or more single-stranded nucleic acid molecules are contained within a single reaction mixture.

91. A method of processing a plurality of different single-stranded nucleic acid molecules comprising a first target molecule and a second target molecule, said method comprising:(a) contacting said first target molecule with a first guide complex comprising a first guide polynucleotide under conditions sufficient to allow said first guide polynucleotide to hybridize to said first target molecule, wherein said first guide polynucleotide comprises:(i) a first non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and(ii) a first target binding region that hybridizes to said first target molecule;(b) contacting said second target molecule with a second guide complex comprising a second guide polynucleotide under conditions sufficient to allow said second guide polynucleotide to hybridize to said second target molecule, wherein said second guide polynucleotide comprises:(i) a second non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and(ii) a second target binding region that hybridizes to said second target molecule, wherein said first non-target binding region and said second non-target binding region have a different sequence or a different length; and(c) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bind to said restriction endonuclease recognition sequence and cut within said first target molecule or said second target molecule, wherein contacting in (a) and contacting in (b) are in a same mixture.

92. The method of claim 91, wherein said first guide polynucleotide further comprises (iii) a first blocked 3' end non-extendable by a polymerase.

93. The method of claim 91, wherein said first guide polynucleotide further comprises (iii) a first unblocked 3' end.

94. The method of claim 92 or 93, wherein said second guide polynucleotide further comprises (iii) a second blocked 3' end non-extendable by a polymerase.

95. The method of claim 92 or 93, wherein said second guide polynucleotide further comprises (iii) a second unblocked 3' end.

96. The method of claim 92 or 94, wherein said first blocked 3' end and / or said second blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof.

97. The method of any one of claims 91-96, wherein said cut within said first target molecule generates a first extendable 3’ end.

98. The method of any one of claims 91-97, wherein said cut within said second target molecule generates a second extendable 3’ end.

99. The method of any one of claims 91-98, wherein said first non-target binding region and said second non-target binding region have at most about 98% sequence identity.

100. The method of any one of claims 91-99, wherein said first non-target binding region has a length that is at least two nucleotides longer than a length of said second non-target binding region.

101. The method of any one of claims 91-99, wherein said first non-target binding region has a length that is at least two nucleotides shorter than a length of said second non-target binding region.

102. The method of any one of claims 91-101, wherein said first non-target binding region comprises a first modified nucleotide.

103. The method of claim 102, wherein said first modified nucleotide comprises 2’-O- methoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base.

104. The method of claim 103, wherein the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP).

105. The method of claim 103, wherein the universal base comprises deoxyinosine, nitroindole, 2’-deoxynebularine, or 3 -nitropyrrole.

106. The method of claim 102, wherein said first modified nucleotide does not comprise adenine, guanine, thymine, or cytosine.

107. The method of any one of claims 91-106, wherein said second non-target binding region comprises a second modified nucleotide.

108. The method of claim 107, wherein said second modified nucleotide comprises 2’-O- methoxy-ethyl modified nucleotide, aminoethyl-phenoxazine-deoxycytosine (AP-dC), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), a fluorinated nucleic acid, a universal base, a phosphorothioate linkage, a C3 spacer, or an unnatural base.

109. The method of claim 108, wherein the unnatural base comprises a a-thiol deoxynucleotide triphosphate (dNTP) or a dideoxyribonucleotide triphosphate (ddNTP).

110. The method of claim 108, wherein the universal base comprises deoxyinosine, nitroindole, 2’-deoxynebularine, or 3 -nitropyrrole.

111. The method of claim 107, wherein said second modified nucleotide does not comprise adenine, guanine, thymine, or cytosine.

112. The method of any one of claims 91-111, wherein a reaction launch rate of said type Ils restriction enzyme on said first target molecule is different from a reaction launch rate of said type Ils restriction enzyme on said second target molecule, and wherein said reaction launch rate is a rate for generating copies of extendable products of said first target molecule or said second target molecule with an extendable 3' end per second.

113. The method of any one of claims 91-112, further comprising amplifying said plurality of single-stranded nucleic acid molecules comprising said first target molecule and said second target molecule, comprising:(d) extending said first extendable 3' end of said first target molecule and said second extendable 3' end of said second target molecule using said polymerase.

114. The method of any one of claims 91-113, further comprising amplifying said plurality of single-stranded nucleic acid molecules comprising said first target molecule and said second target molecule, comprising:(e) extending said first extendable 3' end of said first target molecule and said second extendable 3' end of said second target molecule using said polymerase to generate a plurality of extension products, wherein said extension products displace said first guide polynucleotide and said second guide polynucleotide;(f) cutting said first guide polynucleotide within said first target binding region to expose a first extendable 3' end of said first guide polynucleotide;(g) cutting said second guide polynucleotide within said second target binding region to expose a second extendable 3' end of said second guide polynucleotide;(h) extending said first extendable 3' end of said first guide polynucleotide using said polymerase to generate a first complementary molecule of said first target molecule of said plurality of single-stranded nucleic acid molecules; and(i) extending said second extendable 3' end of said second guide polynucleotide using said polymerase to generate a second complementary molecule of said second target molecule of said plurality of single-stranded nucleic acid molecules, thereby amplifying said plurality of single-stranded nucleic acid molecules.

115. The method of claim 113 or 114, wherein an amplification rate of said first target molecule is different from an amplification rate of said second target molecule.

116. The method of claim 113 or 114, wherein said amplifying shortens a cycle threshold value or a time to result value in a nucleic acid amplification compared to a cycle threshold value or a time to result value in an existing nucleic acid amplification method.

117. The method of claim 116, wherein said existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA).

118. The method of claim 116 or 117, wherein said cycle threshold value is at most 20 minutes.

119. The method of any one of claims 114-118, wherein an additional guide complex binds to said first complementary molecule and / or said second complementary molecule.

120. The method of claim 119, further comprising using said first complementary molecule with said additional guide complex bound thereto as a starting template to generate copies of said first target molecule.

121. The method of claim 119 or 120, further comprising using said second complementary molecule with said additional guide complex bound thereto as a starting template to generate copies of said second target molecule.

122. The method of any one of claims 119-121, wherein said additional guide complex for said first complementary molecule is different than a secondary guide complex for said second complementary molecule.

123. The method of any one of claims 91-122, wherein said type Ils restriction enzyme comprises N.BstNBI, N.Bst9 I, N.BspD6I, a functional fragment thereof, or a combination thereof.

124. The method of any one of claims 92 and 94-123, wherein said first blocked 3' end and / or said second blocked 3’ end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof.

125. The method of claim 124, wherein said ddNTP is ddATP, ddGTP, ddCTP, or ddTTP.

126. The method of any one of claims 91-125, wherein said plurality of single-stranded nucleic acid molecules with said cut and said first guide polynucleotide and said second guide polynucleotide bound thereto are used as starting templates for an amplification.

127. The method of claim 126, wherein said amplification is an isothermal amplification.

128. The method of any one of claims 91-127, wherein said enzyme exhibits a high- frequency endonuclease activity.

129. The method of claim 128, wherein said high-frequency endonuclease activity is from a large subunit of said enzyme.

130. The method of any one of claims 91-129, wherein said enzyme exhibits a low-frequency endonuclease activity.

131. The method of claim 130, wherein said low-frequency endonuclease activity is from a small subunit of said enzyme.

132. The method of any one of claims 91-131, wherein said enzyme exhibits at least two differential enzymatic activity rates.

133. The method of any one of claims 91-131, wherein said enzyme comprises at least two or more subunits.

134. The method of claim 133, wherein each subunit of said at least two or more subunits exhibits a different enzymatic activity rates.

135. The method of any one of claims 91-130, wherein said enzyme is a multimeric enzyme.

136. The method of claim 132, wherein said at least two differential enzymatic activity rates comprise two differential endonuclease activity rates when cutting two different cutting sites.

137. The method of claim 132, wherein one of said at least two differential endonuclease activity rates comprises cutting said first target molecule and / or second target molecule of said plurality of single-stranded nucleic acid molecules with low frequency.

138. The method of claim 132, wherein one of said two differential endonuclease activity rates comprises cutting said first target binding region of said first guide polynucleotide and / or said second target binding region of said second guide polynucleotide with high frequency.

139. The method of claim 132, wherein said two differential endonuclease activity rates are asymmetric or non-equal.

140. The method of claim 132, wherein said enzyme comprises BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nucleases, zinc finger nucleases (ZFNs), N.Bst9 I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt. Alwl, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Mval269I, Nb.BpulOI, and Nt.BpulOI, a functional fragment thereof, or a combination thereof.

141. The method of any one of claims 132-140, wherein a temperature is changed over a course of said method.

142. The method of claim 141, wherein a first activity rate of said at least two differential enzymatic activity rates is favored at a first temperature, and a second activity rate of said at least two differential enzymatic activity rates is favored at a second temperature different from said first temperature.

143. The method of any one of claims 132-142, wherein said enzyme comprises two different active sites or endonuclease domains conferring at least two differential enzymatic activities.

144. The method of any one of claims 91-143, wherein said first target binding region and said second target binding region are each at least about 12 to about 25 nucleotides in length.

145. The method of any one of claims 91-144, wherein a concentration of said first guide polynucleotide and / or said second guide polynucleotide is at least about 0.1 pM, at least about 1 pM, or about 0.1 pM to about 4 pM.

146. The method of any one of claims 91-145, wherein said first non-target binding region and / or said second non-target binding region comprises a palindromic sequence.

147. The method of any one of claims 91-146, wherein said first non-target binding region and / or said second non-target binding region is self-complementary.

148. The method of any one of claims 91-147, wherein said plurality of single-stranded nucleic acid molecules are a plurality of single-stranded deoxyribonucleic acid (ssDNA) molecules or a plurality of single-stranded ribonucleic acid (ssRNA) molecules.

149. The method of claim 113 or 114, wherein said polymerase has strand displacement activity.

150. The method of any one of claims 91-149, wherein said plurality of single-stranded nucleic acid molecules are contained within a single reaction mixture.

151. A polynucleotide-polypeptide complex comprising:a single-stranded nucleic acid molecule having bound thereto a guide complex, wherein said guide complex comprises:(i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of said single-stranded nucleic acid molecule, wherein said non-target binding region comprises a modified nucleotide, and(ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme.

152. A polynucleotide-polypeptide complex comprising:(a) a single-stranded nucleic acid molecule having bound thereto a guide complex and a non-target binding molecule, wherein said guide complex comprises:(i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of said single-stranded nucleic acid molecule, and(ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme.

153. The polynucleotide-polypeptide complex of claim 152, wherein said non-target binding molecule is separated from said guide complex.

154. The polynucleotide-polypeptide complex of claim 152 or 153, wherein said non-target binding molecule is immobilized on a solid surface.

155. The polynucleotide-polypeptide complex of any one of claims 152-154, wherein said non-target binding molecule is soluble.

156. A polynucleotide-polypeptide complex comprising: a plurality of single-stranded nucleic acid molecules having bound thereto a first guide complex and a second guide complex, wherein said first guide complex comprises:(i) a first primary guide polynucleotide comprising, from 5' to 3', a first non-target binding region and a first target binding region that hybridizeswith a first target molecule of said plurality of single-stranded nucleic acid molecules; and(ii) a first secondary guide polynucleotide that hybridizes with said first nontarget binding region of said first target molecule to form a first doublestranded binding region, wherein said first double-stranded binding region comprises a first restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme; and wherein said second guide complex comprises:(i) a second primary guide polynucleotide comprising, from 5' to 3', a second non-target binding region and a second target binding region that hybridizes with a second target molecule of said plurality of singlestranded nucleic acid molecules; and(ii) a second secondary guide polynucleotide that hybridizes with said second non-target binding region of said second target molecule to form a second double-stranded binding region, wherein said second doublestranded binding region comprises a second restriction endonuclease recognition sequence for said enzyme, wherein said first non-target binding region and said second non-target binding region have a different sequence or a different length.

157. The polynucleotide-polypeptide complex of claim 156, wherein said first non-target binding region and said second non-target binding region are configured to be recognized by a same enzyme.

158. A kit comprising a guide complex or a guide polynucleotide of any one of claims 1-157.

159. The kit of claim 158, wherein said kit further comprises a probe or a dye for detecting an amplification product generated using said kit.

160. The kit of claim 158 or 159, wherein said kit further comprises an informational material describing an instruction of using said kit.

161. A method of adjusting a reaction rate of a nucleic acid amplification, said method comprising:(a) contacting a single-stranded nucleic acid molecule comprising a target sequence with a guide complex comprising a guide polynucleotide in a reaction under conditions sufficient to allow said guide polynucleotide to hybridize to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises:(i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, and(ii) a target binding region that hybridizes to said target sequence;(b) introducing said type Ils restriction enzyme under conditions sufficient to allow said type Ils restriction enzyme to bind said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end;(c) changing a sequence or a length of said non-target binding region to provide a changed non-target binding region, introducing a modified nucleotide into said non-target binding region to provide a changed non-target binding region, or adding in a non-target binding molecule in said reaction to adjust said reaction rate; and(d) repeating (a)-(b) with said guide polynucleotide comprising said changed non- target binding region or with said non-target binding molecule in said reaction.

162. The method of claim 161, wherein said guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

163. The method of claim 161, wherein said guide polynucleotide further comprises an unblocked 3' end.

164. A method of processing a single-stranded nucleic acid molecule comprising a target sequence, said method comprising:(a) contacting said single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where said guide polynucleotide hybridizes to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises:(i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, and(ii) a target binding region configured to hybridize to said target sequence, and(b) introducing said type Ils restriction enzyme under conditions sufficient to cause said type Ils restriction enzyme to bind said restriction endonuclease recognition sequence and cut within said target sequence, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

165. The method of claim 164, wherein said guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

166. The method of claim 164, wherein said guide polynucleotide further comprises an unblocked 3' end.

167. A method of amplifying a single-stranded nucleic acid molecule comprising a target sequence, said method comprising:(a) contacting said single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where said guide polynucleotide hybridizes to said single-stranded nucleic acid molecule, wherein said guide polynucleotide comprises:(i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type Ils restriction enzyme, and(ii) a target binding region configured to hybridize to said target sequence, and(b) introducing said type Ils restriction enzyme under conditions sufficient to cause said type Ils restriction enzyme to bind said restriction endonuclease recognition sequence and cut within said target sequence to generate an extendable 3' end; and(c) extending said extendable 3' end using a polymerase, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

168. The method of claim 167, wherein said guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

169. The method of claim 167, wherein said guide polynucleotide further comprises an unblocked 3' end.

170. A method of amplifying a single-stranded nucleic acid molecule comprising a target sequence, said method comprising:(a) contacting a guide complex with said single-stranded nucleic acid molecule, wherein said guide complex comprises:(i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with said target sequence of said single-stranded nucleic acid molecule, and(ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region binds to an enzyme;(b) cutting said target sequence using said enzyme to expose an extendable 3' end;(c) extending said extendable 3' end with a polymerase to generate an extension product, wherein said extension product displaces said second guide polynucleotide;(d) cutting said first guide polynucleotide within said target binding region to expose an extendable 3' end of said first guide polynucleotide; and(e) extending said extendable 3' end of said first guide polynucleotide using said polymerase to generate a complementary molecule of said target sequence of said single-stranded nucleic acid molecule, thereby amplifying said single-stranded nucleic acid molecule, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

171. The method of claim 170, wherein said first guide polynucleotide and / or said second guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

172. The method of claim 170 or 171, wherein said first guide polynucleotide and / or said second guide polynucleotide further comprises an unblocked 3' end.

173. A polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule having bound thereto a guide complex, wherein said guide complex comprises:(i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes with a target sequence of said singlestranded nucleic acid molecule, and(ii) a second guide polynucleotide that hybridizes with said non-target binding region of said first guide molecule to form a double-stranded binding region, wherein said double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

174. The method of claim 173, wherein said first guide polynucleotide and / or said second guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

175. The method of claim 173 or 174, wherein said first guide polynucleotide and / or said second guide polynucleotide further comprises an unblocked 3' end.

176. A system of processing a single-stranded nucleic acid molecule comprising a target sequence, said system comprising: said single-stranded nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide, wherein said guide polynucleotide comprises:(i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme,(ii) a target binding region configured to hybridize to said target sequence, and said enzyme bound to said restriction endonuclease recognition sequence of said nontarget binding region, wherein said single-stranded nucleic acid molecule or said target sequence is from Bacillus anthracis.

177. The system of claim 176, wherein said guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

178. The system of claim 176, wherein said guide polynucleotide further comprises an unblocked 3' end.

179. A system for processing a plurality of single-stranded nucleic acid molecules, each comprising a different target sequence, said system comprising: a first single-stranded nucleic acid molecule wherein said first single-stranded nucleic acid molecule is bound to a first guide complex comprising a first guide polynucleotide, wherein said first guide polynucleotide comprises:(i) a first non-target binding region comprising a first restriction endonuclease recognition sequence for an enzyme that is a type Ils restriction enzyme;(ii) a first target binding region configured to hybridize to a first target sequence; and a second single-stranded nucleic acid molecule wherein said second single-stranded nucleic acid molecule is bound to a second guide complex comprising a second guide polynucleotide, wherein said second guide polynucleotide comprises:(i) a second non-target binding region comprising a second restriction endonuclease recognition sequence for said enzyme that is a type Ils restriction enzyme;(ii) a second target binding region configured to hybridize to a second target sequence; and wherein said enzyme that is a type Ils restriction enzyme binds to said first restriction endonuclease recognition sequence of said first non-target binding region or said second restriction endonuclease recognition sequence of said second non-target binding region, wherein said first single-stranded nucleic acid molecule or said first target sequence is from Bacillus anthracis, and said second single-stranded nucleic acid molecule or said second target sequence is from Bacillus anthracis.

180. The system of claim 179, wherein said first guide polynucleotide and / or said second guide polynucleotide further comprises a blocked 3' end non-extendable by a polymerase.

181. The system of claim 179 or 180, wherein said first guide polynucleotide and / or said second guide polynucleotide further comprises an unblocked 3' end.

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