Compositions Comprising CRISPR Nucleases and Uses Thereof

Abstract

The present disclosure relates to mutant polypeptides, methods of preparing mutant polypeptides, processes for characterizing mutant polypeptides, compositions and cells comprising mutant polypeptides, and methods of using mutant polypeptides. The present disclosure further relates to complexes comprising mutant polypeptides, methods for producing the complexes, processes for characterizing the complexes, cells comprising the complexes, and methods of using the complexes.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 241,821, filed September 8, 2021, and U.S. Provisional Patent Application No. 63 / 296,741, filed January 5, 2022, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as the CRISPR-Cas or CRISPR / Cas system, are an adaptive immune system in archaea and bacteria that protects certain species from foreign genetic elements. Summary of the Invention [Means for solving the problem]

[0003] SUMMARY OF THE INVENTION Over the above background, the present invention offers certain advantages and advancements over the prior art.

[0004] The invention disclosed herein provides, without being limited to a particular benefit or function, variant polypeptides and / or compositions comprising the variant polypeptides, wherein the variant polypeptide comprises a modification relative to a parent polypeptide, wherein the parent polypeptide comprises SEQ ID NO: 3, wherein the variant polypeptide is capable of binding to an RNA guide and a target nucleic acid, and wherein the variant polypeptide or a complex comprising the variant polypeptide exhibits enhanced enzymatic activity, enhanced binding activity, enhanced binding specificity, and / or enhanced stability compared to the parent polypeptide or a complex comprising the parent polypeptide.

[0005] In some embodiments, a variant polypeptide of the invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100%, identity to SEQ ID NO:3.

[0006] In one aspect, the disclosure provides a variant polypeptide, wherein the variant polypeptide comprises modifications relative to the amino acid sequence of SEQ ID NO: 3, the modifications being D509R, S511R, I521R, D535G, Q514G, L516R, L516G, E198R, S527R, D509K, D509G, L580G, V359R, D535K, Y381R, R354G, M380K, M380R, V383R, L580R, E367R, I521K, E367G, E507R, I521G, W350R, D535R, R528K, S527K, L385G, W350G, L516K, Y381G, H532 R, D129R, P615R, G578R, M380G, V595G, R531G, D129G, R531K, D158G, N476G, G578K, A512R, P618R, V595R, V595K, V383G, A597G, Y381K, P618G, E198K, T288R, E507K, L385R, C538G, P615G, D386R, S511K, C371G, K136G, N220R, S78K, K141G, K240R, D277R, T165R or K374R, and the variant polypeptide comprises at least 90% identity to the amino acid sequence of SEQ ID NO:3.

[0007] In one aspect, the disclosure provides a variant polypeptide, wherein the variant polypeptide comprises a modification relative to the amino acid sequence of SEQ ID NO: 3, the modifications being D509R, S511R, I521R, D535G, Q514G, L516R, L516G, E198R, S527R, D509K, D509G, L580G, V359R, D535K, Y381R, R354G, M380K, M380R, V383R, L580R, E367R, I521K, E367G, E507R, I521G, W350R, D535R, R528K, S527K, L385G, W350G, L516K, Y381G, H532R , D129R, P615R, G578R, M380G, V595G, R531G, D129G, R531K, D158G, N476G, G578K, A512R, P618R, V595R, V595K, V383G, A597G, Y381K, P618G, E198K, T288R, E507K, L385R, C538G, P615G, D386R, S511K or C371G, K136G, N220R, S78K, K141G, K240R, D277R, T165R or K374R, and the variant polypeptide differs from the amino acid sequence of SEQ ID NO: 3 by 1 to 20 amino acid residues.

[0008] In some embodiments, the variant polypeptide differs from the amino acid sequence of SEQ ID NO: 3 by 1 to 50 amino acid residues (e.g., 1 to 45, 1 to 35, 1 to 25, 1 to 25, 1 to 15, and 10, as well as 1 to 5, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 10 to 15, 15 to 50, 15 to 40, 15 to 30, 15 to 20, 20 to 50, 20 to 40, 20 to 30, 20 to 35, 25 to 50, 25 to 40, 25 to 30, 30 to 50, 30 to 40, 30 to 35, or 40 to 50 amino acid residues). In some embodiments, the variant polypeptide differs from the amino acid sequence of SEQ ID NO:3 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue.

[0009] In some embodiments, the variant polypeptide further comprises a second modification to the amino acid sequence of SEQ ID NO: 3. In particular embodiments, i) the modification comprises L580G and the second modification comprises L385G ii) the modification comprises D509R and a second modification comprises M380R; iii) the modification comprises L580G and a second modification comprises A512R; iv) the modification comprises S527R and a second modification comprises C538G; v) the modification comprises D129R and the second modification comprises P618R; or vi) The modification comprises Q514G and the second modification comprises A512R.

[0010] In some embodiments, the variant polypeptide further comprises a third modification to the amino acid sequence of SEQ ID NO: 3. i) the modification comprises D535G, the second modification comprises L516R, and the third modification comprises I521R; ii) the modification comprises L516R, the second modification comprises Q514GR, and the third modification comprises I521R; iii) the modification comprises D509R, the second modification comprises L516R, and the third modification comprises I521R; iv) the modification comprises D535G, the second modification comprises L516R, and the third modification comprises Q514G; i) the modification comprises D535G, the second modification comprises Q514G, and the third modification comprises I521R; vi) the modification comprises K136G, the second modification comprises N220R, and the third modification comprises M380R; vii) the modification comprises S78K, the second modification comprises E198R, and the third modification comprises R354G; viii) the modification comprises K141G, the second modification comprises N220R, and the third modification comprises M380R; ix) the modification comprises K141G, the second modification comprises K240R, and the third modification comprises M380R; and x) The modification comprises K141G, the second modification comprises D277R, and the third modification comprises M380R.

[0011] In some embodiments, the modification comprises D535G, the second modification comprises L516R, and the third modification comprises I521R. In some embodiments, the modification comprises L516R, the second modification comprises Q514G, and the third modification comprises I521R. In some embodiments, the modification comprises D509R, the second modification comprises L516R, and the third modification comprises I521R. In some embodiments, the modification comprises D535G, the second modification comprises L516R, and the third modification comprises Q514G. In some embodiments, the modification comprises D535G, the second modification comprises Q514GR, and the third modification comprises I521R. In some embodiments, the modification comprises D509R, the second modification comprises D535G, and the third modification comprises L516R. In some embodiments, the modification comprises D509R, the second modification comprises L516R, and the third modification comprises Q514G. In some embodiments, the modification comprises D509R, the second modification comprises Q514G, and the third modification comprises I521R. In some embodiments, the modification comprises D509R, the second modification comprises D535G, and the third modification comprises I521R. In some embodiments, the modification comprises D509R, the second modification comprises D535G, and the third modification comprises Q514G. In some embodiments, the modification comprises K136G, the second modification comprises N220R, and the third modification comprises M380R; in some embodiments, the modification comprises S78K, the second modification comprises E198R, and the third modification comprises R354G; in some embodiments, the modification comprises K141G, the second modification comprises N220R, and the third modification comprises M380R; in some embodiments, the modification comprises K141G, the second modification comprises K240R, and the third modification comprises M380R. in some embodiments, the modification comprises K141G, the second modification comprises D277R, and the third modification comprises M380R; in some embodiments, the modification comprises K136G, the second modification comprises D277R, and the third modification comprises M380R; in some embodiments, the modification comprises S78K, the second modification comprises E198R, and the third modification comprises L385R; in some embodiments, the modification comprises S78K, the second modification comprises E198R, and the third modification comprises M380R;In some embodiments, the modification comprises K136G, the second modification comprises K240R, and the third modification comprises M380R; in some embodiments, the modification comprises T165R, the second modification comprises N220R, and the third modification comprises M380R; in some embodiments, the modification comprises S78K, the second modification comprises E198R, and the third modification comprises K374R; in some embodiments, the modification comprises T165R, the second modification comprises D277R, and the third modification comprises M380R; in some embodiments, the modification comprises T165R, the second modification comprises K240R, and the third modification comprises M380R; in some embodiments, the modification comprises K141G, the second modification comprises K240R, and the third modification comprises L385R; in some embodiments, the modification comprises K136G, the second modification comprises E198R, and the third modification comprises K374R. In some embodiments, the modification comprises N220R, and the third modification comprises L385R; in some embodiments, the modification comprises K141G, the second modification comprises N220R, and the third modification comprises L385R; in some embodiments, the modification comprises K141G, the second modification comprises D277R, and the third modification comprises L385R; in some embodiments, the modification comprises K136G, the second modification comprises N220R, and the third modification comprises K374R; in some embodiments, the modification comprises K136G, the second modification comprises D277R, and the third modification comprises L385R; in some embodiments, the modification comprises K136G, the second modification comprises K240R, and the third modification comprises L385R; in some embodiments, the modification comprises T165R, the second modification comprises K240R, and the third modification comprises L385R;

[0012] In certain embodiments, the variant polypeptide further comprises a fourth modification relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the modification comprises Q514G, the second modification comprises I521R, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises D535G, the second modification comprises Q514G, the third modification comprises I521R, and the fourth modification comprises E198R. In some embodiments, the modification comprises D535G, the second modification comprises L516R, the third modification comprises Q514G, and the fourth modification comprises I521R. In some embodiments, the modification comprises Q514G, the second modification comprises I521R, the third modification comprises S527R, and the fourth modification comprises E198R. In some embodiments, the modification comprises D535G, the second modification comprises L516R, the third modification comprises I521R, and the fourth modification comprises S527R. In some embodiments, the modification comprises D535G, the second modification comprises I521R, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises I521R, the second modification comprises S527R, the third modification comprises E198R, and the fourth modification comprises M380R. In some embodiments, the modification comprises D535G, the second modification comprises L516R, the third modification comprises Q514G, and the fourth modification comprises S511R. In some embodiments, the modification comprises D535G, the second modification comprises L516R, the third modification comprises Q514G, and the fourth modification comprises S527R. In some embodiments, the modification comprises I521R, the second modification comprises S527R, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises D535G, the second modification comprises L516R, the third modification comprises S527R, and the fourth modification comprises M380R. In some embodiments, the modification comprises Q514G, the second modification comprises S527R, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises L516R, the second modification comprises Q514G, the third modification comprises I521R, and the fourth modification comprises M380R.In some embodiments, the modification comprises D509R, the second modification comprises L516R, the third modification comprises Q514G, and the fourth modification comprises S527R. In some embodiments, the modification comprises D509R, the second modification comprises I521R, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises S527R, the second modification comprises L580G, the third modification comprises E198R, and the fourth modification comprises M380R. In some embodiments, the modification comprises L516R, the second modification comprises S527R, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises D509R, the second modification comprises Q514G, the third modification comprises I521R, and the fourth modification comprises E198R. In some embodiments, the modification comprises D535G, the second modification comprises Q514G, the third modification comprises I521R, and the fourth modification comprises S527R. In some embodiments, the modification comprises Q514G, the second modification comprises L580G, the third modification comprises E198R, and the fourth modification comprises M380R. In some embodiments, the modification comprises D509R, the second modification comprises L516R, the third modification comprises I521R, and the fourth modification comprises E198R. In some embodiments, the modification comprises L516R, the second modification comprises Q514G, the third modification comprises I521R, and the fourth modification comprises L580G. In some embodiments, the modification comprises D509R, the second modification comprises D535G, the third modification comprises Q514G, and the fourth modification comprises E198R. In some embodiments, the modification comprises L516R, the second modification comprises I521R, the third modification comprises S527R, and the fourth modification comprises L580G. In some embodiments, the modification comprises D535G, the second modification comprises I521R, the third modification comprises S527R, and the fourth modification comprises M380R. In some embodiments, the modification comprises L516R, the second modification comprises I521R, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises D535G, the second modification comprises S527R, the third modification comprises L580G, and the fourth modification comprises E198R.In some embodiments, the modification comprises S527R, the second modification comprises L580G, the third modification comprises E198R, and the fourth modification comprises R354G. In some embodiments, the modification comprises Q514G, the second modification comprises I521R, the third modification comprises S527R, and the fourth modification comprises L580G. In some embodiments, the modification comprises D535G, the second modification comprises Q514G, the third modification comprises E198R, and the fourth modification comprises M380R. In some embodiments, the modification comprises L516R, the second modification comprises Q514G, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises I521R, the second modification comprises S527R, the third modification comprises E198R, and the fourth modification comprises R354G. In some embodiments, the modification comprises I521R, the second modification comprises S527R, the third modification comprises R354G, and the fourth modification comprises M380R. In some embodiments, the modification comprises I521R, the second modification comprises L580G, the third modification comprises E198R, and the fourth modification comprises M380R. In some embodiments, the modification comprises L516R, the second modification comprises Q514G, the third modification comprises S527R, and the fourth modification comprises L580G. In some embodiments, the modification comprises D535G, the second modification comprises Q514G, the third modification comprises S527R, and the fourth modification comprises E198R. In some embodiments, the modification comprises D509R, the second modification comprises Q514G, the third modification comprises L580G, and the fourth modification comprises E198R. In some embodiments, the modification comprises L580G, the second modification comprises L385G, the third modification comprises S511R, and the fourth modification comprises A512R. In some embodiments, the modification comprises L580G, the second modification comprises L385G, the third modification comprises Q514G, and the fourth modification comprises A512R. In some embodiments, the modification comprises D129R, the second modification comprises P618R, the third modification comprises S511R, and the fourth modification comprises A512R. In some embodiments, the modification comprises L580G, the second modification comprises L385G, the third modification comprises G578R, and the fourth modification comprises D158G.In some embodiments, the modification comprises D129R, the second modification comprises P618R, the third modification comprises R354G, and the fourth modification comprises A512R. In some embodiments, the modification comprises D129R, the second modification comprises P618R, the third modification comprises Q514G, and the fourth modification comprises A512R. In some embodiments, the modification comprises D129R, the second modification comprises P618R, the third modification comprises S527R, and the fourth modification comprises L385G. In some embodiments, the modification comprises L580G, the second modification comprises L385G, the third modification comprises Q514G, and the fourth modification comprises N476G. In some embodiments, the modification comprises L580G, the second modification comprises L385G, the third modification comprises S512R, and the fourth modification comprises P618R. In some embodiments, the modification comprises D129R, the second modification comprises P618R, the third modification comprises D158G, and the fourth modification comprises A512R. In some embodiments, the modification comprises L580G, the second modification comprises L385G, the third modification comprises S527R, and the fourth modification comprises C538G. In some embodiments, the modification comprises D129R, the second modification comprises P618R, the third modification comprises V359R, and the fourth modification comprises A512R.

[0013] In certain embodiments, the variant polypeptide further comprises a fifth modification relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the modification comprises D509R, the second modification comprises D535G, the third modification comprises L516R, the fourth modification comprises Q514G, and the fifth modification comprises I521R.

[0014] In certain embodiments, the variant polypeptide further comprises a sixth modification, and optionally further comprises a seventh, eighth, ninth, and tenth modification.

[0015] In some embodiments, the second modification comprises a substitution, insertion, or deletion.

[0016] In certain embodiments, the third modification comprises a substitution, insertion, or deletion.

[0017] In some embodiments, the fourth modification comprises a substitution, insertion, or deletion.

[0018] In some embodiments, the fifth modification comprises a substitution, insertion, or deletion.

[0019] In some embodiments, the sixth, optionally seventh, optionally eighth, optionally ninth, or optionally tenth modification each independently comprises a substitution, insertion, or deletion.

[0020] In some embodiments, the mutant polypeptide comprises a modification in Table 6. In some embodiments, the mutant polypeptide comprises a modification in Table 7. In some embodiments, the mutant polypeptide comprises a modification in Table 8. In some embodiments, the mutant polypeptide comprises a modification in Table 9. In some embodiments, the mutant polypeptide comprises two modifications of any cell in Table 7. In some embodiments, the mutant polypeptide comprises two modifications of any cell in Table 8. In some embodiments, the mutant polypeptide comprises two modifications of any cell in Table 9.

[0021] In some embodiments, the variant polypeptide or a complex comprising the variant polypeptide exhibits enhanced enzymatic activity and / or enhanced stability compared to the polypeptide of SEQ ID NO:3.

[0022] In some embodiments, the variant polypeptide or a complex comprising the variant polypeptide comprises at least a 1.5-fold, 2-fold, 2.5-fold, or 3-fold increase in enzymatic activity compared to the polypeptide of SEQ ID NO: 3, e.g., as measured by indel activity, e.g., as described in Examples 9 and 10.

[0023] In some embodiments, the enhanced enzymatic activity is enhanced nuclease activity.

[0024] In some embodiments, the variant polypeptide exhibits enhanced binding activity towards an RNA guide compared to the parent polypeptide.

[0025] In some embodiments, the variant polypeptide exhibits enhanced binding specificity for the RNA guide compared to the parent polypeptide.

[0026] In some embodiments, the mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits one or more of the following characteristics: (i) enhanced binding activity to the target nucleic acid (e.g., on-target binding activity) compared to the parent binary complex; (ii) enhanced binding specificity for the target nucleic acid (e.g., on-target binding specificity) compared to the parent binary complex; (iii) enhanced stability compared to the parent binary complex; and / or (iv) reduced dissociation from the target nucleic acid and / or reduced off-target binding to non-target nucleic acids compared to the parent binary complex.

[0027] In some embodiments, the mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits enhanced binding activity (e.g., on-target binding activity) to a target nucleic acid compared to the parent binary complex.

[0028] In some embodiments, the mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits enhanced binding specificity for a target nucleic acid (e.g., on-target binding specificity) relative to the parent binary complex.

[0029] In some embodiments, the mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits enhanced stability compared to the parent binary complex.

[0030] In some embodiments, the mutant binary complex and the target nucleic acid form a mutant ternary complex, and the mutant ternary complex exhibits increased stability compared to the parent ternary complex.

[0031] In some embodiments, the variant polypeptide further exhibits enhanced binary complex formation, enhanced protein-RNA interaction, and / or reduced dissociation from the RNA guide compared to the parent polypeptide.

[0032] In some embodiments, the mutant binary complex further exhibits reduced dissociation from the target nucleic acid and / or reduced off-target binding to non-target nucleic acids compared to the parent binary complex.

[0033] In some embodiments, the increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability occurs over a temperature range of, for example, 20°C to 65°C.

[0034] In some embodiments, the increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability occurs over a range of incubation times.

[0035] In some embodiments, the increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability occurs in a buffer having a pH in the range of about 7.3 to about 8.6.

[0036] In some embodiments, the enhanced enzymatic activity, enhanced binding activity, enhanced binding specificity, and / or enhanced stability is achieved by increasing the T of the mutant polypeptide, mutant binary complex, or mutant ternary complex. m The value of the T of the parent polypeptide, parent binary complex, or parent ternary complex m occurs when the temperature is at least 8°C higher than the value.

[0037] In other embodiments, the modification comprises a modification of the amino acid sequence compared to the parent polypeptide having the sequence set forth in SEQ ID NO:3, and the modification comprises one or more (e.g., 1, 2, 3, 4, 5, or more) substitutions, insertions, deletions, and / or additions compared to the parent polypeptide having the sequence set forth in SEQ ID NO:3.

[0038] In some embodiments, the modification comprises an amino acid sequence modification relative to the parent polypeptide sequence set forth in SEQ ID NO: 3, wherein the modification comprises one or more of the amino acid substitutions listed in Table 2.

[0039] In some embodiments, the modification comprises an arginine, lysine, glutamine, asparagine, histidine, alanine, or glycine substitution.

[0040] In some embodiments, the mutant polypeptide comprises a RuvC domain or a split RuvC domain.

[0041] In some embodiments, the mutant polypeptide comprises one or more catalytic residues (e.g., aspartic acid or glutamic acid). In some embodiments, the one or more catalytic residues comprise D345, E506, and D594.

[0042] In some embodiments, the mutant polypeptide has reduced or no nuclease activity and is a nuclease dead polypeptide.

[0043] In certain embodiments, the second modification comprises an insertion of a polypeptide domain, optionally at the N-terminus, C-terminus or internally of the sequence of SEQ ID NO:3.

[0044] In some embodiments, the mutant polypeptide further comprises a peptide tag, a fluorescent protein, a base editing domain, a DNA methylation domain, a histone residue modifying domain, a localization factor, a transcriptional modifier, a photogating factor, a chemically inducible factor, or a chromatin visualization factor.

[0045] In one aspect, the disclosure provides a system comprising a variant polypeptide described herein or a first nucleic acid encoding the variant polypeptide, and an RNA guide or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence.

[0046] In some embodiments, the composition or complex comprising the variant polypeptide further comprises an RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence.

[0047] In some embodiments, the direct repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0048] In some embodiments, the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0049] In some embodiments, the direct repeat sequence comprises the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0050] In some embodiments, the spacer sequence comprises 15 to 35 nucleotides in length.

[0051] In certain embodiments, the first nucleic acid is located in a first vector and the second nucleic acid encoding the RNA guide is located in a vector (e.g., the first vector or the second vector), optionally wherein the first and / or second vector is a viral vector.

[0052] In some embodiments, the RNA guide comprises any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, and 23.

[0053] In some embodiments, the target nucleic acid comprises a sequence complementary to a nucleotide sequence in the spacer sequence.

[0054] In some embodiments, the target nucleic acid is flanked by a protospacer adjacent motif (PAM) sequence, wherein the PAM sequence comprises a nucleotide sequence designated as 5'-NTN-3', 5'-HTN-3', or 5'-TNA-3', where N is any nucleotide and H is A, C, or T. In some embodiments, the PAM sequence comprises a nucleotide sequence designated as 5'-CTG-3' or 5'-CTC-3'.

[0055] In some embodiments, the target nucleic acid is single-stranded or double-stranded DNA. In some embodiments, the target nucleic acid is double-stranded DNA.

[0056] In one aspect, the disclosure provides nucleic acids encoding the variant polypeptides described herein.

[0057] In some embodiments, the nucleic acid encoding the variant polypeptide is codon-optimized for expression in the cell.

[0058] In certain embodiments, the nuclear encoding further comprises a sequence encoding a guide RNA.

[0059] In some embodiments, the nucleic acid encoding the variant polypeptide is operably linked to a promoter.

[0060] In certain embodiments, the nucleic acid comprises RNA (eg, mRNA).

[0061] In some embodiments, the nucleic acid encoding the variant polypeptide is in a vector.

[0062] In some embodiments, the vector comprises a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0063] In certain embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0064] In one aspect, the disclosure provides a composition comprising a variant polypeptide, system, nucleic acid, or vector described herein. In some embodiments, the composition is present in a delivery composition comprising a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0065] In one aspect, the disclosure provides a cell comprising a variant polypeptide, system, nucleic acid, or vector described herein.

[0066] In some embodiments, the cells comprise the variant polypeptides and / or compositions disclosed herein.

[0067] In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell or a plant cell. In some embodiments, the cell is a human cell.

[0068] The present disclosure further provides a method for preparing a mutant polypeptide disclosed herein, the method comprising: (i) introducing one or more nucleotide substitutions into a nucleic acid comprising SEQ ID NO:1 or SEQ ID NO:2 to generate a mutant nucleic acid encoding the mutant polypeptide; and (ii) expressing the mutant polypeptide from the mutant nucleic acid.

[0069] The present disclosure further provides a method of forming a mutant binary complex disclosed herein, the method comprising contacting a mutant polypeptide disclosed herein with an RNA guide disclosed herein.

[0070] In one aspect, the disclosure provides a method of producing a variant polypeptide, the method comprising: (i) generating a nucleic acid sequence encoding the variant polypeptide; (ii) introducing the nucleic acid sequence into a suitable host cell capable of expressing the nucleic acid sequence; and (iii) allowing the host cell to express the variant polypeptide.

[0071] In one aspect, the disclosure provides a method of producing a variant polypeptide, the method comprising: (i) introducing one or more nucleotide substitutions into a nucleic acid comprising SEQ ID NO:1 or SEQ ID NO:2 to produce a variant nucleic acid encoding the variant polypeptide; and (ii) expressing the variant polypeptide from the variant nucleic acid.

[0072] The present disclosure further provides a method of forming a mutant ternary complex disclosed herein, the method comprising contacting a mutant polypeptide disclosed herein with an RNA guide disclosed herein and a target nucleic acid disclosed herein.

[0073] In one aspect, the present disclosure provides a method of delivering a mutant polypeptide to a cell, the method comprising introducing into the cell a mutant polypeptide or a nucleic acid encoding the mutant polypeptide as described herein, and optionally introducing an RNA guide or a nucleic acid encoding the RNA guide, wherein the introducing step optionally comprises introducing a nanoparticle, liposome, exosome, microvesicle, viral vector, or any combination thereof.

[0074] In one aspect, the present disclosure provides a method for modifying a target DNA molecule in a cell, the method comprising introducing into the cell a mutant polypeptide or a nucleic acid encoding the mutant polypeptide described herein, and introducing an RNA guide or a nucleic acid encoding the RNA guide, wherein the introducing step optionally comprises introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof. In some embodiments, the introducing into the cell comprises transfecting or transducing the cell. In certain embodiments, the introducing into the cell comprises using electroporation, injection, a gene gun, or any combination thereof. In some embodiments, the nucleic acid encoding the mutant polypeptide comprises RNA (e.g., mRNA).

[0075] In one aspect, the present disclosure provides a method of modifying a target DNA molecule, the method comprising contacting the target DNA molecule with a mutant polypeptide and an RNA guide. In some embodiments, the target DNA molecule is in vitro or in a cell. In certain embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is selected from a prokaryotic cell, a eukaryotic cell, a plant cell, a mammalian cell, and a human cell.

[0076] The present disclosure further provides a method of delivering a mutant polypeptide or composition or mutant binary complex disclosed herein, comprising introducing a mutant polypeptide or a nucleic acid encoding the mutant polypeptide into a cell, and optionally introducing an RNA guide or a nucleic acid encoding an RNA guide as described herein, or introducing a mutant binary complex as described herein, wherein the introducing comprises introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof. In some embodiments, the introducing comprises transfecting or transducing the cell. In some embodiments, the introducing comprises using electroporation, injection, a gene gun, or any combination thereof.

[0077] The present disclosure further provides a composition comprising a variant polypeptide or a complex comprising a variant polypeptide and an RNA guide, wherein the variant polypeptide comprises a modification relative to a parent polypeptide, wherein the parent polypeptide comprises SEQ ID NO: 3, wherein the variant polypeptide is capable of binding to an RNA guide and a target nucleic acid, and wherein the variant polypeptide or complex exhibits increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability compared to the parent polypeptide or the complex comprising the parent polypeptide and an RNA guide.

[0078] In some embodiments, the enhanced enzymatic activity is enhanced nuclease activity.

[0079] In some embodiments, the variant polypeptide exhibits enhanced binding activity towards an RNA guide compared to the parent polypeptide.

[0080] In some embodiments, the variant polypeptide exhibits enhanced binding specificity for the RNA guide compared to the parent polypeptide.

[0081] In some embodiments, the mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits enhanced binding activity to a target nucleic acid (e.g., on-target binding activity) relative to the parent binary complex.

[0082] In some embodiments, the mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits enhanced binding specificity for a target nucleic acid (e.g., on-target binding specificity) relative to the parent binary complex.

[0083] In some embodiments, the mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits enhanced stability compared to the parent binary complex.

[0084] In some embodiments, the mutant binary complex and the target nucleic acid form a mutant ternary complex, and the mutant ternary complex exhibits increased stability compared to the parent ternary complex.

[0085] In some embodiments, the variant polypeptide further exhibits enhanced binary complex formation, enhanced protein-RNA interaction, and / or reduced dissociation from the RNA guide compared to the parent polypeptide.

[0086] In some embodiments, the mutant binary complexes further exhibit reduced dissociation from the target nucleic acid and / or reduced off-target binding to non-target nucleic acids compared to the parent binary complex.

[0087] In some embodiments, the increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability occurs over a range of temperatures, for example, from 20°C to 65°C.

[0088] In some embodiments, the increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability occurs over a given incubation period.

[0089] In some embodiments, the increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability occurs in a buffer having a pH in the range of about 7.3 to about 8.6.

[0090] In some embodiments, the enhanced enzymatic activity, enhanced binding activity, enhanced binding specificity, and / or enhanced stability is achieved by increasing the T of the mutant polypeptide, mutant binary complex, or mutant ternary complex. m The value of the T of the parent polypeptide, parent binary complex, or parent ternary complex m occurs when the temperature is at least 8°C higher than the value.

[0091] In other embodiments, the modification comprises an amino acid sequence modification compared to a parent polypeptide having the sequence set forth in SEQ ID NO:3, wherein the modification comprises one or more (e.g., 1, 2, 3, 4, 5, or more) substitutions, insertions, deletions, and / or additions compared to a parent polypeptide having the sequence set forth in SEQ ID NO:3.

[0092] In some embodiments, the modification comprises an amino acid sequence modification compared to the parent polypeptide sequence set forth in SEQ ID NO:3, wherein the modification comprises one or more of the amino acid substitutions listed in Table 2.

[0093] In some embodiments, the modification comprises an arginine, lysine, glutamine, asparagine, histidine, alanine, or glycine substitution.

[0094] In some embodiments, the modifications include E38R, T60R, D89R, S223R, P353G, L354G, L360G, K368G, E566R, and / or D730R substitutions.

[0095] In some embodiments, the mutant polypeptide comprises a RuvC domain or a split RuvC domain.

[0096] In some embodiments, the mutant polypeptide comprises one or more catalytic residues (e.g., aspartic acid or glutamic acid). In some embodiments, the one or more catalytic residues comprise D345, E506, and D594.

[0097] In some embodiments, the RNA guide comprises a direct repeat sequence and a spacer sequence.

[0098] In some embodiments, the direct repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0099] In some embodiments, the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0100] In some embodiments, the direct repeat sequence comprises the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0101] In some embodiments, the spacer sequence comprises a length of 15 to 35 nucleotides.

[0102] In some embodiments, the target nucleic acid comprises a sequence complementary to a nucleotide sequence in a spacer sequence.

[0103] In some embodiments, the target nucleic acid is flanked by a PAM sequence, and the PAM sequence comprises a nucleotide sequence described as 5'-NTN-3', 5'-HTN-3', or 5'-TNA-3', where N is any nucleotide and H is A, C, or T. In some embodiments, the PAM sequence comprises a nucleotide sequence described as 5'-CTG-3', or 5'-CTC-3'.

[0104] In some embodiments, the target nucleic acid is single-stranded or double-stranded DNA.

[0105] In some embodiments, the mutant polypeptide further comprises a peptide tag, a fluorescent protein, a base editing domain, a DNA methylation domain, a histone residue modifying domain, a localization factor, a transcriptional modifier, a photogating factor, a chemically inducible factor, or a chromatin visualization factor.

[0106] In some embodiments, the nucleic acid encoding the variant polypeptide is codon-optimized for expression in the cell.

[0107] In some embodiments, the nucleic acid encoding the variant polypeptide is operably linked to a promoter.

[0108] In some embodiments, the nucleic acid encoding the variant polypeptide is in a vector.

[0109] In some embodiments, the vector comprises a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated viral vector, or a herpes simplex viral vector.

[0110] In some embodiments, the composition or complex is present in a delivery composition comprising a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0111] The present disclosure further provides an RNA guide or a nucleic acid encoding an RNA guide comprising a direct repeat sequence comprising at least 90% identity to any one of SEQ ID NOs: 4, 5, or 6. In some aspects, the RNA guide or the nucleic acid encoding the RNA guide comprises a spacer sequence adjacent to and bound to a 5'-CTG-3' or 5'-CTC-3' protospacer adjacent motif. In some embodiments, an RNA guide comprising a nucleotide sequence having at least 90% sequence identity to any of SEQ ID NOs: 4-6 binds to a CRISPR nuclease, for example, a CRISPR nuclease according to SEQ ID NO: 3.

[0112] The present disclosure further provides a composition comprising an RNA guide, the composition further comprising a CRISPR nuclease. In some embodiments, the CRISPR nuclease comprises at least 80% identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:3. In some embodiments, the CRISPR nuclease comprises at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:3. In some embodiments, the CRISPR nuclease comprises SEQ ID NO:3.

[0113] The present disclosure further provides cells comprising the variant polypeptides and / or complexes disclosed herein. In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a mammalian cell or a plant cell. In some embodiments, the cell is a human cell.

[0114] The present disclosure further provides a method for preparing a mutant polypeptide disclosed herein, the method comprising: (i) introducing one or more nucleotide substitutions into a nucleic acid comprising SEQ ID NO:1 or SEQ ID NO:2 to generate a mutant nucleic acid encoding the mutant polypeptide; and (ii) expressing the mutant polypeptide from the mutant nucleic acid.

[0115] The present disclosure further provides a method of forming a mutant binary complex, the method comprising contacting a mutant polypeptide disclosed herein with an RNA guide disclosed herein.

[0116] The present disclosure further provides a method of forming a mutant ternary complex disclosed herein, the method comprising contacting a mutant polypeptide disclosed herein with an RNA guide disclosed herein and a target nucleic acid disclosed herein.

[0117] The present disclosure further provides a method of delivering a mutant polypeptide or composition or mutant binary complex disclosed herein to a cell, the method comprising introducing into the cell a mutant polypeptide or a nucleic acid encoding the mutant polypeptide disclosed herein, and optionally introducing an RNA guide or a nucleic acid encoding the RNA guide described herein, or a mutant binary complex described herein, wherein the introducing comprises introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof. In some embodiments, the introducing into the cell comprises transfecting or transducing the cell. In some embodiments, the introducing comprises using electroporation, injection, a gene gun, or any combination thereof. In some embodiments, the cell is selected from a prokaryotic cell, a eukaryotic cell, a plant cell, a mammalian cell, and a human cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo.

[0118] The present disclosure further provides a method for modifying a target DNA molecule in a cell, the method comprising introducing into the cell a mutant polypeptide or a nucleic acid encoding the mutant polypeptide disclosed herein, and an RNA guide or a nucleic acid encoding the RNA guide described herein, or a mutant binary complex described herein, wherein the introducing comprises introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof. In some embodiments, the introducing into the cell comprises transfecting or transducing the cell. In some embodiments, the introducing comprises using electroporation, injection, a gene gun, or any combination thereof. In some embodiments, the cell is selected from a prokaryotic cell, a eukaryotic cell, a plant cell, a mammalian cell, and a human cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo.

[0119] The present disclosure further provides a method of modifying a target DNA molecule, the method comprising contacting the target DNA molecule with a mutant polypeptide disclosed herein and an RNA guide disclosed herein. In some embodiments, the target DNA molecule is in vitro or in a cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is selected from a prokaryotic cell, a eukaryotic cell, a plant cell, a mammalian cell, and a human cell.

[0120] In some aspects, the present disclosure provides a method for modifying a target nucleic acid in a cell, the method comprising: (i) introducing into the cell a polypeptide comprising an amino acid sequence according to SEQ ID NO: 3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or a nucleic acid encoding the polypeptide; and (ii) introducing into the cell an RNA guide (e.g., as described herein) or a nucleic acid encoding the RNA guide, wherein the target nucleic acid is flanked by protospacer adjacent motif (PAM) sequences, and the PAM sequence comprises a nucleotide sequence described as 5'-CTG-3' or 5'-CTC-3'. In some embodiments, the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 3-7. The introducing step optionally comprises introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof. In some embodiments, introducing into a cell comprises transfecting or transducing the cell. In certain embodiments, introducing into a cell comprises using electroporation, injection, a gene gun, or any combination thereof. In some embodiments, the nucleic acid encoding the polypeptide comprises RNA (e.g., mRNA). In some aspects, the method modifies the target DNA at a position adjacent to a 5'-CTG-3' or 5'-CTC-3' protospacer adjacent motif. In some embodiments, the modification comprises nicking or cleaving the target nucleic acid.

[0121] In some aspects, the disclosure provides an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 4-6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein the nucleotide immediately 3' to the direct repeat sequence is selected from C, T, or G. In some aspects, the disclosure provides an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 4-6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein the nucleotide immediately 3' to the direct repeat sequence is other than A. In some aspects, the disclosure provides an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence, and the direct repeat sequence comprises the nucleotide sequence of SEQ ID NO: 5 or 6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some aspects, the disclosure provides an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence, and the direct repeat sequence consists of the nucleotide sequence of any one of SEQ ID NOs: 4-6, or a sequence with one, two, three, or four substitutions thereto. In some embodiments, the spacer is heterologous to the direct repeat sequence. [Brief explanation of the drawings]

[0122] [Figure 1] Indel activity (% raw indels) at eight target sites flanking the 5'-CTG-3'PAM sequence is shown. The sequences of the target sites and crRNAs are shown in Table 4. DETAILED DESCRIPTION OF THE INVENTION

[0123] definition The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Terms as used hereinafter should be understood according to common sense unless otherwise specified.

[0124] Unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. In the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless specifically stated otherwise. The use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0125] In general, the nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The methods and techniques provided herein are performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise specified. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclatures and laboratory procedures and techniques used in connection with synthetic chemistry, synthetic organic chemistry, and medicinal chemistry and drug discovery chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0126] The present disclosure may be more readily understood with the following selection of defined terms.

[0127] The articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the item. By way of example, "an element" means one element or more than one element.

[0128] As used herein, "about" when referring to a measurable value, e.g., amount, duration, etc., is meant to encompass variations from the specified value of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1%, where such variations are appropriate for practicing the disclosed methods.

[0129] As used herein, the term "complex" refers to a grouping of two or more molecules. In some embodiments, a complex comprises a polypeptide and a nucleic acid molecule that interact (e.g., bind, contact, or attach) to each other.

[0130] As used herein, the term "binary complex" refers to a grouping of two molecules (e.g., a polypeptide and a nucleic acid molecule). In some embodiments, a binary complex refers to a grouping of a polypeptide and a targeting moiety (e.g., an RNA guide). In some embodiments, a binary complex refers to a ribonucleoprotein (RNP). As used herein, the term "mutant binary complex" refers to a grouping of a mutant polypeptide and an RNA guide. As used herein, the term "parent binary complex" refers to a grouping of a parent polypeptide and an RNA guide or a reference polypeptide and an RNA guide.

[0131] As used herein, the term "ternary complex" refers to a grouping of three molecules (e.g., a polypeptide and two nucleic acid molecules). In some embodiments, a "ternary complex" refers to a grouping of a polypeptide, an RNA molecule, and a DNA molecule. In some embodiments, a "ternary complex" refers to a grouping of a polypeptide, a targeting moiety (e.g., an RNA guide), and a target nucleic acid (e.g., a target DNA molecule). In some embodiments, a "ternary complex" refers to a grouping of a binary complex (e.g., a ribonucleoprotein) and a third molecule (e.g., a target nucleic acid).

[0132] As used herein, the term "domain" refers to a distinct functional and / or structural unit of a polypeptide. In some embodiments, a domain may comprise a conserved amino acid sequence.

[0133] As used herein, the terms "parent," "parent polypeptide," and "parent sequence" refer to the original polypeptide (e.g., reference or starting polypeptide) from which modifications are made to produce a variant polypeptide of the invention.

[0134] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a DNA sequence adjacent to a target sequence to which a complex comprising an effector (e.g., a CRISPR nuclease) and an RNA guide binds. In some embodiments, the PAM is required for enzymatic activity. As used herein, the term "adjacent" includes cases where the RNA guide of the complex specifically binds, interacts with, or associates with a target sequence directly adjacent to the PAM. In such cases, there are no nucleotides between the target sequence and the PAM. The term "adjacent" also includes cases where there are a small number of nucleotides (e.g., 1, 2, 3, 4, or 5) between the target sequence to which the RNA guide binds and the PAM. In a double-stranded DNA molecule, the strand containing the PAM motif is referred to as the "PAM strand," and the complementary strand is referred to as the "non-PAM strand." The RNA guide binds to a site in the non-PAM strand that is complementary to the target sequence disclosed herein. In some embodiments, the PAM strand is the coding (e.g., sense) strand. In other embodiments, the PAM strand is non-coding (e.g., antisense) strand. Because the RNA guide binds to the non-PAM strand through base pairing, the non-PAM strand is also known as the target strand (TS), while the PAM strand is also known as the non-target strand (NTS).

[0135] As used herein, "reference composition," "reference molecule," "reference sequence," and "reference" refer to a control, such as a negative control or a parent (e.g., a parent sequence, parent protein, or wild-type protein). For example, a reference molecule refers to a polypeptide to which a variant polypeptide is compared. Similarly, a reference RNA guide refers to a target moiety to which a modified RNA guide is compared. A variant or modified molecule can be compared to a reference molecule based on sequence (e.g., a variant or modified molecule can have X% sequence identity or homology with the reference molecule), thermal stability, or activity (e.g., a variant or modified molecule can have X% activity of the reference molecule). For example, a variant or modified molecule can be characterized as having 10% or less of the activity of the reference polypeptide, or as having at least 10% more activity than the reference polypeptide. Examples of reference polypeptides include naturally occurring unmodified polypeptides, such as naturally occurring polypeptides from archaeal or bacterial species. In certain embodiments, a reference polypeptide is a naturally occurring polypeptide that has the closest sequence identity or homology to the variant polypeptide being compared. In certain embodiments, a reference polypeptide is a parent molecule having a naturally occurring or known sequence that has been mutated to arrive at a variant polypeptide.

[0136] As used herein, the term "RNA guide" or "RNA guide sequence" refers to any RNA molecule that facilitates targeting of a polypeptide described herein to a target nucleic acid. For example, an RNA guide can be a molecule that recognizes (e.g., binds to) a target nucleic acid. An RNA guide can be designed to be complementary to a specific nucleic acid sequence. An RNA guide includes a DNA-targeting sequence (also referred to herein as a spacer sequence) and a direct repeat (DR) sequence that facilitates binding of the RNA guide to a polypeptide of the present invention. The terms CRISPR RNA (crRNA), pre-crRNA, and mature crRNA are also used herein to refer to an RNA guide. In some examples, an RNA guide can be a modified RNA molecule that includes one or more deoxyribonucleotides in the DNA-binding sequence included in the RNA guide that binds to the non-PAM strand of a target nucleic acid. In some examples, the DNA-binding sequence can include a DNA sequence or a DNA / RNA hybrid sequence.

[0137] As used herein, the term "substantially identical" refers to a sequence, polynucleotide, or polypeptide having a degree of identity to a reference sequence.

[0138] As used herein, the terms "target nucleic acid," "target sequence," and "target substrate" refer to a nucleic acid to which an RNA guide specifically binds. In some embodiments, the DNA targeting sequence (i.e., spacer sequence) of the RNA guide binds to the target nucleic acid. In some embodiments, the target sequence is a segment of DNA adjacent to the PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. The target sequence may be directly adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separated by a small sequence segment (e.g., up to 5 nucleotides, e.g., up to 4, 3, 2, or 1 nucleotide). The target sequence may be located at the 3' end of the PAM motif or the 5' end of the PAM motif, depending on the CRISPR nuclease that recognizes the PAM motif known in the art. For example, the target sequence is located at the 3' end of the PAM motif of a Cas12i polypeptide (e.g., a Cas12i2 polypeptide such as those disclosed herein). It is understood that DNA is often double-stranded, and the RNA guide binds to one of the two strands to which it is complementary. The location in DNA to which the RNA guide binds can be simply described by providing the sequence of the strand to which the RNA guide binds (the non-PAM strand) or the sequence of the strand to which the RNA guide does not bind (the PAM strand). Thus, as will be apparent from the context of the entire application, a target nucleic acid sequence can be described by providing the nucleic acid sequence of either strand of double-stranded DNA that is targeted by the RNA guides described herein.

[0139] As used herein, the terms "mutant polypeptide," "mutant effector polypeptide," and "mutant CRISPR nuclease polypeptide" refer to a polypeptide that includes a modification, such as, but not limited to, a substitution, insertion, deletion, addition, and / or fusion, at one or more residue positions compared to a parent polypeptide. The positions of the identified modifications provided herein are numbered relative to SEQ ID NO: 3 unless otherwise specified. For example, a modification of D509R indicates a modification of amino acid 509 relative to SEQ ID NO: 3, even if the mutant polypeptide includes an N-terminal, C-terminal, or internal fusion or truncation, such that the modification is not at position 509 relative to the N-terminus in a fusion or truncated polypeptide. The amino acid positions of SEQ ID NO: 3 are further set forth in Table 2. As used herein, the terms "mutant polypeptide," "mutant effector polypeptide," and "mutant CRISPR nuclease polypeptide" refer to a polypeptide that includes modifications relative to the polypeptide of SEQ ID NO:3.

[0140] composition In some aspects, the present invention provides novel variants of an effector (e.g., a CRISPR nuclease) of SEQ ID NO: 3, compositions comprising the variants, and methods of preparation and use thereof. In other aspects, the present invention further provides complexes comprising variants of an effector (e.g., a CRISPR nuclease) of SEQ ID NO: 3, and compositions, and methods of preparation and use thereof. In some aspects, compositions comprising complexes with one or more properties are described herein. In some aspects, methods of delivering compositions comprising complexes are described.

[0141] In some embodiments, compositions of the disclosure comprise variant polypeptides that exhibit increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability compared to the parent polypeptide. In some embodiments, compositions of the disclosure comprise complexes comprising variant polypeptides that exhibit increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability compared to the parent complex. In some embodiments, the variant polypeptide comprises a modification relative to the parent polypeptide, wherein the parent polypeptide comprises SEQ ID NO: 3, and the variant polypeptide is capable of binding to an RNA guide and a target nucleic acid.

[0142] In some embodiments, the compositions of the disclosure comprise a mutant polypeptide and an RNA guide. In some embodiments, the compositions of the disclosure comprise a mutant binary complex comprising the mutant polypeptide and an RNA guide.

[0143] In some embodiments of the compositions, the variant polypeptide has increased complex formation (e.g., increased binary complex formation) with the RNA guide compared to the parent polypeptide. In some embodiments of the compositions, the variant polypeptide and RNA guide have greater binding affinity compared to the parent polypeptide and RNA guide. In some embodiments of the compositions, the variant polypeptide and RNA guide have stronger protein-RNA interactions (e.g., ionic interactions) compared to the parent polypeptide and RNA guide. In some embodiments of the compositions, the variant binary complex is more stable than the parent binary complex.

[0144] In some embodiments, the compositions of the disclosure comprise a mutant polypeptide, an RNA guide, and a target nucleic acid. In some embodiments, the compositions of the disclosure comprise a mutant ternary complex comprising a mutant polypeptide, an RNA guide, and a target nucleic acid.

[0145] In some embodiments of the compositions, the variant polypeptide has increased complex formation (e.g., increased ternary complex formation) with the RNA guide and target nucleic acid compared to the parent polypeptide. In some embodiments of the compositions, the variant polypeptide and RNA guide (e.g., variant binary complex) have greater binding affinity for the target nucleic acid compared to the parent polypeptide and RNA guide (e.g., parent binary complex). In some embodiments of the compositions, the variant ternary complex is more stable than the parent ternary complex.

[0146] In some embodiments, a composition of the disclosure comprises a variant polypeptide described herein.

[0147] Mutant Polypeptides In one embodiment, the mutant polypeptide (e.g., mutant CRISPR nuclease polypeptide) is an isolated or purified polypeptide.

[0148] In some embodiments, a variant polypeptide of the present disclosure is a variant of a parent polypeptide (e.g., a parent CRISPR nuclease), where the parent is encoded by a polynucleotide that includes a nucleotide sequence such as SEQ ID NO: 1 or SEQ ID NO: 2, or that includes an amino acid sequence such as SEQ ID NO: 3. See Table 1.

[0149] [Table 1-1]

[0150] [Table 1-2]

[0151] Nucleic acid sequences encoding parent polynucleotides described herein can be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a variant polypeptide is encoded by a nucleic acid comprising a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, 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 99%, or at least about 99.5% sequence identity to a reference nucleic acid sequence, e.g., the nucleic acid sequence encoding the parent polypeptide, e.g., SEQ ID NO: 1 or SEQ ID NO: 2. The percent identity between two such nucleic acid sequences can be determined manually by inspection of two optimally aligned nucleic acid sequences or by using software or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize under otherwise stringent conditions (e.g., within a range of moderate to high stringency) to a complementary sequence.

[0152] In some embodiments, a variant polypeptide is encoded by a nucleic acid sequence that has at least about 60%, at least about 65%, at least about 70%, at least about 75%, 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 99% or more sequence identity to a reference nucleic acid sequence, e.g., a nucleic acid sequence encoding a parent polypeptide, e.g., SEQ ID NO:1 or SEQ ID NO:2, but not 100% sequence identity.

[0153] In some embodiments, variant polypeptides of the disclosure comprise a polypeptide sequence that is 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but not 100%, to SEQ ID NO: 3. In some embodiments, variant polypeptides of the disclosure comprise a polypeptide sequence that is more than 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, but not 100% identical, to SEQ ID NO: 3.

[0154] In some embodiments, the present disclosure describes variant polypeptides that have a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., parent polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, but not 100% sequence identity. Homology or identity can be determined by amino acid sequence alignment, e.g., using programs such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0155] Also provided are variant polypeptides of the disclosure that have enzymatic activity, e.g., nuclease or endonuclease activity, and comprise an amino acid sequence that differs from the amino acid sequence of any one of the parent polypeptide and SEQ ID NO:3 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue (when aligned using any of the alignment methods described above).

[0156] In some embodiments, the variant polypeptide comprises an alteration at one or more (e.g., several) amino acids of the parent polypeptide, and at least 1, 2, 3, 4, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 , 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210 6, 157, 158, 159, 160, 161, 162, 162, 164, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, 198, 199, 200 or more are modified.

[0157] The modification may include substitution, insertion, deletion, addition, or fusion of amino acids in a peptide or polypeptide, or nucleotides in a nucleotide, compared to a reference sequence. No specific process is involved in the method for producing a sequence containing the modification. For example, a sequence containing the modification can be directly synthesized from individual nucleotides. In another embodiment, the modification is performed by providing a reference sequence and then modifying it.

[0158] Substitutions may include substitutions of amino acids with amino acids that differ from the reference sequence, or substitutions of nucleotides with nucleotides that differ from the reference sequence. No particular process is involved in creating a sequence containing substitutions. For example, a sequence containing substitutions can be directly synthesized from individual amino acids or nucleotides. In other embodiments, the modification is performed by providing a reference sequence and then modifying it. The nucleic acid sequence may be in the genome of an organism. The nucleic acid sequence may be in a cell. The nucleic acid sequence may be a DNA sequence. Nucleic acid substitutions as described herein refer to substitutions of up to several kilobases.

[0159] In some embodiments, the variant polypeptide comprises one or more of the amino acid substitutions listed in Table 2.

[0160] [Table 2-1]

[0161] [Table 2-2]

[0162] [Table 2-3]

[0163] [Table 2-4]

[0164] [Table 2-5]

[0165] [Table 2-6]

[0166] [Table 2-7]

[0167] [Table 2-8]

[0168] [Table 2-9]

[0169] [Table 2-10]

[0170] Some of the beneficial modifications described herein are contained in a hotspot located at approximately residues 500-580 of the nuclease polypeptide of SEQ ID NO: 3, near the RuvC "lid" that separates the TS-spacer helix and the ssDNA NTS. Without wishing to be bound by theory, in some embodiments, the substitutions described herein have one or more of the following benefits to enzyme function: stabilization of the ssDNA NTS in the active site groove; removal of H-bonding interactions with the TS-spacer helix; addition of favorable charge interactions with the TS-spacer helix; and removal of charge repulsion by the ssDNA TS proceeding from the spacer helix.

[0171] In some embodiments, the mutant polypeptide comprises a D509R amino acid substitution. In some embodiments, the mutant polypeptide comprises a S511R amino acid substitution. In some embodiments, the mutant polypeptide comprises a I521R amino acid substitution. In some embodiments, the mutant polypeptide comprises a D535G amino acid substitution. In some embodiments, the mutant polypeptide comprises a Q514G amino acid substitution. In some embodiments, the mutant polypeptide comprises a L516R amino acid substitution. In some embodiments, the mutant polypeptide comprises a L516G amino acid substitution. In some embodiments, the mutant polypeptide comprises an E198R amino acid substitution. In some embodiments, the mutant polypeptide comprises a S527R amino acid substitution. In some embodiments, the mutant polypeptide comprises a D509R amino acid substitution. In some embodiments, the mutant polypeptide comprises a D509G amino acid substitution. In some embodiments, the mutant polypeptide comprises a L580G amino acid substitution. In some embodiments, the mutant polypeptide comprises a V359R amino acid substitution. In some embodiments, the mutant polypeptide comprises a D535K amino acid substitution. In some embodiments, the mutant polypeptide comprises a Y381R amino acid substitution. In some embodiments, the mutant polypeptide comprises a R354G amino acid substitution. In some embodiments, the mutant polypeptide comprises a M380R amino acid substitution. In some embodiments, the mutant polypeptide comprises a M380R amino acid substitution. In some embodiments, the mutant polypeptide comprises a V383R amino acid substitution. In some embodiments, the mutant polypeptide comprises a L580R amino acid substitution. In some embodiments, the mutant polypeptide comprises an E367R amino acid substitution. In some embodiments, the mutant polypeptide comprises a I521K amino acid substitution. In some embodiments, the mutant polypeptide comprises an E367G amino acid substitution. In some embodiments, the mutant polypeptide comprises an E507R amino acid substitution. In some embodiments, the mutant polypeptide comprises a I521G amino acid substitution. In some embodiments, the mutant polypeptide comprises a W350R amino acid substitution.In some embodiments, the mutant polypeptide comprises a D535R amino acid substitution. In some embodiments, the mutant polypeptide comprises a R528K amino acid substitution. In some embodiments, the mutant polypeptide comprises a S527K amino acid substitution. In some embodiments, the mutant polypeptide comprises a L385G amino acid substitution. In some embodiments, the mutant polypeptide comprises a W350G amino acid substitution. In some embodiments, the mutant polypeptide comprises a L516K amino acid substitution. In some embodiments, the mutant polypeptide comprises a Y381G amino acid substitution. In some embodiments, the mutant polypeptide comprises a H532R amino acid substitution. In some embodiments, the mutant polypeptide comprises a D129R amino acid substitution. In some embodiments, the mutant polypeptide comprises a P615R amino acid substitution. In some embodiments, the mutant polypeptide comprises a G578R amino acid substitution. In some embodiments, the mutant polypeptide comprises a M380G amino acid substitution. In some embodiments, the mutant polypeptide comprises a V595G amino acid substitution. In some embodiments, the mutant polypeptide comprises a R531G amino acid substitution. In some embodiments, the mutant polypeptide comprises a D129G amino acid substitution. In some embodiments, the mutant polypeptide comprises a R531K amino acid substitution. In some embodiments, the mutant polypeptide comprises a D158G amino acid substitution. In some embodiments, the mutant polypeptide comprises a N476G amino acid substitution. In some embodiments, the mutant polypeptide comprises a G578K amino acid substitution. In some embodiments, the mutant polypeptide comprises an A512R amino acid substitution. In some embodiments, the mutant polypeptide comprises a P618R amino acid substitution. In some embodiments, the mutant polypeptide comprises a V595R amino acid substitution. In some embodiments, the mutant polypeptide comprises a V595K amino acid substitution. In some embodiments, the mutant polypeptide comprises a V383G amino acid substitution. In some embodiments, the mutant polypeptide comprises an A597G amino acid substitution. In some embodiments, the mutant polypeptide comprises a Y381K amino acid substitution.In some embodiments, the mutant polypeptide comprises a P618G amino acid substitution. In some embodiments, the mutant polypeptide comprises an E198K amino acid substitution. In some embodiments, the mutant polypeptide comprises a T288R amino acid substitution. In some embodiments, the mutant polypeptide comprises an E507K amino acid substitution. In some embodiments, the mutant polypeptide comprises an L385R amino acid substitution. In some embodiments, the mutant polypeptide comprises a C538G amino acid substitution. In some embodiments, the mutant polypeptide comprises a P615G amino acid substitution. In some embodiments, the mutant polypeptide comprises a D386R amino acid substitution. In some embodiments, the mutant polypeptide comprises a S511K amino acid substitution. In some embodiments, the mutant polypeptide comprises a C371G amino acid substitution. In some embodiments, the mutant polypeptide comprises a K136G amino acid substitution. In some embodiments, the mutant polypeptide comprises an N220R amino acid substitution. In some embodiments, the mutant polypeptide comprises a S78K amino acid substitution. In some embodiments, the mutant polypeptide comprises a K141G amino acid substitution. In some embodiments, the mutant polypeptide comprises a K240R amino acid substitution. In some embodiments, the mutant polypeptide comprises a D277R amino acid substitution. In some embodiments, the mutant polypeptide comprises a T165R amino acid substitution. In some embodiments, the mutant polypeptide comprises a K374R amino acid substitution.

[0172] In some embodiments, the variant polypeptide comprises a second modification relative to the amino acid sequence of SEQ ID NO:3. In some embodiments, the modification comprises L580G and the second modification comprises L385G. In some embodiments, the modification comprises L580G and the second modification comprises A512R. In some embodiments, the modification comprises S527R and the second modification comprises C538G. In some embodiments, the modification comprises D129R and the second modification comprises P618R. In some embodiments, the modification comprises Q514G and the second modification comprises A512R. In some embodiments, the modification comprises S527R and the second modification comprises L385G. In some embodiments, the modification comprises G578R and the second modification comprises D158G. In some embodiments, the modification comprises S511R and the second modification comprises P618R. In some embodiments, the modification comprises D158G and the second modification comprises A512R. In some embodiments, the modification comprises R354G and the second modification comprises A512R. In some embodiments, the modification comprises A512R and the second modification comprises P618R. In some embodiments, the modification comprises V359R and the second modification comprises A512R. In some embodiments, the modification comprises E367R and the second modification comprises A512R. In some embodiments, the modification comprises Q514G and the second modification comprises N476G. In some embodiments, the modification comprises S511R and the second modification comprises A512R. In some embodiments, the modification comprises E198R and the second modification comprises R354G. In some embodiments, the modification comprises Q514G and the second modification comprises C538G. In some embodiments, the modification comprises S527R and the second modification comprises T288R. In some embodiments, the modification comprises R354G and the second modification comprises D158G. In some embodiments, the modification comprises R354G and the second modification comprises L385G. In some embodiments, the modification comprises G578R and the second modification comprises A512R. In some embodiments, the modification comprises R354G and the second modification comprises E507R. In some embodiments, the modification comprises S527R and the second modification comprises G578R. In some embodiments, the modification comprises S511R and the second modification comprises C538G.In some embodiments, the modification comprises I521R and the second modification comprises V383R. In some embodiments, the modification comprises S511R and the second modification comprises I521R. In some embodiments, the modification comprises L385G and the second modification comprises G578R. In some embodiments, the modification comprises L580G and the second modification comprises N476G. In some embodiments, the modification comprises Q514G and the second modification comprises T288R. In some embodiments, the modification comprises D535G and the second modification comprises A512R. In some embodiments, the modification comprises D509R and the second modification comprises M380R. In some embodiments, the modification comprises S527R and the second modification comprises C371G. In some embodiments, the modification comprises Q514G and the second modification comprises D158G. In some embodiments, the modification comprises E198R and the second modification comprises C538G. In some embodiments, the modification comprises S511R and the second modification comprises L385G. In some embodiments, the modification comprises S527R and the second modification comprises N476G. In some embodiments, the modification comprises L516R and the second modification comprises A512R. In some embodiments, the modification comprises S511R and the second modification comprises C371G. In some embodiments, the modification comprises L516R and the second modification comprises C538G. In some embodiments, the modification comprises E198R and the second modification comprises C371G. In some embodiments, the modification comprises Q514G and the second modification comprises V383R. In some embodiments, the modification comprises L516R and the second modification comprises N476G. In some embodiments, the modification comprises D509R and the second modification comprises T288R. In some embodiments, the modification comprises D509R and the second modification comprises G578R. In some embodiments, the modification comprises D158G and the second modification comprises C371G. In some embodiments, the modification comprises I521R and the second modification comprises N476G. In some embodiments, the modification comprises E198R and the second modification comprises M380R. In some embodiments, the modification comprises D129R and the second modification comprises N476G. In some embodiments, the modification comprises M380R and the second modification comprises A512R.In some embodiments, the modification comprises R354G and the second modification comprises D129R. In some embodiments, the modification comprises S527R and the second modification comprises L580G. In some embodiments, the modification comprises R354G and the second modification comprises N476G. In some embodiments, the modification comprises Q514G and the second modification comprises P618R. In some embodiments, the modification comprises V383R and the second modification comprises L385G. In some embodiments, the modification comprises E198R and the second modification comprises V359R. In some embodiments, the modification comprises D535G and the second modification comprises N476G. In some embodiments, the modification comprises Q514G and the second modification comprises L385G. In some embodiments, the modification comprises S511R and the second modification comprises N476G. In some embodiments, the modification comprises D158G and the second modification comprises A597G. In some embodiments, the modification comprises L516R and the second modification comprises G578R. In some embodiments, the modification comprises E198R and the second modification comprises L580G. In some embodiments, the modification comprises E198R and the second modification comprises L385G. In some embodiments, the modification comprises L580G and the second modification comprises V383R. In some embodiments, the modification comprises I521R and the second modification comprises C371G. In some embodiments, the modification comprises V359R and the second modification comprises R354G. In some embodiments, the modification comprises Y381R and the second modification comprises G578R. In some embodiments, the modification comprises E198R and the second modification comprises V383R. In some embodiments, the modification comprises L580G and the second modification comprises D386R. In some embodiments, the modification comprises L385G and the second modification comprises P615R. In some embodiments, the modification comprises V383R and the second modification comprises G578R. In some embodiments, the modification comprises D535G and the second modification comprises T288R. In some embodiments, the modification comprises Y381R and the second modification comprises P618R. In some embodiments, the modification comprises A512R and the second modification comprises A597G. In some embodiments, the modification comprises Q514G and the second modification comprises L580G.In some embodiments, the modification comprises S527R and the second modification comprises P615R. In some embodiments, the modification comprises E198R and the second modification comprises P615R. In some embodiments, the modification comprises I521R and the second modification comprises T288R. In some embodiments, the modification comprises Y381R and the second modification comprises C538G. In some embodiments, the modification comprises S527R and the second modification comprises A512R. In some embodiments, the modification comprises Y381R and the second modification comprises C371G. In some embodiments, the modification comprises I521R and the second modification comprises L385G. In some embodiments, the modification comprises L516R and the second modification comprises T288R. In some embodiments, the modification comprises L516R and the second modification comprises D158G. In some embodiments, the modification comprises S527R and the second modification comprises P618R. In some embodiments, the modification comprises S527R and the second modification comprises R354G. In some embodiments, the modification comprises Y381R and the second modification comprises V383R. In some embodiments, the modification comprises A512R and the second modification comprises C371G. In some embodiments, the modification comprises S511R and the second modification comprises T288R. In some embodiments, the modification comprises S511R and the second modification comprises Q514G. In some embodiments, the modification comprises L516R and the second modification comprises V383R. In some embodiments, the modification comprises A512R and the second modification comprises C538G. In some embodiments, the modification comprises I521R and the second modification comprises M380R. In some embodiments, the modification comprises D535G and the second modification comprises C538G. In some embodiments, the modification comprises E198R and the second modification comprises T288R. In some embodiments, the modification comprises Q514G and the second modification comprises M380R. In some embodiments, the modification comprises S527R and the second modification comprises V383R. In some embodiments, the modification comprises L580G and the second modification comprises Y381R. In some embodiments, the modification comprises S511R and the second modification comprises G578R. In some embodiments, the modification comprises Y381R and the second modification comprises R354G.In some embodiments, the modification comprises L516R and the second modification comprises L385G. In some embodiments, the modification comprises D535G and the second modification comprises E198R. In some embodiments, the modification comprises S527R and the second modification comprises M380R. In some embodiments, the modification comprises L580G and the second modification comprises M380R. In some embodiments, the modification comprises N476G and the second modification comprises A597G. In some embodiments, the modification comprises I521R and the second modification comprises C538G. In some embodiments, the modification comprises R354G and the second modification comprises G578R. In some embodiments, the modification comprises S511R and the second modification comprises D386R. In some embodiments, the modification comprises P615R and the second modification comprises C538G. In some embodiments, the modification comprises L580G and the second modification comprises T288R. In some embodiments, the modification comprises E507R and the second modification comprises C538G. In some embodiments, the modification comprises E198R and the second modification comprises A597G. In some embodiments, the modification comprises P615R and the second modification comprises G578R. In some embodiments, the modification comprises S511R and the second modification comprises R354G. In some embodiments, the modification comprises D509R and the second modification comprises D386R. In some embodiments, the modification comprises S511R and the second modification comprises L580G. In some embodiments, the modification comprises E198R and the second modification comprises S527R. In some embodiments, the modification comprises V359R and the second modification comprises G578R. In some embodiments, the modification comprises I521R and the second modification comprises A512R. In some embodiments, the modification comprises D509R and the second modification comprises C538G. In some embodiments, the modification comprises L385G and the second modification comprises P618R. In some embodiments, the modification comprises P615R and the second modification comprises A512R. In some embodiments, the modification comprises V383R and the second modification comprises A512R. In some embodiments, the modification comprises L580G and the second modification comprises C538G. In some embodiments, the modification comprises G578R and the second modification comprises C538G.In some embodiments, the modification comprises D535G and the second modification comprises L385G. In some embodiments, the modification comprises L516R and the second modification comprises D386R. In some embodiments, the modification comprises L516R and the second modification comprises E507R. In some embodiments, the modification comprises S511R and the second modification comprises Y381R. In some embodiments, the modification comprises G578R and the second modification comprises N476G. In some embodiments, the modification comprises E507R and the second modification comprises C371G. In some embodiments, the modification comprises G578R and the second modification comprises T288R. In some embodiments, the modification comprises L385G and the second modification comprises A597G. In some embodiments, the modification comprises I521R and the second modification comprises D535G. In some embodiments, the modification comprises S527R and the second modification comprises A597G. In some embodiments, the modification comprises I521R and the second modification comprises E198R. In some embodiments, the modification comprises S511R and the second modification comprises E198R. In some embodiments, the modification comprises S511R and the second modification comprises M380R. In some embodiments, the modification comprises S511R and the second modification comprises E507R. In some embodiments, the modification comprises D129R and the second modification comprises A512R. In some embodiments, the modification comprises G578R and the second modification comprises C371G. In some embodiments, the modification comprises D509R and the second modification comprises L385G. In some embodiments, the modification comprises I521R and the second modification comprises P618R. In some embodiments, the modification comprises S511R and the second modification comprises V383R. In some embodiments, the modification comprises L385G and the second modification comprises A512R. In some embodiments, the modification comprises D509R and the second modification comprises P618R. In some embodiments, the modification comprises Y381R and the second modification comprises L385G. In some embodiments, the modification comprises E367R and the second modification comprises L385G. In some embodiments, the modification comprises Q514G and the second modification comprises D129R. In some embodiments, the modification comprises E198R and the second modification comprises E367R. In some embodiments, the modification comprises D129R and the second modification comprises C371G. In some embodiments, the modification comprises S527R and the second modification comprises D386R. In some embodiments, the modification comprises Q514G and the second modification comprises E507R. In some embodiments, the modification comprises V359R and the second modification comprises L385G. In some embodiments, the modification comprises M380R and the second modification comprises L385G. In some embodiments, the modification comprises Q514G and the second modification comprises R354G.In some embodiments, the modification comprises E198R and the second modification comprises Y381R. In some embodiments, the modification comprises M380R and the second modification comprises D129R. In some embodiments, the modification comprises L580G and the second modification comprises D129R. In some embodiments, the modification comprises D129R and the second modification comprises G578R. In some embodiments, the modification comprises S511R and the second modification comprises A597G. In some embodiments, the modification comprises D535G and the second modification comprises D158G. In some embodiments, the modification comprises N476G and the second modification comprises A512R. In some embodiments, the modification comprises Q514G and the second modification comprises L516R. In some embodiments, the modification comprises E507R and the second modification comprises P618R. In some embodiments, the modification comprises V383R and the second modification comprises D129R. In some embodiments, the modification comprises L385G and the second modification comprises C538G. In some embodiments, the modification comprises I521R and the second modification comprises D158G. In some embodiments, the modification comprises L516R and the second modification comprises L580G. In some embodiments, the modification comprises S527R and the second modification comprises D158G. In some embodiments, the modification comprises H532R and the second modification comprises A512R. In some embodiments, the modification comprises I521R and the second modification comprises R354G. In some embodiments, the modification comprises I521R and the second modification comprises Y381R. In some embodiments, the modification comprises D129R and the second modification comprises P615R. In some embodiments, the modification comprises V359R and the second modification comprises E507R. In some embodiments, the modification comprises E507R and the second modification comprises G578R. In some embodiments, the modification comprises L516R and the second modification comprises P618R. In some embodiments, the modification comprises R354G and the second modification comprises M380R. In some embodiments, the modification comprises R354G and the second modification comprises D386R. In some embodiments, the modification comprises I521R and the second modification comprises G578R. In some embodiments, the modification comprises Y381R and the second modification comprises A512R.In some embodiments, the modification comprises A512R and the second modification comprises D386R. In some embodiments, the modification comprises M380R and the second modification comprises G578R. In some embodiments, the modification comprises Y381R and the second modification comprises E507R. In some embodiments, the modification comprises L580G and the second modification comprises E507R. In some embodiments, the modification comprises V359R and the second modification comprises Y381R. In some embodiments, the modification comprises P615R and the second modification comprises N476G. In some embodiments, the modification comprises D158G and the second modification comprises N476G. In some embodiments, the modification comprises Y381R and the second modification comprises P615R. In some embodiments, the modification comprises V595G and the second modification comprises A512R. In some embodiments, the modification comprises V383R and the second modification comprises D158G. In some embodiments, the modification comprises E507R and the second modification comprises D158G. In some embodiments, the modification comprises V383R and the second modification comprises C538G. In some embodiments, the modification comprises V383R and the second modification comprises E507R. In some embodiments, the modification comprises E198R and the second modification comprises D386R. In some embodiments, the modification comprises D129R and the second modification comprises D386R. In some embodiments, the modification comprises M380R and the second modification comprises D158G. In some embodiments, the modification comprises E198R and the second modification comprises P618R. In some embodiments, the modification comprises Q514G and the second modification comprises E198R. In some embodiments, the modification comprises I521R and the second modification comprises S527R. In some embodiments, the modification comprises E198R and the second modification comprises A512R. In some embodiments, the modification comprises L385G and the second modification comprises C371G. In some embodiments, the modification comprises H532R and the second modification comprises C538G. In some embodiments, the modification comprises N476G and the second modification comprises C371G. In some embodiments, the modification comprises Y381R and the second modification comprises N476G. In some embodiments, the modification comprises M380R and the second modification comprises V383R.In some embodiments, the modification comprises H532R and the second modification comprises T288R. In some embodiments, the modification comprises Q514G and the second modification comprises D386R. In some embodiments, the modification comprises R354G and the second modification comprises T288R. In some embodiments, the modification comprises Y381R and the second modification comprises D158G. In some embodiments, the modification comprises M380R and the second modification comprises E507R. In some embodiments, the modification comprises P615R and the second modification comprises C371G. In some embodiments, the modification comprises S527R and the second modification comprises D129R. In some embodiments, the modification comprises M380R and the second modification comprises C538G. In some embodiments, the modification comprises L580G and the second modification comprises R354G. In some embodiments, the modification comprises L385G and the second modification comprises T288R. In some embodiments, the modification comprises L580G and the second modification comprises G578R. In some embodiments, the modification comprises E507R and the second modification comprises L385G. In some embodiments, the modification comprises E507R and the second modification comprises P615R. In some embodiments, the modification comprises E198R and the second modification comprises E507R. In some embodiments, the modification comprises S511R and the second modification comprises D158G. In some embodiments, the modification comprises G578R and the second modification comprises P618R. In some embodiments, the modification comprises D509R and the second modification comprises P615R. In some embodiments, the modification comprises D158G and the second modification comprises D386R. In some embodiments, the modification comprises I521R and the second modification comprises Q514G. In some embodiments, the modification comprises D535G and the second modification comprises D386R. In some embodiments, the modification comprises P615R and the second modification comprises D386R. In some embodiments, the modification comprises D535G and the second modification comprises G578R. In some embodiments, the modification comprises D535G and the second modification comprises S527R. In some embodiments, the modification comprises Y381R and the second modification comprises D386R. In some embodiments, the modification comprises D129R and the second modification comprises D158G.In some embodiments, the modification comprises Q514G and the second modification comprises G578R. In some embodiments, the modification comprises L516R and the second modification comprises D129R. In some embodiments, the modification comprises L385G and the second modification comprises D129R. In some embodiments, the modification comprises L385G and the second modification comprises D158G. In some embodiments, the modification comprises E198R and the second modification comprises H532R. In some embodiments, the modification comprises Q514G and the second modification comprises C371G. In some embodiments, the modification comprises G578R and the second modification comprises D386R. In some embodiments, the modification comprises R354G and the second modification comprises A597G. In some embodiments, the modification comprises Q514G and the second modification comprises A597G. In some embodiments, the modification comprises E367R and the second modification comprises E507R. In some embodiments, the modification comprises L580G and the second modification comprises C371G. In some embodiments, the modification comprises E367R and the second modification comprises G578R. In some embodiments, the modification comprises L516R and the second modification comprises M380R. In some embodiments, the modification comprises H532R and the second modification comprises N476G. In some embodiments, the modification comprises V359R and the second modification comprises D129R. In some embodiments, the modification comprises S511R and the second modification comprises P615R. In some embodiments, the modification comprises Q514G and the second modification comprises Y381R. In some embodiments, the modification comprises M380R and the second modification comprises N476G. In some embodiments, the modification comprises Q514G and the second modification comprises P615R. In some embodiments, the modification comprises D158G and the second modification comprises C538G. In some embodiments, the modification comprises Y381R and the second modification comprises E367R. In some embodiments, the modification comprises I521R and the second modification comprises E507R. In some embodiments, the modification comprises I521R and the second modification comprises L516R. In some embodiments, the modification comprises L385G and the second modification comprises N476G. In some embodiments, the modification comprises E198R and the second modification comprises N476G.In some embodiments, the modification comprises R531G and the second modification comprises T288R. In some embodiments, the modification comprises Y381R and the second modification comprises T288R. In some embodiments, the modification comprises Y381R and the second modification comprises V595G. In some embodiments, the modification comprises P615R and the second modification comprises D158G. In some embodiments, the modification comprises D509R and the second modification comprises E198R. In some embodiments, the modification comprises E507R and the second modification comprises V595G. In some embodiments, the modification. In some embodiments, the modification comprises L580G and the second modification comprises P615R. In some embodiments, the modification comprises D509R and the second modification comprises L580G. In some embodiments, the modification comprises V359R and the second modification comprises D158G. In some embodiments, the modification comprises S527R and the second modification comprises Y381R. In some embodiments, the modification comprises D509R and the second modification comprises C371G. In some embodiments, the modification comprises E198R and the second modification comprises G578R. In some embodiments, the modification comprises D535G and the second modification comprises D129R. In some embodiments, the modification comprises L516R and the second modification comprises P615R. In some embodiments, the modification comprises D129R and the second modification comprises T288R. In some embodiments, the modification comprises R354G and the second modification comprises V383R. In some embodiments, the modification comprises E507R and the second modification comprises D129R. In some embodiments, the modification comprises E507R and the second modification comprises A512R. In some embodiments, the modification comprises N476G and the second modification comprises C538G. In some embodiments, the modification comprises I521R and the second modification comprises L580G. In some embodiments, the modification comprises L516R and the second modification comprises C371G. In some embodiments, the modification comprises D535G and the second modification comprises L580G. In some embodiments, the modification comprises D535G and the second modification comprises E507R. In some embodiments, the modification comprises D129R and the second modification comprises C538G. In some embodiments, the modification comprises R354G and the second modification comprises C538G. In some embodiments, the modification comprises I521R and the second modification comprises D386R. In some embodiments, the modification comprises Y381R and the second modification comprises D129R. In some embodiments, the modification comprises V383R and the second modification comprises N476G. In some embodiments, the modification comprises M380R and the second modification comprises P615R. In some embodiments, the modification comprises E507R and the second modification comprises A597G. In some embodiments, the modification comprises S527R and the second modification comprises E507R. In some embodiments, the modification comprises I521R and the second modification comprises D129R.In some embodiments, the modification comprises Q514G and the second modification comprises V359R. In some embodiments, the modification comprises Y381R and the second modification comprises A597G. In some embodiments, the modification comprises S511R and the second modification comprises D535G. In some embodiments, the modification comprises L580G and the second modification comprises D158G. In some embodiments, the modification comprises D509R and the second modification comprises D158G. In some embodiments, the modification comprises E198R and the second modification comprises D129R. In some embodiments, the modification comprises L385G and the second modification comprises V595G. In some embodiments, the modification comprises P615R and the second modification comprises T288R. In some embodiments, the modification comprises M380R and the second modification comprises P618R. In some embodiments, the modification comprises V383R and the second modification comprises C371G. In some embodiments, the modification comprises D509R and the second modification comprises N476G. In some embodiments, the modification comprises M380R and the second modification comprises T288R. In some embodiments, the modification comprises S511R and the second modification comprises S527R. In some embodiments, the modification comprises D158G and the second modification comprises T288R. In some embodiments, the modification comprises Q514G and the second modification comprises S527R. In some embodiments, the modification comprises L516R and the second modification comprises Y381R. In some embodiments, the modification comprises R354G and the second modification comprises E367R. In some embodiments, the modification comprises A512R and the second modification comprises T288R. In some embodiments, the modification comprises V359R and the second modification comprises D386R. In some embodiments, the modification comprises S527R and the second modification comprises V595G. In some embodiments, the modification comprises D509R and the second modification comprises A512R. In some embodiments, the modification comprises D535G and the second modification comprises Q514G. In some embodiments, the modification comprises W350R and the second modification comprises N476G. In some embodiments, the modification comprises S511R and the second modification comprises D129R. In some embodiments, the modification comprises V359R and the second modification comprises P615R.In some embodiments, the modification comprises I521R and the second modification comprises P615R. In some embodiments, the modification comprises M380R and the second modification comprises A597G. In some embodiments, the modification comprises E367R and the second modification comprises D386R. In some embodiments, the modification comprises M380R and the second modification comprises D386R. In some embodiments, the modification comprises L516R and the second modification comprises S527R. In some embodiments, the modification comprises L385G and the second modification comprises D386R. In some embodiments, the modification comprises V383R and the second modification comprises T288R. In some embodiments, the modification comprises V359R and the second modification comprises M380R. In some embodiments, the modification comprises L385G and the second modification comprises H532R. In some embodiments, the modification comprises S511R and the second modification comprises V359R. In some embodiments, the modification comprises R354G and the second modification comprises P615R. In some embodiments, the modification comprises L516R and the second modification comprises A597G. In some embodiments, the modification comprises E507R and the second modification comprises D386R. In some embodiments, the modification comprises I521R and the second modification comprises A597G. In some embodiments, the modification comprises V595G and the second modification comprises D386R. In some embodiments, the modification comprises N476G and the second modification comprises P618R. In some embodiments, the modification comprises E367R and the second modification comprises D129R. In some embodiments, the modification comprises D535G and the second modification comprises M380R. In some embodiments, the modification comprises S527R and the second modification comprises H532R. In some embodiments, the modification comprises D509R and the second modification comprises I521R. In some embodiments, the modification comprises E198R and the second modification comprises V595G. In some embodiments, the modification comprises E198R and the second modification comprises D158G. In some embodiments, the modification comprises D509R and the second modification comprises A597G. In some embodiments, the modification comprises A597G and the second modification comprises T288R. In some embodiments, the modification comprises D509R and the second modification comprises D129R.In some embodiments, the modification comprises P615R and the second modification comprises A597G. In some embodiments, the modification comprises S511R and the second modification comprises E367R. In some embodiments, the modification comprises L516R and the second modification comprises E198R. In some embodiments, the modification comprises D535G and the second modification comprises A597G. In some embodiments, the modification comprises H532R and the second modification comprises D158G. In some embodiments, the modification comprises H532R and the second modification comprises D129R. In some embodiments, the modification comprises D535G and the second modification comprises Y381R. In some embodiments, the modification comprises E507R and the second modification comprises T288R. In some embodiments, the modification comprises H532R and the second modification comprises G578R. In some embodiments, the modification comprises D509R and the second modification comprises L516R. In some embodiments, the modification comprises G578R and the second modification comprises A597G. In some embodiments, the modification comprises L580G and the second modification comprises A597G. In some embodiments, the modification comprises R531G and the second modification comprises C371G. In some embodiments, the modification comprises D535G and the second modification comprises C371G. In some embodiments, the modification comprises E507R and the second modification comprises N476G. In some embodiments, the modification comprises L516R and the second modification comprises H532R. In some embodiments, the modification comprises V383R and the second modification comprises A597G. In some embodiments, the modification comprises G578R and the second modification comprises V595G. In some embodiments, the modification comprises D509R and the second modification comprises Y381R. In some embodiments, the modification comprises D158G and the second modification comprises P618R. In some embodiments, the modification comprises P618R and the second modification comprises C538G. In some embodiments, the modification comprises P618R and the second modification comprises C371G. In some embodiments, the modification comprises N476G and the second modification comprises D386R. In some embodiments, the modification comprises V359R and the second modification comprises P618R. In some embodiments, the modification comprises D509R and the second modification comprises Q514G.In some embodiments, the modification comprises H532R and the second modification comprises D386R. In some embodiments, the modification comprises L580G and the second modification comprises W350R. In some embodiments, the modification comprises S527R and the second modification comprises V359R. In some embodiments, the modification comprises T288R and the second modification comprises C371G. In some embodiments, the modification comprises I521R and the second modification comprises H532R. In some embodiments, the modification comprises W350R and the second modification comprises A512R. In some embodiments, the modification comprises V595G and the second modification comprises D158G. In some embodiments, the modification comprises Y381R and the second modification comprises M380R. In some embodiments, the modification comprises M380R and the second modification comprises R531G. In some embodiments, the modification comprises D535G and the second modification comprises L516R. In some embodiments, the modification comprises M380R and the second modification comprises C371G. In some embodiments, the modification comprises V595G and the second modification comprises C538G. In some embodiments, the modification comprises L516R and the second modification comprises R354G. In some embodiments, the modification comprises D129R and the second modification comprises V595G. In some embodiments, the modification comprises D509R and the second modification comprises S527R. In some embodiments, the modification comprises V383R and the second modification comprises P615R. In some embodiments, the modification comprises Y381R and the second modification comprises H532R. In some embodiments, the modification comprises D129R and the second modification comprises A597G. In some embodiments, the modification comprises L516R and the second modification comprises R531G. In some embodiments, the modification comprises D386R and the second modification comprises C371G. In some embodiments, the modification comprises H532R and the second modification comprises C371G. In some embodiments, the modification comprises W350R and the second modification comprises C538G. In some embodiments, the modification comprises V383R and the second modification comprises W350R. In some embodiments, the modification comprises P615R and the second modification comprises P618R. In some embodiments, the modification comprises S511R and the second modification comprises H532R.In some embodiments, the modification comprises R354G and the second modification comprises P618R. In some embodiments, the modification comprises G578R and the second modification comprises R531G. In some embodiments, the modification comprises E367R and the second modification comprises D158G. In some embodiments, the modification comprises V359R and the second modification comprises A597G. In some embodiments, the modification comprises L580G and the second modification comprises P618R. In some embodiments, the modification comprises I521R and the second modification comprises E367R. In some embodiments, the modification comprises V359R and the second modification comprises A597G. In some embodiments, the modification comprises L580G and the second modification comprises P618R. In some embodiments, the modification comprises I521R and the second modification comprises E367R. In some embodiments, the modification comprises E367R and the second modification comprises D158G ... In some embodiments, the modification comprises W350R. In some embodiments, the modification comprises W350R and the second modification comprises G578R. In some embodiments, the modification comprises Q514G and the second modification comprises E367R. In some embodiments, the modification comprises S511R and the second modification comprises V595G. In some embodiments, the modification comprises C538G and the second modification comprises D386R. In some embodiments, the modification comprises A597G and the second modification comprises D386R. In some embodiments, the modification comprises H532R and the second modification comprises P615R. In some embodiments, the modification comprises V359R and the second modification comprises V595G. In some embodiments, the modification comprises M380R and the second modification comprises H532R. In some embodiments, the modification comprises W350R and the second modification comprises P618R. In some embodiments, the modification comprises H532R and the second modification comprises P618R. In some embodiments, the modification comprises Q514G and the second modification comprises H532R. In some embodiments, the modification comprises L580G and the second modification comprises H532R. In some embodiments, the modification comprises W350R and the second modification comprises T288R. In some embodiments, the modification comprises D509R and the second modification comprises E507R. In some embodiments, the modification comprises P618R and the second modification comprises D386R. In some embodiments, the modification comprises E507R and the second modification comprises W350R. In some embodiments, the modification comprises A597G and the second modification comprises C538G. In some embodiments, the modification comprises S511R and the second modification comprises L516R. In some embodiments, the modification comprises W350R and the second modification comprises L385G. In some embodiments, the modification comprises W350R and the second modification comprises D158G. In some embodiments, the modification comprises P618R and the second modification comprises A597G. In some embodiments, the modification comprises Q514G and the second modification comprises V595G. In some embodiments, the modification comprises E198R and the second modification comprises W350R. In some embodiments, the modification comprises D509R and the second modification comprises V383R. In some embodiments, the modification comprises D509R and the second modification comprises R354G.

[0173] In some embodiments, the mutant polypeptide comprises D535G, L516R, and I521R substitutions. In some embodiments, the mutant polypeptide comprises D509R, L516R, and I521R substitutions. In some embodiments, the mutant polypeptide comprises D535G, L516R, and Q514G substitutions. In some embodiments, the mutant polypeptide comprises D535G, Q514G, and I521R substitutions. In some embodiments, the mutant polypeptide comprises Q514G, I521R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D535G, Q514G, I521R, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D535G, L516R, Q514G, and I521R substitutions. In some embodiments, the mutant polypeptide comprises Q514G, I521R, S527R, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D509R, D535G, L516R, Q514G, and I521R substitutions. In some embodiments, the mutant polypeptide comprises D535G, L516R, I521R, and S527R substitutions. In some embodiments, the mutant polypeptide comprises D535G, I521R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D509R, D535G, and L516R substitutions. In some embodiments, the mutant polypeptide comprises I521R, S527R, E198R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises D509R, L516R, and Q514G substitutions. In some embodiments, the mutant polypeptide comprises D509R, Q514G, and I521R substitutions. In some embodiments, the mutant polypeptide comprises D535G, L516R, Q514G, and S511R substitutions. In some embodiments, the mutant polypeptide comprises D535G, L516R, Q514G, and S527R substitutions. In some embodiments, the mutant polypeptide comprises I521R, S527R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D535G, L516R, S527R, and M380R substitutions.In some embodiments, the mutant polypeptide comprises Q514G, S527R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises L516R, Q514G, I521R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises D509R, L516R, Q514G, and S527R substitutions. In some embodiments, the mutant polypeptide comprises D509R, I521R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises S527R, L580G, E198R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises L516R, S527R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D509R, Q514G, I521R, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D535G, Q514G, I521R, and S527R substitutions. In some embodiments, the mutant polypeptide comprises Q514G, L580G, E198R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises D509R, L516R, I521R, and E198R substitutions. In some embodiments, the mutant polypeptide comprises L516R, Q514G, I521R, and L580G substitutions. In some embodiments, the mutant polypeptide comprises D509R, D535G, Q514G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises L516R, I521R, S527R, and L580G substitutions. In some embodiments, the mutant polypeptide comprises D535G, I521R, S527R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises L516R, I521R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D509R, D535G, and I521R substitutions. In some embodiments, the mutant polypeptide comprises D535G, S527R, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D509R, D535G, and Q514G substitutions.In some embodiments, the mutant polypeptide comprises S527R, L580G, E198R, and R354G substitutions. In some embodiments, the mutant polypeptide comprises Q514G, I521R, S527R, and L580G substitutions. In some embodiments, the mutant polypeptide comprises D535G, Q514G, E198R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises L516R, Q514G, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises I521R, S527R, E198R, and R354G substitutions. In some embodiments, the mutant polypeptide comprises I521R, S527R, R354G, and M380R substitutions. In some embodiments, the mutant polypeptide comprises I521R, L580G, E198R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises L516R, Q514G, S527R, and L580G substitutions. In some embodiments, the mutant polypeptide comprises D535G, Q514G, S527R, and E198R substitutions. In some embodiments, the mutant polypeptide comprises D509R, Q514G, L580G, and E198R substitutions. In some embodiments, the mutant polypeptide comprises K136G, N220R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises S78K, E198R, and R354G substitutions. In some embodiments, the mutant polypeptide comprises K141G, N220R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises K141G, K240R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises K141G, D277R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises K136G, D277R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises S78K, E198R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises L580G, L385G, S511R, and A512R substitutions. In some embodiments, the mutant polypeptide comprises S78K, E198R, and M380R substitutions.In some embodiments, the mutant polypeptide comprises K136G, K240R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises T165R, N220R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises L580G, L385G, Q514G, and A512R substitutions. In some embodiments, the mutant polypeptide comprises D129R, P618R, S511R, and A512R substitutions. In some embodiments, the mutant polypeptide comprises S78K, E198R, and K374R substitutions. In some embodiments, the mutant polypeptide comprises T165R, D277R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises L580G, L385G, G578R, and D158G substitutions. In some embodiments, the mutant polypeptide comprises D129R, P618R, R354G, and A512R substitutions. In some embodiments, the mutant polypeptide comprises T165R, K240R, and M380R substitutions. In some embodiments, the mutant polypeptide comprises K141G, K240R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises K136G, N220R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises D129R, P618R, Q514G, and A512R substitutions. In some embodiments, the mutant polypeptide comprises D129R, P618R, S527R, and L385G substitutions. In some embodiments, the mutant polypeptide comprises K141G, N220R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises L580G, L385G, Q514G, and N476G substitutions. In some embodiments, the mutant polypeptide comprises L580G, L385G, A512R, and P618R substitutions. In some embodiments, the mutant polypeptide comprises K141G, D277R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises D129R, P618R, D158G, and A512R substitutions. In some embodiments, the mutant polypeptide comprises K136G, N220R, and K374R substitutions.In some embodiments, the mutant polypeptide comprises K136G, D277R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises K136G, K240R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises L580G, L385G, S527R, and C538G substitutions. In some embodiments, the mutant polypeptide comprises T165R, K240R, and L385R substitutions. In some embodiments, the mutant polypeptide comprises D129R, P618R, V359R, and A512R substitutions.

[0174] In some embodiments, the mutant polypeptide comprises a modification that increases the interaction of the mutant polypeptide with an RNA guide. In some embodiments, the modification that increases the interaction with an RNA guide is an arginine, lysine, glutamine, asparagine, or histidine substitution. In some embodiments, the mutant polypeptide comprises a modification that increases the interaction of the mutant polypeptide with a target nucleic acid. In some embodiments, the modification that increases the interaction with a target nucleic acid is an arginine, lysine, glutamine, asparagine, or histidine substitution. In some embodiments, the mutant polypeptide comprises an alanine substitution. In some embodiments, the alanine substitution does not affect the geometry of the mutant polypeptide backbone. In some embodiments, the mutant polypeptide comprises a glycine substitution. In some embodiments, the glycine substitution changes the Ramachandran bond angle of the mutant polypeptide backbone.

[0175] In some embodiments, a variant polypeptide comprises at least one RuvC motif or RuvC domain. As used herein, a "biologically active portion" is a portion that retains (e.g., completely, partially, minimally) at least one function of a parent polypeptide (e.g., a "minimal" or "core" domain). In some embodiments, a variant polypeptide retains enzymatic activity at least as active as the parent polypeptide. Thus, in some embodiments, a variant polypeptide has enzymatic activity that exceeds that of the parent polypeptide.

[0176] In some embodiments, the mutant polypeptide has reduced or no nuclease activity. As used herein, catalytic residues of the polypeptides disclosed herein include D345, E506, and D594. In some embodiments, a mutant polypeptide comprising a substitution at D345 and / or E506 and / or D594 (e.g., D345A and / or E506A and / or D594A) exhibits reduced or no nuclease activity compared to the parent polypeptide. In some embodiments, residue R593 contributes to nuclease activity. In some embodiments, a substitution at R593 alters nuclease activity, e.g., results in reduced nuclease activity.

[0177] In some embodiments, variant polypeptides of the disclosure have enzymatic activity that is equal to or greater than that of the parent polypeptide. In some embodiments, variant polypeptides of the disclosure have enzymatic activity in a temperature range of about 20° C. to about 90° C. In some embodiments, variant polypeptides of the disclosure have enzymatic activity at a temperature of about 20° C. to about 25° C., or at a temperature of about 37° C.

[0178] In some embodiments, the variant polypeptide comprises at least one modification that results in enhanced affinity for RNA (e.g., RNA affinity) compared to the parent polypeptide. In some embodiments, the variant polypeptide exhibits enhanced RNA affinity compared to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits enhanced RNA affinity compared to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant polypeptide comprises a T m The value is the T of the parent polypeptide. m In one embodiment, the variant polypeptide exhibits enhanced RNA affinity when the T of the variant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the variant polypeptide. m The value is the T of the parent polypeptide. m A value at least 8°C greater than this indicates enhanced RNA affinity.

[0179] In some embodiments, the variant polypeptide comprises at least one modification that results in enhanced complex formation (e.g., binary complex formation) with an RNA guide relative to the parent polypeptide. In some embodiments, the variant polypeptide exhibits enhanced binary complex formation relative to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits enhanced binary complex formation compared to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the variant polypeptide exhibits enhanced binary complex formation compared to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6. m The value is the T of the parent polypeptide. m In one embodiment, the variant polypeptide exhibits enhanced binary complex formation when the T of the variant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the variant polypeptide. m The value is the T of the parent polypeptide. m A value at least 8°C greater than this indicates enhanced binary complex formation.

[0180] In some embodiments, the variant polypeptide comprises at least one modification that enhances its RNA guide binding activity relative to the parent polypeptide. In some embodiments, the variant polypeptide exhibits enhanced RNA guide binding activity relative to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits enhanced RNA guide binding activity relative to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant polypeptide comprises a T m The value is the T of the parent polypeptide. m In one embodiment, the variant polypeptide exhibits enhanced RNA guide binding activity compared to the parent polypeptide if the T of the variant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the variant polypeptide. m The value is the T of the parent polypeptide. m A value at least 8°C greater than this indicates enhanced RNA guide binding activity.

[0181] In some embodiments, the variant polypeptide comprises at least one modification that enhances binding specificity for an RNA guide relative to the parent polypeptide. In some embodiments, the variant polypeptide exhibits enhanced RNA guide binding specificity relative to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits enhanced RNA guide binding specificity relative to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant polypeptide comprises a T m The value is the T of the parent polypeptide. m In one embodiment, the variant polypeptide exhibits enhanced RNA guide binding specificity when the T of the variant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the variant polypeptide. m The value is the T of the parent polypeptide. m A value at least 8°C greater than the ΔΨ value indicates enhanced RNA guide binding specificity.

[0182] In some embodiments, the variant polypeptide comprises at least one modification that enhances protein-RNA interaction compared to the parent polypeptide. In some embodiments, the variant polypeptide exhibits enhanced protein-RNA interaction compared to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits enhanced protein-RNA interaction compared to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant polypeptide comprises a T m The value is the T of the parent polypeptide. m A mutant polypeptide exhibits enhanced protein-RNA interaction when compared to the parent polypeptide if the T of the mutant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the mutant polypeptide. m The value is the T of the parent polypeptide. m A value at least 8°C greater than this indicates an enhancement of the protein-RNA interaction.

[0183] In some embodiments, the variant polypeptide comprises at least one modification that enhances protein stability relative to the parent polypeptide. In some embodiments, the variant polypeptide exhibits enhanced protein stability relative to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits enhanced protein stability relative to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6 ... m The value is the T of the parent polypeptide. m In one embodiment, the variant polypeptide exhibits enhanced protein stability when the T of the variant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the variant polypeptide. m The value is the T of the parent polypeptide. m A value of at least 8°C greater than this indicates enhanced protein stability.

[0184] In some embodiments, the variant polypeptide comprises at least one modification that reduces dissociation from the RNA guide (e.g., binary complex dissociation) compared to the parent polypeptide. In some embodiments, the variant polypeptide exhibits reduced dissociation from the RNA guide compared to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits reduced dissociation from the RNA guide in a buffer having a pH in the range of about 7.3 to about 8.6, as compared to the parent polypeptide. In some embodiments, the variant polypeptide exhibits reduced dissociation from the RNA guide in a buffer having a pH in the range of about 7.3 to about 8.6, as compared to the parent polypeptide. m The value is the T of the parent polypeptide. m In one embodiment, the variant polypeptide exhibits reduced dissociation from the RNA guide when the T of the variant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C higher than the T of the variant polypeptide. m The value is the T m A mutant polypeptide exhibits reduced dissociation from the RNA guide when its temperature is at least 8°C higher than a value of 0.05°C. In some embodiments, the mutant polypeptide exhibits reduced dissociation from the RNA guide compared to the parent polypeptide over an incubation period of at least about any one of 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more. In some embodiments, the mutant CRISPR nuclease ribonucleoprotein (RNP) complex does not exchange the RNA guide for a different RNA.

[0185] In some embodiments, the variant polypeptide comprises at least one modification that enhances the formation of a ternary complex between the RNA guide and the target nucleic acid compared to the parent polypeptide. In some embodiments, the variant polypeptide exhibits enhanced ternary complex formation compared to the parent polypeptide at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the variant polypeptide exhibits enhanced ternary complex formation compared to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant polypeptide comprises a T m The value is the T of the parent polypeptide. m In one embodiment, the variant polypeptide exhibits enhanced ternary complex formation when the T of the variant polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the variant polypeptide. m The value is the T of the parent polypeptide. m A value at least 8°C greater than this indicates enhanced ternary complex formation.

[0186] In some embodiments, the variant polypeptide comprises at least one modification such that a binary complex (e.g., the variant binary complex) comprising the variant polypeptide exhibits enhanced binding affinity for a target nucleic acid when compared to the parent binary complex. In some embodiments, the variant binary complex exhibits enhanced binding affinity for a target nucleic acid when compared to the parent binary complex at temperatures below any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the mutant binary complex exhibits enhanced binding affinity for the target nucleic acid compared to the parent binary complex in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant binary complex exhibits enhanced binding affinity for the target nucleic acid compared to the parent binary complex. m The value is the T of the parent binary complex m In one embodiment, the mutant binary complex exhibits enhanced binding affinity for the target nucleic acid when the T of the mutant binary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the mutant binary complex. m The value is the T of the parent binary complex m A value at least 8°C greater than this indicates an enhancement of binding affinity to the parent nucleic acid.

[0187] In some embodiments, the mutant polypeptide comprises at least one modification such that a binary complex (e.g., mutant binary complex) comprising the mutant polypeptide exhibits enhanced on-target binding activity compared to the parent binary complex. In some embodiments, the mutant binary complex exhibits enhanced on-target binding activity compared to the parent binary complex at temperatures below any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the mutant binary complex exhibits enhanced on-target binding activity compared to the parent binary complex in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant binary complex exhibits enhanced on-target binding activity compared to the parent binary complex. m The value is the T of the parent binary complex m In one embodiment, the mutant binary complex exhibits enhanced on-target binding activity when the T of the mutant binary complex is at least 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., or 20° C. greater than the T of the mutant binary complex. m The value is the T of the parent binary complex m A value at least 8°C greater than this indicates enhanced on-target binding activity.

[0188] In some embodiments, the mutant polypeptide comprises at least one modification such that a binary complex (e.g., mutant binary complex) comprising the mutant polypeptide exhibits enhanced on-target binding specificity when compared to the parent binary complex. In some embodiments, the mutant binary complex exhibits enhanced on-target binding specificity when compared to the parent binary complex at temperatures below about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the mutant binary complex exhibits enhanced on-target binding specificity compared to the parent binary complex in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant binary complex exhibits enhanced on-target binding specificity compared to the parent binary complex. m The value is the T of the parent binary complex m In one embodiment, the mutant binary complex exhibits enhanced on-target binding specificity when the T of the mutant binary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the mutant binary complex. m The value is the T of the parent binary complex m A value at least 8°C greater than this indicates enhanced on-target binding specificity.

[0189] In some embodiments, the mutant polypeptide comprises at least one modification such that a binary complex (e.g., mutant binary complex) comprising the mutant polypeptide exhibits reduced off-target binding to non-target nucleic acids when compared to the parent binary complex. In some embodiments, the mutant binary complex exhibits reduced off-target binding to non-target nucleic acids when compared to the parent binary complex at temperatures below any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the mutant binary complex exhibits reduced off-target binding to non-target nucleic acids compared to the parent binary complex in a buffer having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant binary complex exhibits reduced off-target binding to non-target nucleic acids compared to the parent binary complex in a buffer having a pH in the range of about 7.3 to about 8.6. m The value is the T of the parent polypeptide. m In one embodiment, the mutant binary complex exhibits reduced off-target binding to non-target nucleic acids when the T of the mutant binary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the mutant binary complex. m The value is the T of the parent polypeptide. m A value at least 8°C greater than this indicates reduced off-target binding to non-target nucleic acids.

[0190] In some embodiments, the mutant polypeptide comprises at least one modification such that a binary complex (e.g., mutant binary complex) comprising the mutant polypeptide exhibits reduced dissociation from a target nucleic acid compared to the parent binary complex. In some embodiments, the mutant binary complex exhibits reduced dissociation from a target nucleic acid compared to the parent binary complex at temperatures below any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the mutant binary complex exhibits reduced dissociation from the target nucleic acid in a buffer having a pH in the range of about 7.3 to about 8.6, as compared to the parent binary complex. In some embodiments, the mutant binary complex exhibits reduced dissociation from the target nucleic acid in a buffer having a pH in the range of about 7.3 to about 8.6, as compared to the parent binary complex. m The value is the T of the parent polypeptide. m In one embodiment, the mutant binary complex exhibits reduced dissociation from the target nucleic acid when the T of the mutant binary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C higher than the T of the mutant binary complex. m The value is the T of the parent polypeptide. m A temperature at least 8°C higher than this value indicates a decrease in dissociation from the target nucleic acid.

[0191] In some embodiments, the variant polypeptide comprises at least one modification such that a ternary complex (e.g., variant ternary complex) comprising the variant polypeptide exhibits enhanced stability compared to the parent ternary complex. In some embodiments, the variant ternary complex exhibits enhanced stability compared to the parent ternary complex at temperatures below any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C. In some embodiments, the mutant ternary complex exhibits enhanced stability compared to the parent ternary complex in buffers having a pH in the range of about 7.3 to about 8.6. In some embodiments, the mutant ternary complex exhibits enhanced stability compared to the parent ternary complex. m The value is the T of the parent ternary complex m In one embodiment, the mutant ternary complex exhibits enhanced stability when the T of the mutant ternary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than the T of the mutant ternary complex. m The value is the T of the parent ternary complex m A value of at least 8°C greater than this indicates enhanced stability.

[0192] While the changes described herein can be the change of one or more amino acids, changes to the variant polypeptides can also be of a substantial nature, such as the fusion of a polypeptide as an amino-terminal extension and / or a carboxyl-terminal extension. For example, the variant polypeptides can contain additional peptides, such as one or more peptides. Examples of additional peptides can include epitope peptides for labeling, such as polyhistidine tags (His tags), Myc, and FLAG. In some embodiments, the variant polypeptides described herein can be fused to a detectable moiety, such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).

[0193] In some embodiments, the variant polypeptide comprises at least one (e.g., 2, 3, 4, 5, 6, or more) nuclear localization signal (NLS). In some embodiments, the variant polypeptide comprises at least one (e.g., 2, 3, 4, 5, 6, or more) nuclear export signal (NES). In some embodiments, the variant polypeptide comprises at least one (e.g., 2, 3, 4, 5, 6, or more) NLS and at least one (e.g., 2, 3, 4, 5, 6, or more) NES.

[0194] In some embodiments, the mutant polypeptides described herein are capable of self-inactivating. See Epstein et al., "Engineering a Self-Inactivating CRISPR System for AAV Vectors," Mol. Ther., 24(2016):S50, which is incorporated herein by reference in its entirety.

[0195] In some embodiments, nucleotide sequences encoding the variant polypeptides described herein may be codon-optimized for use in a particular host cell or organism, for example, the nucleic acid may be codon-optimized for any non-human eukaryote, including mouse, rat, rabbit, dog, livestock, or non-human primate. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www.kazusa.orjp / Codon / , and these tables can be adapted in many ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, PA).

[0196] RNA guide In some embodiments, the compositions or complexes described herein comprise a targeting moiety (e.g., an RNA guide, an antisense, an oligonucleotide, a peptide-oligonucleotide conjugate) that binds to a target nucleic acid and interacts with a mutant polypeptide. The targeting moiety is capable of binding to the target nucleic acid (e.g., with specific binding affinity for the target nucleic acid).

[0197] In some embodiments, the targeting moiety comprises or is an RNA guide. In some embodiments, the RNA guide targets a variant polypeptide described herein to a specific nucleic acid sequence. Those skilled in the art will understand from reading the examples of specific types of RNA guides below that in some embodiments the RNA guide is site-specific. That is, in some embodiments, the RNA guide specifically associates with one or more target nucleic acid sequences (e.g., specific DNA or genomic DNA sequences) but does not associate with non-target nucleic acid sequences (e.g., non-specific DNA or random sequences).

[0198] In some embodiments, the compositions described herein comprise an RNA guide that associates with a variant polypeptide described herein and directs the variant polypeptide to a target nucleic acid sequence (e.g., DNA). In some embodiments, the RNA guide can associate with a polypeptide described herein (e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 3, or a variant thereof). In some embodiments, the RNA guide directs the polypeptide to a target nucleic acid sequence (e.g., DNA).

[0199] The RNA guide can target (e.g., associate with, be directed to, contact, or bind to) one or more nucleotides of a target sequence, e.g., a site-specific sequence or site-specific target. In some embodiments, the mutant ribonucleoprotein (e.g., a mutant CRISPR nuclease polypeptide plus an RNA guide) is activated upon binding to a target nucleic acid (e.g., a sequence-specific substrate or target nucleic acid) that is complementary to a DNA-targeting sequence in the RNA guide.

[0200] In some embodiments, the spacer or spacer sequence is part of an RNA guide that is the RNA equivalent of a target sequence (DNA sequence). Typically, the spacer contains a sequence that can bind to the non-PAM strand by base pairing at a site complementary to the target sequence (in the PAM strand). In some examples, the spacer can be at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the target sequence, where T is considered equivalent to U for purposes of this comparison. In some examples, the spacer can be 100% identical to the target sequence, where T is considered equivalent to U for purposes of this comparison.

[0201] In some examples, when a first polynucleotide (e.g., a spacer sequence of an RNA guide) has a certain level of complementarity to a second polynucleotide (e.g., a complementary sequence of a target sequence), the polynucleotide is complementary to another polynucleotide, and the first and second polynucleotides may base pair to form a double-stranded complex, allowing an effector polypeptide complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide may be substantially complementary to the second polynucleotide. In some embodiments, the first polynucleotide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide. In some embodiments, the first polynucleotide is fully complementary to the second polynucleotide, i.e., has 100% complementarity to the second polynucleotide.

[0202] In some embodiments, the RNA guide comprises a spacer having a length of about 11 nucleotides to about 100 nucleotides. For example, the DNA-targeting segment can have a length of about 11 nucleotides to about 80 nucleotides, about 11 nucleotides to about 50 nucleotides, about 11 nucleotides to about 40 nucleotides, about 11 nucleotides to about 30 nucleotides, about 11 nucleotides to about 25 nucleotides, about 11 nucleotides to about 20 nucleotides, or about 11 nucleotides to about 19 nucleotides. For example, the spacer can have a length of about 19 nucleotides to about 20 nucleotides, about 19 nucleotides to about 25 nucleotides, about 19 nucleotides to about 30 nucleotides, about 19 nucleotides to about 35 nucleotides, about 19 nucleotides to about 40 nucleotides, about 19 nucleotides to about 45 nucleotides, about 19 nucleotides to about 50 nucleotides, about 19 nucleotides to about 60 nucleotides, about 19 nucleotides to about 70 nucleotides, about 19 nucleotides to about 80 nucleotides, about 19 nucleotides to about 90 nucleotides, about 19 nucleotides to about 100 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 35 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 45 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 90 nucleotides, or about 20 nucleotides to about 100 nucleotides.

[0203] In some embodiments, the RNA guide generally has a length of 11 to 50 nucleotides (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides) and can be designed to be complementary to a specific target nucleic acid sequence. In some particular embodiments, the RNA guide can be designed to be complementary to a specific DNA strand of, for example, a genomic locus. In some embodiments, a DNA targeting sequence is designed to be complementary to a specific DNA strand of, for example, a genomic locus.

[0204] The RNA guide may be substantially identical to the complementary strand of a reference nucleic acid sequence. In some embodiments, the RNA guide comprises a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, 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 99%, or at least about 99.5% sequence identity to the complementary strand of a reference nucleic acid sequence, e.g., a target nucleic acid. The percent identity between two such nucleic acids can be determined manually by inspection of two optimally aligned nucleic acid sequences or by using software or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.

[0205] In some embodiments, the RNA guide has at least about 60%, at least about 65%, at least about 70%, at least about 75%, 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 99%, or at least about 99.5% sequence identity to the complementary strand of the target nucleic acid.

[0206] In some embodiments, the RNA guide comprises a spacer that is 11-50 nucleotides in length (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides) and is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target nucleic acid. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target DNA sequence. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target genomic sequence. In some embodiments, the RNA guide comprises an RNA sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target nucleic acid, e.g., up to 50 nucleotides in length. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target DNA sequence. In some embodiments, the RNA guide comprises a sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target genomic sequence.

[0207] In certain embodiments, the RNA guide comprises, consists essentially of, or consists of a direct repeat sequence linked to a DNA targeting sequence. In some embodiments, the RNA guide comprises a direct repeat sequence and a DNA targeting sequence, or a direct repeat-DNA targeting sequence-direct repeat sequence. In some embodiments, the RNA guide comprises a truncated direct repeat sequence and a DNA targeting sequence typical of processed or mature crRNA. In some embodiments, the polypeptides described herein form a complex with the RNA guide, and the RNA guide directs the complex to associate with a site-specific target nucleic acid that is complementary to at least a portion of the RNA guide.

[0208] In some embodiments, the direct repeat sequence is at least 90% identical to a sequence set forth in Table 3 or a portion of a sequence set forth in Table 3. In some embodiments, the direct repeat sequence is at least 95% (e.g., at least 97%, at least 99%, or at least 100%) identical to a sequence set forth in Table 3 or a portion of a sequence set forth in Table 3. In some embodiments, the direct repeat sequence is identical to a sequence set forth in Table 3 or a portion of a sequence set forth in Table 3. In some embodiments, the direct repeat sequence comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 4-6.

[0209] [Table 3]

[0210] In some embodiments, the direct repeat sequence comprises a sequence having at least 90% identity to UGUGGUUCCGAC (SEQ ID NO: 7). In some embodiments, the direct repeat sequence comprises the sequence set forth in SEQ ID NO: 7.

[0211] In some embodiments, a PAM corresponding to a mutant polypeptide of the disclosure comprises 5'-NTN-3', 5'-HTN-3', or 5'-TNA-3'. As used herein, each N can be any nucleotide (e.g., A, G, T, or C) or a subset thereof (e.g., R (A or G), Y (C or T), K (G or T), B (G, T, or C), H (A, C, or T). In some embodiments, a PAM comprises 5'-CTG-3'. In some embodiments, a PAM comprises 5'-CTG-3'. In some embodiments, a binary complex comprising a mutant polypeptide of the disclosure binds to a target nucleic acid adjacent to a 5'-NTN-3', 5'-HTN-3', or 5'-TNA-3' sequence. In some embodiments, a binary complex comprising a mutant polypeptide of the disclosure binds to a target nucleic acid adjacent to a 5'-CTG-3' or 5'-CTC-3' sequence.

[0212] In some embodiments, a composition or complex described herein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) RNA guides, e.g., a plurality of RNA guides.

[0213] In some embodiments, the RNA guide has a structure similar to the RNA guides of WO 2014 / 093622 and WO 2015 / 070083 (the entire contents of each of which are incorporated herein by reference).

[0214] Unless otherwise specified, all compositions and complexes, and polypeptides provided herein are made with reference to the activity level of the composition or complex or polypeptide and do not include impurities, such as residual solvents or by-products, that may be present in commercially available sources. The weight of the enzyme component is based on total active protein. All percentages and ratios are calculated by weight unless otherwise specified. All percentages and ratios are calculated based on the total composition unless otherwise specified. In the exemplified compositions, enzyme levels are expressed by pure enzyme by weight of the total composition, and ingredients are expressed by weight of the total composition unless otherwise specified.

[0215] qualification Either the nucleic acid sequence encoding the RNA guide or the variant polypeptide may contain one or more covalent modifications relative to the reference sequence, particularly the parent polyribonucleotide, and this is included within the scope of the present disclosure.

[0216] Exemplary modifications can include, for example, modifications to any of the sugars, nucleobases, internucleoside linkages (e.g., to the phosphate linkage / to the phosphodiester linkage / to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.

[0217] Any nucleic acid sequence encoding a component of an RNA guide or variant polypeptide can include any useful modifications, for example, to the sugar, nucleobase, internucleoside linkage (e.g., to the phosphate linkage / phosphodiester linkage / phosphodiester backbone), and any combination thereof. One or more atoms of a pyrimidine nucleobase can be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of the sugar and the internucleoside linkage. The modification can be to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Additional modifications are described herein.

[0218] In some embodiments, the modification can include a chemical modification or a cell-induced modification. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions" in Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0219] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) may be present at various positions in the sequence. One of skill in the art will understand that nucleotide analogs or other modifications may be placed at any position in the sequence without substantially diminishing the function of the sequence. The sequence may contain from about 1% to about 100% modified nucleotides (either with respect to the overall nucleotide content or with respect to one or more types of nucleotides, i.e., A, G, U, or C), or any percentage therebetween (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100% modified nucleotides.

[0220] In some embodiments, sugar modifications (e.g., at the 2' or 4' position) or sugar replacements in one or more ribonucleotides of the sequence, as well as backbone modifications, may include modifications or replacements of phosphodiester linkages. Specific examples of sequences include sequences containing modified backbones or non-natural internucleoside linkages, such as internucleoside modifications, including, but not limited to, modifications or replacements of phosphodiester linkages. Sequences with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes of this application, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In certain embodiments, the sequence will include ribonucleotides that have a phosphorus atom in their internucleoside backbone.

[0221] Modified sequence backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments, the sequences may be negatively or positively charged.

[0222] Modified nucleotides that can be incorporated into a sequence can be modified at the internucleoside linkage (e.g., the phosphate backbone). Thus, the terms "phosphate" and "phosphodiester" are used interchangeably with respect to polynucleotide backbones. The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can contain large-scale replacement of unmodified phosphate moieties with alternative internucleoside linkages as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced by sulfur. Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0223] The α-thio-substituted phosphate moieties are provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease accessibility and subsequently have longer half-lives in the intracellular environment.

[0224] In specific embodiments, the modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine).

[0225] Other internucleoside linkages that can be utilized in accordance with the present disclosure are described herein, including internucleoside linkages that do not contain a phosphorus atom.

[0226] In some embodiments, the sequence may include one or more cytotoxic nucleosides, for example, cytotoxic nucleosides may be incorporated into the sequence as a bifunctional modification. Cytotoxic nucleosides can include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenesidicine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidate).

[0227] In some embodiments, the sequence comprises one or more post-translational modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequences, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol and thiosine residues). The one or more post-translational modifications can be any post-transcriptional modification, such as any of the over 100 different nucleoside modifications identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27:196-197). In some embodiments, the initially isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA is selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5- The compound comprises at least one nucleoside selected from the group consisting of methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.In some embodiments, the mRNA is selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4 2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA is selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)ade ... and 2-methoxy-adenine.In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0228] The sequence may be uniformly modified or unmodified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines, or pyrimidines, or any one or more or all of A, G, U, C, I, pU) may be uniformly modified or unmodified in the sequence or in a given sequence region thereof. In some embodiments, the sequence contains pseudouridine. In some embodiments, the sequence contains inosine, which helps the immune system characterize the sequence as endogenous RNA versus viral RNA. Incorporation of inosine may also mediate improved RNA stability / reduced degradation. See, e.g., Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as "self". Cell Res. 25, 1283-1284, incorporated by reference in its entirety.

[0229] target nucleic acid The methods disclosed herein are applicable to a variety of target nucleic acids. In some embodiments, the target nucleic acid is DNA, such as a DNA locus. In some embodiments, the target nucleic acid is RNA, such as an RNA locus or mRNA. In some embodiments, the target nucleic acid is single-stranded (e.g., single-stranded DNA). In some embodiments, the target nucleic acid is double-stranded (e.g., double-stranded DNA). In some embodiments, the target nucleic acid contains both single-stranded and double-stranded regions. In some embodiments, the target nucleic acid is linear. In some embodiments, the target nucleic acid is circular. In some embodiments, the target nucleic acid contains one or more modified nucleotides, such as methylated nucleotides, damaged nucleotides, or nucleotide analogs. In some embodiments, the target nucleic acid is unmodified.

[0230] The target nucleic acid may be of any length, for example, at least one of about 100 bp, 200 bp, 500 bp, 1000 bp, 2000 bp, 5000 bp, 10 kb, 20 kb, 50 kb, 100 kb, 200 kb, 500 kb, 1 Mb, or more. The target nucleic acid may also comprise any sequence. In some embodiments, the target nucleic acid is GC-rich, for example, having a GC content of at least about 40%, 45%, 50%, 55%, 60%, 65%, or more. In some embodiments, the target nucleic acid has a GC content of at least about 70%, 80%, or more. In some embodiments, the target nucleic acid is a GC-rich fragment of a non-GC-rich target nucleic acid. In some embodiments, the target nucleic acid is not GC-rich. In some embodiments, the target nucleic acid has one or more secondary or higher order structures. In some embodiments, the target nucleic acid is not in a condensed state, such as chromatin, to render the target nucleic acid inaccessible by the mutant polypeptide / RNA guide complex.

[0231] In some embodiments, the target nucleic acid is present in a cell. In some embodiments, the target nucleic acid is present in the nucleus of a cell. In some embodiments, the target nucleic acid is endogenous to the cell. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is chromosomal DNA. In one embodiment, the target nucleic acid is an extrachromosomal nucleic acid. In some embodiments, the target nucleic acid is a protein-coding gene or a functional region thereof, e.g., a coding region, or a regulatory element, e.g., a promoter, an enhancer, a 5' or 3' untranslated region, etc. In some embodiments, the target nucleic acid is a non-coding gene, e.g., a transposon, miRNA, tRNA, ribosomal RNA, ribozyme, or lincRNA. In some embodiments, the target nucleic acid is a plasmid.

[0232] In some embodiments, the target nucleic acid is exogenous to the cell. In some embodiments, the target nucleic acid is a viral nucleic acid, e.g., viral DNA or viral RNA. In some embodiments, the target nucleic acid is a horizontally transferred plasmid. In some embodiments, the target nucleic acid is integrated into the genome of the cell. In some embodiments, the target nucleic acid is not integrated into the genome of the cell. In some embodiments, the target nucleic acid is a plasmid in the cell. In some embodiments, the target nucleic acid is present in an extrachromosomal array.

[0233] In some embodiments, the target nucleic acid is an isolated nucleic acid, e.g., isolated DNA or isolated RNA. In some embodiments, the target nucleic acid is present in a cell-free environment. In some embodiments, the target nucleic acid is an isolated vector, e.g., a plasmid. In some embodiments, the target nucleic acid is an ultra-pure plasmid.

[0234] The target nucleic acid is a segment of the target nucleic acid that hybridizes to the RNA guide. In some embodiments, the target nucleic acid has only one copy of the target nucleic acid. In some embodiments, the target nucleic acid has two or more copies of the target nucleic acid, for example, about 2, 3, 4, 5, 10, 100, or more copies. For example, a target nucleic acid containing a repetitive sequence in a viral nucleic acid or bacterial genome can be targeted by a mutant ribonucleoprotein.

[0235] In some embodiments, the target nucleic acid is present in an easily accessible region of the target nucleic acid. In some embodiments, the target nucleic acid is present in an exon of the target gene. In some embodiments, the target nucleic acid spans an exon-intron junction of the target gene. In some embodiments, the target nucleic acid is present in a non-coding region, for example, a regulatory region of a gene. In some embodiments, the target nucleic acid is exogenous to the cell, the target nucleic acid comprises a sequence not found in the genome of the cell.

[0236] Suitable DNA / RNA binding conditions include physiological conditions normally present in cells. Other suitable DNA / RNA binding conditions (e.g., conditions in cell-free systems) are known in the art, see, for example, Sambrook, supra. The strand of the target nucleic acid that is complementary to and hybridizes with the RNA guide is referred to as the "complementary strand," and the strand of the target nucleic acid that is complementary to the "complementary strand" (and therefore not complementary to the RNA guide) is referred to as the "noncomplementary strand" or "non-complementary strand."

[0237] In some embodiments, the target nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, and 22.

[0238] binary complex The binary complexes described herein, in some embodiments, comprise a mutant polypeptide associated with at least one RNA guide, each RNA guide targeting a target nucleic acid, such as DNA. In some embodiments, the mutant polypeptide / RNA guide complex (e.g., mutant binary complex) comprises an enzymatic activity, such as a nuclease activity, capable of nicking or cleaving the target nucleic acid. The mutant polypeptide and the RNA guide, alone or together, do not occur in nature. Complex formation between the mutant polypeptide and the RNA guide can enhance stability and / or protein-RNA interaction between the two, compared to the parent polypeptide and RNA guide.

[0239] Generally, the variant polypeptide and the targeting moiety (e.g., RNA guide) are bound to each other in a molar ratio of about 1:1 to form the variant binary complex. The binding of the mutant polypeptide and the targeting moiety (e.g., RNA guide) to form the mutant binary complex is referred to as loading the RNA guide onto the polypeptide.

[0240] In some embodiments, the binary complex follows the one-guide rule, i.e., the mutant polypeptide does not dissociate from the bound RNA guide in the complex or switch the RNA guide with free, unbound RNA. In some embodiments, the ternary complex follows the one-binary complex rule, i.e., the mutant binary complex does not dissociate from the bound target nucleic acid (e.g., target DNA substrate) or switch the target nucleic acid with free, unbound nucleic acid.

[0241] Functionality of the binary complex In some embodiments, a mutant binary complex comprises a mutant polypeptide having at least one modification or mutation that enhances at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability.

[0242] In some aspects, a mutant binary complex comprises a mutant polypeptide having at least one modification or mutation, wherein the mutant binary complex has reduced off-target binding to non-target nucleic acids, activity at non-target loci of target nucleic acids, binary complex dissociation, or dissociation from the target nucleic acid.

[0243] In some embodiments, the variant polypeptide and the targeting moiety (e.g., RNA guide) form a variant binary complex that exhibits increased enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability compared to the parent binary complex.

[0244] In some embodiments, the mutant binary complex is characterized by comparing at least one of the enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability of the mutant binary complex with the enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability of the parent binary complex. Variant polypeptides include those having at least one modification or mutation that increases on-target binding activity, on-target binding specificity, and / or stability by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0245] In some embodiments, the mutant binary complex exhibits enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability at a temperature ranging from about 20°C to about 65°C, e.g., at any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C, relative to the parent binary complex.

[0246] In some embodiments, the variant binary complex exhibits enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in a buffer having a pH in the range of about 7.3 to about 8.6 (e.g., 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, or any value in the range incorporating any combination of these values), relative to the parent binary complex.

[0247] In some embodiments, the mutant binary complex comprises a T m The value is the T of the parent binary complex m In one embodiment, the mutant binary complex exhibits enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability relative to the parent binary complex when the T of the mutant binary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C higher than a value of mThe value is the T of the parent binary complex m A temperature at least 8°C higher than the value indicates enhancement of at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability.

[0248] In some embodiments, the variant binary complexes exhibit increased enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability over a range of incubation times, relative to the parent binary complex, of at least about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more. In some embodiments, the variant binary complexes exhibit increased enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability over a range of incubation times, relative to the parent binary complex.

[0249] In some embodiments, the variant polypeptide and the targeting moiety (e.g., RNA guide) form a variant binary complex, and the variant binary complex exhibits reduced off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, dissociation of the binary complex, and / or dissociation from the target nucleic acid, compared to the parent binary complex. In some embodiments, the mutant binary complex exhibits a decrease in at least one of off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, dissociation of the binary complex, and / or dissociation from the target nucleic acid that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than the off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, dissociation of the binary complex, and / or dissociation from the target nucleic acid of the parent binary complex.

[0250] In some embodiments, the mutant binary complex exhibits a decrease in at least one of off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, dissociation of the binary complex, and / or dissociation from the target nucleic acid that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than the off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, dissociation of the binary complex, and / or dissociation from the target nucleic acid of the parent binary complex.

[0251] In some embodiments, the mutant binary complex exhibits at least one of decreased off-target binding to non-target nucleic acids, activity at non-target loci of target nucleic acids, dissociation of the binary complex, and / or dissociation from the target nucleic acid, relative to the parent binary complex, at a temperature ranging from about 20°C to about 65°C, e.g., at any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C.

[0252] In some embodiments, the mutant binary complex exhibits reduced off-target binding to non-target nucleic acids, activity at non-target loci of target nucleic acids, dissociation of the binary complex, and / or dissociation from the target nucleic acid in a buffer having a pH in the range of about 7.3 to about 8.6 (e.g., 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, or any value in the range incorporating any combination of these values), compared to the parent binary complex.

[0253] In some embodiments, the mutant binary complex comprises a T m The value is the T of the parent binary complex m

[0023] In one embodiment, the mutant binary complex exhibits a decrease in at least one of off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, dissociation of the binary complex, and / or dissociation from the target nucleic acid, relative to the parent binary complex, when the T of the mutant binary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C above the T of the mutant binary complex. m The value is the T of the parent binary complex m A temperature at least 8°C higher than this value indicates a decrease in at least one of off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, dissociation of the binary complex, and / or dissociation from the target nucleic acid.

[0254] In some embodiments, the mutant binary complex exhibits at least one of decreased dissociation of the complex (mutant binary complex or mutant ternary complex) and / or dissociation from the target locus at about 37° C. over an incubation period of at least about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more, compared to the complex formed by the parent polypeptide and RNA guide. In some embodiments, the mutant binary complex exhibits at least one of decreased dissociation of the complex (mutant binary complex or mutant ternary complex) and / or dissociation from the target locus over a range of incubation periods, compared to the complex formed by the parent polypeptide and RNA guide.

[0255] In some embodiments, the mutant binary complex exhibits at least one of decreased dissociation of the complex (mutant binary complex or mutant ternary complex) and / or dissociation from the target locus over an incubation period of at least about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more at about 37° C. compared to the parent binary complex. In some embodiments, the mutant binary complex exhibits at least one of decreased dissociation of the complex (mutant binary complex or mutant ternary complex) and / or dissociation from the target locus over a range of incubation periods compared to the parent binary complex.

[0256] ternary complex The ternary complexes described herein, in some embodiments, comprise a mutant polypeptide associated with at least one RNA guide (i.e., forming a mutant binary complex), where each RNA guide targets and associates with (i.e., forms a mutant ternary complex with) a target nucleic acid, such as DNA. In some embodiments, the mutant polypeptide / RNA guide complex (e.g., mutant binary complex) comprises an enzymatic activity, such as a nuclease activity, that can nick or cleave the target nucleic acid of the mutant ternary complex. In some embodiments, the mutant ternary complex comprises an enzymatic activity, such as a nuclease activity. The mutant polypeptide, RNA guide, and target nucleic acid, either alone or together, do not occur in nature.

[0257] Generally, the mutant binary complex, e.g., the mutant polypeptide and the targeting moiety, binds to the target nucleic acid in a molar ratio of about 1:1 to form the mutant ternary complex. Binding of the mutant binary complex to the target nucleic acid, e.g., the target DNA substrate, to form the mutant ternary complex is referred to as loading the mutant binary complex onto the target nucleic acid.

[0258] Generally, the variant polypeptide, targeting moiety (e.g., RNA guide), and target nucleic acid associate with each other in a molar ratio of about 1:1:1 to form the variant ternary complex.

[0259] In some embodiments, the target nucleic acid comprises one or more target loci of the mutant binary complex or plurality of mutant binary complexes, hi some embodiments, the target nucleic acid comprises one or more non-target loci of the mutant binary complex or plurality of mutant binary complexes.

[0260] Functionality of the ternary complex In some aspects, a mutant ternary complex comprises a mutant polypeptide having at least one modification or mutation, wherein the mutant ternary complex has enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability.

[0261] In some aspects, a mutant ternary complex comprises a mutant polypeptide having at least one modification or mutation, wherein the mutant ternary complex has reduced at least one of dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or dissociation of the ternary complex.

[0262] In some embodiments, the mutant binary complex and the target nucleic acid (e.g., DNA) form a mutant ternary complex that exhibits increased enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability compared to the parent binary complex.

[0263] In some embodiments, the mutant ternary complex has at least 1%, 2%, 3%, 4%, 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119%, 120%, The mutant ternary complex exhibits at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability that is 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater.

[0264] In some embodiments, the variant ternary complex exhibits enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability at a temperature ranging from about 20°C to about 65°C, e.g., at any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C, relative to the parent ternary complex.

[0265] In some embodiments, the variant ternary complex exhibits enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in a buffer having a pH in the range of about 7.3 to about 8.6 (e.g., 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, or any value in the range incorporating any combination of these values), relative to the parent ternary complex.

[0266] In some embodiments, the mutant ternary complex comprises a T m The value is the T of the parent ternary complex m In one embodiment, the mutant ternary complex exhibits enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability compared to the parent ternary complex when the T of the mutant ternary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C greater than a value of m The value is the T of the parent ternary complex m A temperature at least 8°C higher than the value indicates enhancement of at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability.

[0267] In some embodiments, the variant ternary complex exhibits enhanced at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability over a range of incubation times, relative to the parent ternary complex, of at least about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more. In some embodiments, the variant ternary complex exhibits increased at least one of enzymatic activity, target nucleic acid complex formation, target nucleic acid binding activity, target nucleic acid affinity, target nucleic acid binding specificity, protein-nucleic acid interaction, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability over a range of incubation times, relative to the parent ternary complex.

[0268] In some embodiments, the mutant binary complex and the target nucleic acid (e.g., DNA) form a mutant ternary complex, and the mutant ternary complex exhibits reduced at least one of dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or dissociation of the ternary complex compared to the parent binary complex.

[0269] In some embodiments, the mutant ternary complex exhibits a decrease in at least one of dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or dissociation of the ternary complex that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than the dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or dissociation of the ternary complex of the parent binary complex.

[0270] In some embodiments, the mutant ternary complex exhibits at least one of a decrease in dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or dissociation of the ternary complex compared to the parent ternary complex at any one of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, or 65°C.

[0271] In some embodiments, the mutant ternary complex exhibits, relative to the parent ternary complex, at least one of decreased dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or ternary complex dissociation over a temperature range of about 20° C. to about 65° C. In some embodiments, the mutant ternary complex exhibits, relative to the parent ternary complex, at least one of decreased dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or ternary complex dissociation over an incubation period of at least about any one of 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more at about 37° C. In some embodiments, the mutant ternary complex exhibits reduced at least one of dissociation from the target locus, off-target binding to non-target nucleic acids, activity at the non-target locus of the target nucleic acid, and / or ternary complex dissociation at various incubation times compared to the parent ternary complex.

[0272] In some embodiments, the mutant ternary complex exhibits reduced dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or ternary complex dissociation compared to the parent ternary complex in a buffer having a pH in the range of about 7.3 to about 8.6. In one embodiment, the mutant ternary complex exhibits reduced dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or ternary complex dissociation compared to the parent ternary complex at a pH of about 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or 8.6.

[0273] In some embodiments, the mutant ternary complex comprises a T m The value is the T of the reference molecule m value or reference value T m , e.g., T of the parent ternary complex m

[0023] In one embodiment, the mutant ternary complex exhibits a decrease in at least one of dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or ternary complex dissociation when the T of the mutant ternary complex is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C higher than the T of the mutant ternary complex. m The value is the T of the reference molecule m value or reference value T m , e.g., T of the parent ternary complex m A temperature at least 8°C above the target locus indicates a decrease in at least one of dissociation from the target locus, off-target binding to non-target nucleic acids, activity of the target nucleic acid at the non-target locus, and / or ternary complex dissociation.

[0274] In some embodiments, the mutant ternary complex exhibits increased stability compared to the parent ternary complex over an incubation period of at least about any one of 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more at about 37° C. In some embodiments, the mutant ternary complex exhibits increased stability over a range of incubation periods compared to the parent ternary complex.

[0275] In some aspects, the mutant binary complex exhibits reduced activity at a non-target locus of the target nucleic acid compared to the parent binary complex. In some embodiments, the non-target activity is assessed at a PAM-flanking sequence at a particular Levenshtein distance (e.g., 1, 2, 3, or 4 edit distance) from the on-target locus sequence. In some embodiments, the activity at the non-target locus by the mutant binary complex may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than the activity at the non-target locus by the parent binary complex.

[0276] preparation In some embodiments, mutant polypeptides of the present disclosure can be prepared by (a) culturing bacteria that produce the mutant polypeptides of the present disclosure, isolating the mutant polypeptides, optionally purifying the mutant polypeptides, and complexing the mutant polypeptides with an RNA guide. Mutant polypeptides can also be prepared by (b) known genetic engineering techniques, specifically by isolating genes encoding the mutant polypeptides of the present disclosure from bacteria, constructing recombinant expression vectors, and then transferring the vectors into appropriate host cells that express the RNA guides for expression of recombinant proteins complexed with the RNA guides in the host cells. Alternatively, mutant polypeptides can be prepared by (c) an in vitro coupled transcription-translation system followed by complexing with the RNA guides. Bacteria that can be used to prepare mutant polypeptides of the present disclosure are not particularly limited, as long as they are capable of producing the mutant polypeptides of the present disclosure. Some non-limiting examples of bacteria include the Escherichia coli (E. coli) cells described herein.

[0277] vector The present disclosure provides vectors for expressing the mutant polypeptides described herein, or nucleic acids encoding the mutant polypeptides described herein can be incorporated into vectors. In some embodiments, the vectors of the present disclosure comprise a nucleotide sequence encoding the mutant polypeptide.

[0278] The present disclosure also provides vectors that can be used to prepare mutant polypeptides as described herein, or compositions comprising the mutant polypeptides. In some embodiments, the present disclosure includes a composition or vector described herein in a cell. In some embodiments, the present disclosure includes a method of expressing a composition comprising a mutant polypeptide, or a vector or nucleic acid encoding a mutant polypeptide, in a cell. The method can include providing a composition, e.g., a vector or nucleic acid, and delivering the composition to the cell.

[0279] Expression of a natural or synthetic polynucleotide is typically achieved by operably linking a polynucleotide encoding a gene of interest, for example, a nucleotide sequence encoding a mutant polypeptide, to a promoter and incorporating the construct into an expression vector. The expression vector is not particularly limited, so long as it contains a polynucleotide encoding a mutant polypeptide of the present disclosure and can be suitable for replication and integration in eukaryotic cells.

[0280] A typical expression vector may include transcription and translation terminators, initiation sequences, and promoters useful for expressing a desired polynucleotide. For example, a plasmid vector (e.g., pSP64, pBluescript) carrying a recognition sequence for RNA polymerase may be used. Vectors, including those derived from retroviruses such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term and stable integration of the transgene and its propagation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. An expression vector may be provided to cells in the form of a viral vector.

[0281] Viral vector technology is well known in the art and is described in various virology and molecular biology manuals.Viruses that can be used as vectors include, but are not limited to, phage virus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.Generally, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers.

[0282] The type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. More specifically, depending on the type of host cell, a promoter sequence is appropriately selected to ensure expression of the mutant polypeptide from a polynucleotide, and this promoter sequence and polynucleotide are inserted into any of various plasmids, etc. to prepare an expression vector.

[0283] Additional promoter elements, such as enhancer sequences, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although a number of bacterial promoters have been shown to contain functional elements downstream of the start site as well. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription.

[0284] Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when expression of the polynucleotide sequence is desired, or can turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0285] The introduced expression vector can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells to be transfected or infected with a viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection technique. Both the selectable marker and the reporter gene can be flanked by appropriate transcription control sequences to enable expression in the host cells. Examples of such markers include the dihydrofolate reductase gene and neomycin resistance gene for eukaryotic cell culture, and the tetracycline resistance gene and ampicillin resistance gene for culture in Escherichia coli (E. coli) and other bacteria. The use of such selectable markers can confirm whether a polynucleotide encoding a variant polypeptide of the present disclosure has been transferred into a host cell and then correctly expressed.

[0286] The preparation method for the recombinant expression vector is not particularly limited, and examples thereof include methods using a plasmid, a phage, or a cosmid.

[0287] Method of Expression The present disclosure includes methods for protein expression comprising translating the variant polypeptides described herein.

[0288] In some embodiments, the host cells described herein are used to express the mutant polypeptides. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of host cells include bacteria such as Escherichia coli (E. coli), yeast (Saccharomyces cerevisiae, Saccharomyces cerevisiae, and Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (e.g., CHO cells, COS cells, and HEK293 cells). The method for transferring the expression vector into the host cell, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, calcium phosphate transformation, liposome transformation, and DEAE-dextran transformation can be used.

[0289] After a host has been transformed with an expression vector, the host cells can be cultured, cultivated, or bred to produce the mutant polypeptide. After expression of the mutant polypeptide, the host cells can be harvested and the mutant polypeptide can be purified from the culture or the like according to conventional methods (e.g., filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).

[0290] In some embodiments, methods for variant polypeptide expression comprise translating at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of the variant polypeptide. In some embodiments, methods for protein expression include translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids, or more of a variant polypeptide.

[0291] Various methods can be used to determine the level of production of a mature variant polypeptide in a host cell. Such methods include, but are not limited to, methods utilizing either polyclonal or monoclonal antibodies specific for the variant polypeptide, or labeling tags, as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and fluorescence-activated cell sorting (FACS). These and other assays are well known in the art (see, e.g., Maddox et al., J. Exp. Med. 158:1211

[1983] ).

[0292] The present disclosure provides a method for in vivo expression of a mutant polypeptide in a cell, the method comprising providing a host cell with a polyribonucleotide encoding the mutant polypeptide, wherein the polyribonucleotide encodes the mutant polypeptide, expressing the mutant polypeptide in the cell, and obtaining the mutant polypeptide from the cell.

[0293] Introduction of modifications or mutations Nucleic acid sequences encoding variant polypeptides or variant polypeptides can be produced by synthetic methods known in the art. The nucleic acid sequence encoding the parent polypeptide itself can be used as a framework to insert one or more modifications or mutations to modify the nucleic acid sequence encoding the parent polypeptide. Along similar lines, the parent polypeptide can also be modified or mutated by introducing changes into the polypeptide sequence as it is effectively synthesized. This can be accomplished by methods well known in the art.

[0294] The generation and introduction of modifications or mutations into a parent polypeptide sequence can be accomplished using any method known to those of skill in the art. In particular, in some embodiments, oligonucleotide primers for PCR can be used for the rapid synthesis of DNA templates containing one or more modifications or mutations in the nucleic acid sequence encoding the variant polypeptide. Site-directed mutagenesis can also be used as a useful technique for the preparation of individual peptides or biologically functional equivalent proteins or peptides through specific mutagenesis of the underlying DNA. Techniques that incorporate one or more of the aforementioned considerations by introducing one or more nucleotide sequence changes into DNA further provide an easy ability to prepare and test variants. Site-directed mutagenesis enables the generation of variants through the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation, as well as a sufficient number of flanking nucleotides, providing primer sequences of sufficient size and sequence complexity to form stable duplexes on both sides of the deletion junction to be passed. Typically, primers of approximately 17-25 nucleotides in length are preferred, with approximately 5-10 residues on either side of the junction of the sequence to be modified.

[0295] Introduction of structural changes, such as polypeptide fusions, such as amino- and / or carboxyl-terminal extensions, can be accomplished in a manner similar to the introduction of a modified or mutated parent polypeptide. Additional peptides can be added to a parent or variant polypeptide by including appropriate nucleic acid sequences encoding the additional peptides in the nucleic acid sequence encoding the parent or variant polypeptide. Optionally, additional peptides can be added directly to a variant polypeptide through synthetic polypeptide production.

[0296] In aspects, the present disclosure also provides methods for introducing modifications or mutations into a parent polypeptide sequence to produce a variant polypeptide that has increased on-target binding to two or more loci (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) of a target nucleic acid compared to the parent polypeptide sequence.

[0297] In aspects, the present disclosure also provides methods for introducing modifications or mutations into a parent polypeptide sequence to produce a plurality of variant polypeptides (e.g., distinct variant polypeptides having the same amino acid sequence) that, when individually complexed with a plurality of distinct RNA guides, have increased on-target binding to two or more loci of a target sequence compared to a plurality of parent polypeptides and RNA guides.

[0298] In aspects, the present disclosure also provides methods for introducing modifications or mutations into a parent polypeptide sequence to produce a variant polypeptide that has increased on-target ternary complex formation with two or more target loci of a target nucleic acid compared to the parent polypeptide.

[0299] In aspects, the present disclosure also provides methods for introducing modifications or mutations into a parent polypeptide sequence to produce a plurality of variant polypeptides (e.g., distinct variant polypeptides having the same amino acid sequence) that, when individually complexed with a plurality of distinct RNA guides, have increased ternary complex formation with two or more loci of a target nucleic acid compared to a plurality of parent polypeptides and RNA guides.

[0300] In aspects, the present disclosure also provides methods for introducing modifications or mutations into a parent polypeptide sequence to produce a variant polypeptide that exhibits increased targeting of a target nucleic acid or target locus when compared to the parent polypeptide.

[0301] In aspects, the present disclosure also provides methods for introducing modifications or mutations into a parent polypeptide sequence to produce a plurality of variant polypeptides (e.g., distinct variant polypeptides having the same amino acid sequence) that, when individually complexed with a plurality of distinct RNA guides, exhibit increased numbers of target nucleic acids or target loci when compared to a plurality of parent polypeptides and RNA guides.

[0302] In aspects, the present disclosure also provides methods for introducing modifications or mutations into a parent polypeptide sequence to enhance the stability of a variant polypeptide. The stability of a variant polypeptide may be determined by or may include, but is not limited to, thermal denaturation assays, thermal shift assays, differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), pulse chase, bleach chase, cycloheximide chase, circular dichroism (CD) spectroscopy, crystallization, and fluorescence-based activity assays.

[0303] Mutant selection for functionality In one aspect, the present disclosure provides methods for introducing modifications or mutations into a parent polypeptide sequence to enhance binary complex formation, RNA-guided binding activity, and / or RNA-guided binding specificity.

[0304] In one aspect, the present disclosure also provides a method for introducing modifications or mutations into a parent polypeptide sequence to enhance ternary complex formation, on-target binding affinity, on-target binding activity, on-target binding, and / or on-target binding specificity. In one aspect, the present disclosure also provides a method for introducing modifications or mutations into a parent polypeptide sequence to enhance on-target binding affinity (e.g., affinity or time to interact with target), on-target binding activity (e.g., nuclease activity upon interacting with target), on-target binding (e.g., strength of interaction with target), and / or on-target binding specificity (e.g., preference for a specific target) of a binary complex (e.g., a ribonucleoprotein).

[0305] In some embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide with increased on-target binding and / or activity. In such embodiments, the variant polypeptide may also have decreased off-target binding and / or activity compared to the parent polypeptide. Furthermore, specificity may be increased or decreased with respect to on-target versus off-target binding.

[0306] In some embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide that has increased on-target binding when complexed with an RNA guide. Such embodiments may also reduce off-target binding in complexes comprising the variant polypeptide and an RNA guide. Furthermore, specificity may be increased or decreased with respect to on-target versus off-target binding / activity.

[0307] In certain embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide that has enhanced stability and / or protein-RNA interaction, hi certain embodiments, the variant polypeptide comprises at least one modification that enhances the stability and / or RNA interaction and enzymatic activity of the variant polypeptide compared to the parent polypeptide.

[0308] In certain embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide that (a) lacks enzymatic activity but (b) retains enhanced stability and / or protein-RNA interaction. In certain embodiments, the variant polypeptide comprises at least one modification that enhances the stability and / or RNA interaction, but not the enzymatic activity, of the variant polypeptide relative to the parent polypeptide.

[0309] In certain embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide that (a) enhances enzymatic activity, and (b) enhances binary complex formation, RNA-guided binding activity, and / or RNA-guided binding specificity. In certain embodiments, the variant polypeptide comprises at least one modification that enhances RNA-guided complex formation, RNA-guided binding activity, and / or RNA-guided binding specificity, and enzymatic activity of the variant polypeptide compared to the parent polypeptide.

[0310] In certain embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide that (a) lacks enzymatic activity but (b) retains enhanced binary complex formation, RNA-guided binding activity, and / or RNA-guided binding specificity. In certain embodiments, the variant polypeptide comprises at least one modification that enhances binary complex formation, RNA-guided binding activity, and / or RNA-guided binding specificity of the variant polypeptide, but does not enhance enzymatic activity, compared to the parent polypeptide.

[0311] In certain embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide that (a) enhances enzymatic activity, and (b) enhances on-target ternary complex formation, on-target binding affinity, on-target binding activity, and / or on-target binding specificity. In certain embodiments, the variant polypeptide comprises at least one modification that enhances on-target ternary complex formation, on-target binding affinity, on-target binding activity, and / or on-target binding specificity, and enzymatic activity of the variant polypeptide compared to the parent polypeptide.

[0312] In certain embodiments, modifications or mutations are introduced into a parent polypeptide sequence to generate a variant polypeptide that (a) lacks enzymatic activity but (b) retains enhanced on-target ternary complex formation, on-target binding affinity, on-target binding activity, and / or on-target binding specificity. In certain embodiments, the variant polypeptide comprises at least one modification that enhances on-target ternary complex formation, on-target binding affinity, on-target binding activity, and / or on-target binding specificity, but does not enhance enzymatic activity, of the variant polypeptide relative to the parent polypeptide.

[0313] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) decreased enzymatic activity and (b) enhanced RNA affinity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) enhanced RNA affinity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) enhanced RNA affinity relative to the parent polypeptide of SEQ ID NO: 3.

[0314] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) decreased enzymatic activity and (b) enhanced RNA affinity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) enhanced RNA affinity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) enhanced binary complex formation relative to the parent polypeptide of SEQ ID NO: 3.

[0315] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) decreased enzymatic activity and (b) enhanced RNA-guided binding activity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) enhanced RNA-guided binding activity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) enhanced RNA-guided binding activity relative to the parent polypeptide of SEQ ID NO: 3.

[0316] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) reduced enzymatic activity and (b) enhanced RNA-guided binding specificity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) enhanced RNA-guided binding specificity relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) enhanced RNA-guided binding specificity relative to the parent polypeptide of SEQ ID NO: 3.

[0317] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) decreased enzymatic activity and (b) enhanced protein-RNA interaction relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) enhanced protein-RNA interaction relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) enhanced protein-RNA interaction relative to the parent polypeptide of SEQ ID NO: 3.

[0318] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) decreased enzymatic activity and (b) enhanced protein stability relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) enhanced protein stability relative to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) enhanced protein stability relative to the parent polypeptide of SEQ ID NO: 3.

[0319] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) decreased enzymatic activity and (b) decreased dissociation from the RNA guide compared to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) decreased dissociation from the RNA guide compared to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) decreased dissociation from the RNA guide compared to the parent polypeptide of SEQ ID NO: 3.

[0320] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) decreased enzymatic activity and (b) enhanced ternary complex formation compared to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) increased enzymatic activity and (b) enhanced ternary complex formation compared to the parent polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a variant polypeptide that exhibits (a) retained enzymatic activity and (b) enhanced ternary complex formation compared to the parent polypeptide of SEQ ID NO: 3.

[0321] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) reduced enzymatic activity and (b) enhanced binding affinity for a target nucleic acid relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) increased enzymatic activity and (b) enhanced binding affinity for a target nucleic acid relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) retained enzymatic activity and (b) enhanced binding affinity for a target nucleic acid relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3.

[0322] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) reduced enzymatic activity and (b) enhanced on-target binding activity relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) increased enzymatic activity and (b) enhanced on-target binding activity relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) retained enzymatic activity and (b) enhanced on-target binding activity relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3.

[0323] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) reduced enzymatic activity and (b) enhanced on-target binding specificity relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) increased enzymatic activity and (b) enhanced on-target binding specificity relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) retained enzymatic activity and (b) enhanced on-target binding specificity relative to the parent binary complex comprising the polypeptide of SEQ ID NO: 3.

[0324] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) reduced enzymatic activity and (b) reduced off-target binding to non-target nucleic acids compared to a parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) increased enzymatic activity and (b) reduced off-target binding to non-target nucleic acids compared to a parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) retained enzymatic activity and (b) reduced off-target binding to non-target nucleic acids compared to a parent binary complex comprising the polypeptide of SEQ ID NO: 3.

[0325] In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) reduced enzymatic activity and (b) decreased dissociation from a target nucleic acid compared to a parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) increased enzymatic activity and (b) decreased dissociation from a target nucleic acid compared to a parent binary complex comprising the polypeptide of SEQ ID NO: 3. In some embodiments, at least one modification is introduced into a parent polypeptide of SEQ ID NO: 3 to generate a mutant polypeptide that forms a mutant binary complex that exhibits (a) retained enzymatic activity and (b) decreased dissociation from a target nucleic acid compared to a parent binary complex comprising the polypeptide of SEQ ID NO: 3.

[0326] Mutant binary complex formation Generally, the mutant polypeptide and RNA guide bind to each other in a molar ratio of about 1:1 to form a mutant binary complex, and the mutant polypeptide and RNA guide do not occur in nature either alone or together.

[0327] In some embodiments, the mutant polypeptide is overexpressed in a host cell, purified as described herein, and then complexed (e.g., in a test tube) with an RNA guide to form a mutant ribonucleoprotein (RNP) (e.g., a mutant binary complex).

[0328] In some embodiments, the mutant binary complexes exhibit increased binding affinity for the target nucleic acid, increased on-target binding activity, increased on-target binding specificity, increased ternary complex formation with the target nucleic acid, and / or increased stability over a range of incubation times. In some embodiments, the mutant binary complexes exhibit decreased off-target binding to non-target nucleic acids and / or decreased dissociation from the target nucleic acid over a range of incubation times. In some embodiments, the mutant binary complexes exhibit increased target nucleic acid complex formation, target nucleic acid activity, and / or target nucleic acid specificity over a range of incubation times.

[0329] In some embodiments, complexation of the binary complex occurs at a temperature below any one of about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 50° C., or 55° C. In some embodiments, the variant polypeptide does not dissociate from the RNA guide or bind to free RNA over at least any one of about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more incubation times at about 37° C. In some embodiments, after binary complex formation, the mutant ribonucleoprotein complex does not exchange the RNA guide for a different RNA.

[0330] In some embodiments, the mutant polypeptide and the RNA guide are complexed in a binary complexation buffer. In some embodiments, the mutant polypeptide is stored in a buffer that is replaced with the binary complexation buffer to form a complex with the RNA guide. In some embodiments, the mutant polypeptide is stored in the binary complexation buffer.

[0331] In some embodiments, the binary complexation buffer has a pH in the range of about 7.3 to 8.6. In one embodiment, the pH of the binary complexation buffer is about 7.3. In one embodiment, the pH of the binary complexation buffer is about 7.4. In one embodiment, the pH of the binary complexation buffer is about 7.5. In one embodiment, the pH of the binary complexation buffer is about 7.6. In one embodiment, the pH of the binary complexation buffer is about 7.7. In one embodiment, the pH of the binary complexation buffer is about 7.8. In one embodiment, the pH of the binary complexation buffer is about 7.9. In one embodiment, the pH of the binary complexation buffer is about 8.0. In one embodiment, the pH of the binary complexation buffer is about 8.1. In one embodiment, the pH of the binary complexation buffer is about 8.2. In one embodiment, the pH of the binary complexation buffer is about 8.3. In one embodiment, the pH of the binary complexation buffer is about 8.4. In one embodiment, the pH of the binary complexation buffer is about 8.5. In one embodiment, the pH of the binary complexation buffer is about 8.6.

[0332] The thermal stability of the mutant polypeptide can be increased under favorable conditions, such as the addition of an RNA guide, e.g., binding to an RNA guide.

[0333] In some embodiments, the mutant polypeptide can be overexpressed in a host cell and complexed with an RNA guide prior to purification as described herein. In some embodiments, mRNA or DNA encoding the mutant polypeptide is introduced into a cell, thereby expressing the mutant polypeptide in the cell. An RNA guide that directs the mutant polypeptide to a desired target nucleic acid is also introduced into the cell, either simultaneously with a single mRNA or DNA construct, separately, or sequentially, thereby allowing the necessary ribonucleoprotein complex to bind in the cell.

[0334] Assessment of stability and functionality of mutant binary complexes In certain embodiments, provided herein are methods for identifying optimal mutant polypeptide / RNA guide complexes (referred to herein as mutant binary complexes), the methods comprising: (a) combining a mutant polypeptide and an RNA guide in a sample to form a mutant binary complex; (b) measuring a value for the mutant binary complex; and (c) determining that the mutant binary complex is more optimal than the reference molecule if the value for the mutant binary complex is greater than the value for the reference molecule. In some embodiments, the value includes a stability measurement (e.g., T m These may include, but are not limited to, the following: binding activity, activity (e.g., binding rate, thermal stability), rate of binary complex formation, RNA guide binding specificity, and / or complex activity.

[0335] In some embodiments, an optimal mutant polypeptide / RNA guide complex (i.e., a mutant binary complex) can be identified by (a) combining a mutant polypeptide and an RNA guide in a sample to form a mutant binary complex; and (b) determining the T m (c) detecting the T value of the mutant binary complex. m The value is the T of the reference molecule m Greater than value or T m determining that the mutant binary complex is stable if its temperature is at least 8° C. greater than the reference value.

[0336] Methods involving measuring the thermal stability of mutant polypeptide / RNA guide complexes (i.e., mutant binary complexes) can include, but are not limited to, methods for determining the stability of mutant binary complexes, methods for determining conditions that promote stable mutant binary complexes, methods for screening stable mutant binary complexes, and methods for identifying optimal RNA guides to form stable mutant binary complexes. In certain embodiments, the thermal stability values ​​of mutant binary complexes may be measured.

[0337] Additionally, in certain embodiments, the thermostability value of a reference molecule may also be measured. In certain embodiments, a mutant binary complex may be determined to be stable if the measured thermostability value of the mutant binary complex is greater than the measured thermostability value of the reference molecule or a thermostability reference value measured under the same experimental conditions as described herein. In certain embodiments, the reference molecule may be a mutant polypeptide in the absence of an RNA guide.

[0338] In certain embodiments, the measured thermostability value may be a denaturation temperature value. In these embodiments, the thermostability reference value is a denaturation temperature reference value. In certain embodiments, the measured thermostability value is a T m In these embodiments, the thermal stability reference value can be a T m The thermostability value may be a reference value. In certain embodiments, the thermostability value may be measured using a thermal shift assay. In certain embodiments, the assay used to measure thermostability may involve techniques described herein, including, but not limited to, thermal denaturation assay, thermal shift assay, differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), pulse chase, bleach chase, cycloheximide chase, circular dichroism (CD) spectroscopy, crystallization, and fluorescence-based activity assay.

[0339] In certain embodiments, a mutant binary complex may be identified when the rate of mutant polypeptide / RNA guide complex formation, RNA guide binding specificity, and / or complex activity of the mutant binary complex is greater than the value for a reference molecule or reference value (e.g., the value for a parent polypeptide / RNA guide complex, referred to herein as the parent binary complex). For example, in certain embodiments, a mutant binary complex may be identified when the value for the rate of mutant polypeptide / RNA guide complex formation, RNA guide binding specificity, and / or complex activity of the mutant binary complex is at least X% greater than the value for a reference molecule or reference value (e.g., the value for the parent binary complex). In certain embodiments, the methods described herein may further comprise a step comprising measuring the activity of the mutant binary complex as described herein.

[0340] Mutant ternary complex formation In some embodiments, a mutant polypeptide, an RNA guide, and a target nucleic acid as described herein form a mutant ternary complex (e.g., in a test tube or in a cell). Generally, the mutant polypeptide, the RNA guide, and the target nucleic acid associate with each other in a molar ratio of about 1:1:1 to form the mutant ternary complex. The mutant polypeptide, the RNA guide, and the target nucleic acid do not exist in nature, either alone or together.

[0341] In some embodiments, a mutant binary complex as described herein (e.g., a complex of a mutant polypeptide and an RNA guide) is further complexed with a target nucleic acid (e.g., in a test tube or in a cell) to form a mutant ternary complex.

[0342] In some embodiments, ternary complexation occurs at a temperature below any one of about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 50° C., or 55° C. In some embodiments, the mutant binary complex does not dissociate from the target nucleic acid or bind to free nucleic acid (e.g., free DNA) over at least any one of about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more incubation times at about 37° C. In some embodiments, after formation of the ternary complex, the mutant binary complex does not exchange the target nucleic acid for a different nucleic acid.

[0343] In some embodiments, the mutant polypeptide, the RNA guide, and the target nucleic acid are complexed in a ternary complexing buffer. In some embodiments, the mutant polypeptide is stored in a buffer that replaces the ternary complexing buffer to form a complex between the RNA guide and the target nucleic acid. In some embodiments, the mutant polypeptide is stored in the ternary complexing buffer.

[0344] In some embodiments, the mutant binary complex and the target nucleic acid are complexed in a ternary complexation buffer. In some embodiments, the mutant binary complex is stored in a buffer that is replaced with the ternary complexation buffer to form a complex with the target nucleic acid. In some embodiments, the mutant binary complex is stored in the ternary complexation buffer.

[0345] In some embodiments, the ternary complexation buffer has a pH in the range of about 7.3 to 8.6. In one embodiment, the pH of the ternary complexation buffer is about 7.3. In one embodiment, the pH of the ternary complexation buffer is about 7.4. In one embodiment, the pH of the ternary complexation buffer is about 7.5. In one embodiment, the pH of the ternary complexation buffer is about 7.6. In one embodiment, the pH of the ternary complexation buffer is about 7.7. In one embodiment, the pH of the ternary complexation buffer is about 7.8. In one embodiment, the pH of the ternary complexation buffer is about 7.9. In one embodiment, the pH of the ternary complexation buffer is about 8.0. In one embodiment, the pH of the ternary complexation buffer is about 8.1. In one embodiment, the pH of the ternary complexation buffer is about 8.2. In one embodiment, the pH of the ternary complexation buffer is about 8.3. In one embodiment, the pH of the ternary complexation buffer is about 8.4. In one embodiment, the pH of the ternary complexation buffer is about 8.5. In one embodiment, the pH of the ternary complexation buffer is about 8.6.

[0346] The thermal stability of the mutant polypeptide can be increased under favorable conditions, such as the addition of RNA guide and target nucleic acids.

[0347] Assessment of stability and functionality of mutant ternary complexes In certain embodiments, provided herein are methods for identifying optimal mutant ternary complexes, the methods comprising: (a) combining a mutant polypeptide, an RNA guide, and a target nucleic acid in a sample to form a mutant ternary complex; (b) measuring a value for the mutant ternary complex; and (c) determining that the mutant ternary complex is more optimal than the reference molecule if the value for the mutant ternary complex is greater than the value for the reference molecule. In some embodiments, the value includes a stability measurement (e.g., T m These may include, but are not limited to, measurements of DNA binding affinity, DNA binding specificity, and / or complex activity (e.g., nuclease activity).

[0348] In some embodiments, an optimal mutant ternary complex can be prepared by (a) combining a mutant polypeptide, an RNA guide, and a target nucleic acid in a sample to form a mutant ternary complex; and (b) determining the T of the mutant ternary complex. m (c) detecting the T value of the mutant ternary complex. m The value is the T of the reference molecule m Greater than value or T m determining that the mutant ternary complex is stable if its temperature is at least 8° C. greater than the reference value.

[0349] Methods involving measuring the thermostability of mutant ternary complexes can include, but are not limited to, methods for determining the stability of mutant ternary complexes, methods for determining conditions that promote stable mutant ternary complexes, methods for screening for stable mutant ternary complexes, and methods for identifying optimal binary complexes to form stable mutant ternary complexes. In certain embodiments, the thermostability values ​​of mutant ternary complexes may be measured.

[0350] Additionally, in certain embodiments, the thermostability value of a reference molecule may also be measured. In certain embodiments, a mutant ternary complex may be determined to be stable if the measured thermostability value of the mutant ternary complex is greater than the measured thermostability value of the reference molecule or a thermostability reference value measured under the same experimental conditions as described herein. In certain embodiments, the reference molecule may be a mutant polypeptide in the absence of the RNA guide and / or target nucleic acid.

[0351] In certain embodiments, the measured thermostability value may be a denaturation temperature value. In these embodiments, the thermostability reference value is a denaturation temperature reference value. In certain embodiments, the measured thermostability value is a T m In these embodiments, the thermal stability reference value can be a T mThe thermostability value may be a reference value. In certain embodiments, the thermostability value may be measured using a thermal shift assay. In certain embodiments, the assay used to measure thermostability may involve techniques described herein, including but not limited to, differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), or isothermal titration calorimetry (ITC).

[0352] In certain embodiments, a mutant ternary complex may be identified when the rate of ternary complex formation, DNA binding affinity, DNA binding specificity, and / or complex activity (e.g., nuclease activity) of the mutant ternary complex is greater than that of a reference molecule or reference value (e.g., that of the parent ternary complex). For example, in certain embodiments, a mutant ternary complex may be identified when the rate of ternary complex formation, DNA binding affinity, DNA binding specificity, and / or complex activity value of the mutant ternary complex is at least X% greater than that of a reference molecule or reference value (e.g., that of the parent ternary complex). In certain embodiments, the methods described herein may further comprise a step comprising measuring the activity of the mutant ternary complex as described herein.

[0353]

[0010] In one aspect, a method of modifying a target DNA molecule is provided, the method comprising contacting the target DNA molecule with a mutant polypeptide disclosed herein and an RNA guide disclosed herein. In some embodiments, the target DNA molecule is in vitro. In some embodiments, the target DNA molecule is in a cell. In certain embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is selected from a prokaryotic cell, a eukaryotic cell, a plant cell, a mammalian cell, and a human cell.

[0354] delivery The compositions or complexes described herein can be formulated with carriers, such as carriers and / or polymeric carriers, e.g., liposomes, and delivered to cells (e.g., prokaryotes, eukaryotes, plants, mammals, etc.) by known methods, including, but not limited to, transfection (e.g., lipid-mediated, cationic polymer, calcium phosphate, dendrimers), electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, particle bombardment ("gene gun"), fugene, direct acoustic loading, cell compression, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.

[0355] In some embodiments, the methods include delivering one or more nucleic acids (e.g., nucleic acids encoding mutant polypeptides, RNA guides, donor DNA, etc.), one or more transcripts thereof, and / or preformed mutant polypeptide / RNA guide complexes (i.e., mutant binary complexes) into cells. Exemplary intracellular delivery methods include, but are not limited to, viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as microinjection, electroporation, cell compaction, sonoporation, optical transfection, impalement, protoplast fusion, bacterial conjugation, plasmid or transposon delivery; particle-based methods, such as gene guns, magnetofection or magnet-assisted transfection, particle guns; and hybrid methods, such as nucleofection. In some embodiments, the present application further provides cells produced by such methods and organisms (such as animals, plants, or fungi) comprising or produced from such cells.

[0356] cell The polypeptides, compositions, or complexes described herein can be delivered to a variety of cells. In some embodiments, the cells are isolated cells. In some embodiments, the cells are in cell culture. In some embodiments, the cells are ex vivo. In some embodiments, the cells are obtained from a living organism and maintained in cell culture. In some embodiments, the cells are unicellular organisms.

[0357] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is or is derived from a bacterial cell. In some embodiments, the bacterial cell is unrelated to the bacterial species from which the parent polypeptide is derived. In some embodiments, the cell is or is derived from an archaeal cell. In some embodiments, the bacterium is a eukaryotic cell. In some embodiments, the cell is or is derived from a plant cell. In some embodiments, the cell is or is derived from a fungal cell. In some embodiments, the cell is or is derived from a fungal cell. In some embodiments, the cell is or is derived from an animal cell. In some embodiments, the cell is or is derived from an invertebrate cell. In some embodiments, the cell is or is derived from an invertebrate cell. In some embodiments, the cell is or is derived from a vertebrate cell. In some embodiments, the cell is or is derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebrafish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically produced, sometimes referred to as an artificial cell.

[0358] In some embodiments, the cells are derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, cells transfected with one or more nucleic acids (e.g., Ago-encoding vectors and gDNAs) or Ago-gDNA complexes described herein are used to establish new cell lines containing modifications to target nucleic acids, including one or more vector-derived cells. In some embodiments, cells transiently or non-transiently transfected with one or more nucleic acids (e.g., vectors and RNA guides encoding mutant polypeptides) or mutant polypeptide / RNA guide complexes (i.e., mutant binary complexes) described herein, or cell lines derived from such cells, are used to evaluate one or more test compounds.

[0359] In some embodiments, the methods include introducing one or more nucleic acids comprising a nucleotide sequence encoding a DNA-targeting RNA (e.g., RNA guide) and / or a mutant polypeptide into a host cell. In one embodiment, the cell comprising the target DNA is in vitro, in vivo, or ex vivo. In other embodiments, the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA (e.g., RNA guide) and / or a mutant polypeptide is comprised in a recombinant expression vector, such as, but not limited to, an adeno-associated virus construct, a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, etc.

[0360] In some embodiments, the cells are primary cells. For example, a culture of primary cells may be passaged 0, 1, 2, 4, 5, 10, 15, or more times. In some embodiments, primary cells are harvested from an individual by any known method. For example, leukocytes may be harvested by apheresis, leukopheresis, or density gradient separation. Cells from tissues, such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, or stomach, may be harvested by biopsy. An appropriate solution may be used to disperse or suspend the harvested cells. Such solutions are generally balanced salt solutions (e.g., saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.), which are advantageously supplemented with fetal bovine serum or other naturally occurring factors along with low concentrations of an acceptable buffer. Buffers may include HEPES, phosphate buffer, lactate buffer, etc. The cells may be used immediately, or they may be preserved (e.g., by freezing). Frozen cells can be thawed and reused. Cells can be frozen in DMSO, serum, media buffer (e.g., 10% DMSO, 50% serum, 40% buffered media), and / or some other such common solutions can be used to preserve cells at freezing temperatures.

[0361] In some embodiments, the mutant polypeptide has nuclease activity that induces double-strand or single-strand breaks in target nucleic acids (e.g., genomic DNA). The double-strand break can stimulate cell-intrinsic DNA repair pathways, including homologous recombination (HDR), non-homologous end joining (NHEJ), or non-homologous end rejoining (A-NHEJ). NHEJ can repair the cleaved target nucleic acid without the need for a homologous template. This can result in the deletion or insertion of one or more nucleotides into the target nucleic acid. HDR can occur with a homologous template, such as donor DNA. The homologous template can include a sequence homologous to a sequence adjacent to the target nucleic acid cleavage site. In some cases, HDR can insert an exogenous polynucleotide sequence into the cleaved target nucleic acid. The modification of the target DNA resulting from NHEJ and / or HDR can lead to, for example, mutations, deletions, alterations, recombinations, gene corrections, gene replacements, gene tagging, transgene knock-ins, gene disruption, and / or gene knockouts.

[0362] In some embodiments, cell cultures are synchronized to increase the efficiency of the method. In some embodiments, cells in S phase and G2 phase are used for HDR-mediated gene editing. In some embodiments, cells at any stage of the cell cycle may be subjected to the method. In some embodiments, overplating cells significantly reduces the efficiency of the method. In some embodiments, the method is applied to cell cultures at a confluency of about 40%, 45%, 50%, 55%, 60%, 65%, or 70% or less.

[0363] In some embodiments, binding of the mutant polypeptide / RNA guide complex (i.e., mutant binary complex) to a target nucleic acid in a cell recruits one or more endogenous cellular molecules or pathways other than DNA repair pathways to modify the target nucleic acid. In some embodiments, binding of the mutant binary complex blocks access of one or more endogenous cellular molecules or pathways to the target nucleic acid, thereby modifying the target nucleic acid. For example, binding of the mutant binary complex can block endogenous transcription or translation machinery, resulting in reduced expression of the target nucleic acid.

[0364] In some embodiments, a method for modifying a target DNA molecule in a cell is provided. The method includes contacting the target DNA molecule inside the cell with a mutant polypeptide described herein and a single-molecule DNA-targeting RNA comprising, in 5' to 3' order, a first nucleotide segment that hybridizes to a target sequence of the target DNA molecule, a nucleotide linker, and a second nucleotide segment that hybridizes to the first nucleotide segment to form a double-stranded RNA duplex. The mutant polypeptide forms a complex with the single-molecule DNA-targeting RNA inside the cell, and the target DNA molecule is modified.

[0365] In one aspect, a method for modifying a target DNA molecule in a cell is provided. The method includes introducing into a cell a mutant polypeptide or a nucleic acid encoding the mutant polypeptide disclosed herein, and an RNA guide or a nucleic acid encoding the RNA guide described herein, or a mutant binary complex described herein, wherein the introducing step comprises introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof. In some embodiments, the introducing step comprises transfecting or transducing the cell. In some embodiments, the introducing step comprises using electroporation, injection, a gene gun, or any combination thereof. In some embodiments, the cell is selected from a prokaryotic cell, a eukaryotic cell, a plant cell, a mammalian cell, and a human cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo.

[0366] kit The present disclosure also provides kits that can be used, for example, to practice the methods described herein. In some embodiments, the kits include a variant polypeptide of the present disclosure, e.g., a variant containing at least one amino acid substitution in Table 2. In some embodiments, the kits include a polynucleotide encoding such a variant polypeptide, optionally contained in a vector, e.g., as described herein. The kits can also optionally include an RNA guide, e.g., as described herein. The RNA guide of the kits of the present disclosure can be designed to target a sequence of interest, as known in the art. The CRISPR nuclease mutant and the RNA guide can be packaged in the same vial or other container within the kit, or in separate vials or other containers, the contents of which can be mixed prior to use. The kits can further optionally include buffers and / or instructions for using the CRISPR nuclease mutant and / or the RNA guide.

[0367] All references and publications cited herein are hereby incorporated by reference. [Example]

[0368] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure, and it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0369] Example 1 - Engineering of mutant constructs In this example, mutant constructs were generated.

[0370] A DNA template containing a single mutation was constructed through two PCR steps using mutagenic forward and reverse primers ordered from IDT. In the first step, two sets of PCR reactions were performed in a 384-well plate to generate two fragments. The overlapping region of the two PCR fragments contained the desired single mutation, allowing assembly of the entire DNA template via a second PCR. In the second step, the purified fragment from the first step was used as a template for overlapping PCR (OL PCR), with Fw and Rv oligos annealed to the vector backbone as OL PCR primers. The resulting linear DNA template contained a T7 promoter, a T7 terminator, and an open reading frame for CRISPR nuclease.

[0371] These linear DNA templates were directly used in a cell-free transcription and translation system to express CRISPR nuclease mutants containing single mutations. The mutant constructs were then individually transferred into transient transfection vectors. Furthermore, DNA templates containing combination mutations were prepared by PCR and then transferred into transient transfection vectors.

[0372] Example 2 - Fluorescence Polarization Assay for Detection of Mutant Binary Complexes In this example, the ability of a CRISPR nuclease polypeptide and an RNA guide to form a binary complex is assessed through a fluorescence polarization assay.

[0373] A linear ssDNA fragment containing the reverse complement of the T7 RNA polymerase promoter sequence upstream of the direct repeat sequence and the desired 20-bp RNA guide target was synthesized by IDT. A universal T7 forward oligo was then annealed to the reverse complement ssDNA (95°C to 4°C at 5°C / min) and filled in with Klenow fragment (New England Biolabs®) for 15 minutes at 25°C to generate a linear dsDNA in vitro transcription (IVT) template. The resulting IVT template was then transcribed into RNA guides using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs®) at 37°C for 4 hours. After transcription, each RNA guide was purified using an RNA Clean and Concentrator Kit (Zymo) and stored at -20°C until use.

[0374] The RNA guide is then labeled with 6-carboxyfluorescein (6-FAM) (IDT). 25 nM of CRISPR nuclease polypeptide (wild-type or mutant polypeptide) in 1x assay buffer (20 mM Tris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl, 1 mM DTT) is titrated with increasing concentrations of labeled RNA guide (7.5-250 nM). The complex is incubated at 37 °C for 30 min, after which fluorescence polarization measurements are taken using a microplate reader (Infinite200 Pro, Tecan).

[0375] Binary complex formation at different temperatures is also examined. Furthermore, binding experiments as described above are carried out isothermally at 25, 50, 60, and 70°C.

[0376] Formation of a binary complex upon titration of a CRISPR nuclease polypeptide (wild-type or mutant polypeptide) with increasing concentrations of the RNA guide (or formation of a binary complex upon titration of an RNA guide with increasing concentrations of the CRISPR nuclease polypeptide) results in a change in the fluorescence polarization signal in millipolarization (mP). A binding curve is generated by plotting the change in fluorescence polarization signal over a range of RNA guide concentrations.

[0377] This example demonstrates how the binding affinity of a CRISPR nuclease polypeptide (wild-type or mutant polypeptide) to an RNA guide can be determined and compared.

[0378] Example 3 - RNA electrophoretic mobility shift assay for detection of mutant binary complexes This example describes the use of an RNA electrophoretic mobility shift assay (EMSA) to determine the ability of a CRISPR nuclease polypeptide (wild-type or mutant) to bind to an RNA guide.

[0379] Synthetic RNA guides (IDT™) are labeled with 5'IRDye® 800CW using the 5'EndTag Labeling Kit (Vector® Labs) and IRDye® 800CW Maleimide (LI-COR® Biosciences), as previously detailed in Yan et al., 2018. After labeling, the RNA guides are purified and concentrated via phenol-chloroform extraction. The concentration is quantified by Nanodrop™.

[0380] For RNA binding assays, CRISPR nuclease polypeptides (wild-type or mutant polypeptides) are diluted to 2.5 μM in 1x binding buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9). The polypeptides are then serially diluted from 2.5 μM to 37.5 μM in 1x binding buffer. The polypeptides are again diluted 1:10 in 1x binding buffer plus 50 nM IR800-labeled RNA guide and mixed thoroughly. These reactions can also contain 0.5–5 μg of tRNA, which acts as a competitive inhibitor to reduce nonspecific binding of the polypeptide to RNA, thus facilitating accurate determination of specific binding. The reactions are incubated at 37°C for 1 hour. For full stain visualization, 1 μL of 100× bromophenol blue was added to the reaction, and the entire reaction was loaded onto a 6% DNA Retardation Gel (ThermoFisher®) and run for 90 minutes at 80 V. Gels were imaged on a Licor® Odyssey® CLx.

[0381] This assay is based on the principle that the rate at which RNA migrates through a gel is determined by its size. An RNA-only sample can migrate a specific distance. However, if the RNA binds to a polypeptide, a larger, slower-migrating band representing the RNA complex appears, which is "upshifted" on the gel.

[0382] Therefore, two bands are measured: 1) an RNA-only band, and 2) an "upshifted" RNA band bound to the polypeptide. If all RNA is bound to the polypeptide, only the upshifted band is observed. As the concentration of the polypeptide decreases, the intensity of the upshifted band decreases, while the intensity of the RNA-only band increases. In comparing the RNA binding affinity of a CRISPR nuclease polypeptide (wild-type or mutant polypeptide), higher polypeptide / RNA affinity is characterized by more specific binding at lower concentrations of the polypeptide.

[0383] This example demonstrates how the binding affinity of a wild-type CRISPR nuclease polypeptide to an RNA guide and the binding affinity of a mutant polypeptide to an RNA guide can be determined and compared.

[0384] Example 4 - In vitro cleavage assay for mutant binary complexes This example describes methods for preparing CRISPR nuclease RNPs and determining the in vitro biochemical activity of CRISPR nuclease (wild-type or mutant) RNPs.

[0385] The CRISPR nuclease vector was transformed into E. coli BL21(DE3) (New England Biolabs®) and expressed under the T7 promoter. Transformed cells were first grown overnight in 5 mL of Luria Broth (TEKNOVA) + 50 μg / mL kanamycin and subsequently inoculated into 1 L of Terrific Broth (TEKNOVA) + 50 μg / mL kanamycin. Cells were grown at an OD of 0.6-0.8. 600The cells are grown at 37°C until 500µL, and then protein expression is induced with 0.5mM IPTG. The culture is then grown at 18°C ​​for an additional 14-18 hours. The culture is harvested, pelleted by centrifugation, and resuspended in 1mL of extraction buffer (50mM HEPES, pH 7.5, 500mM NaCl, 5% glycerol, 0.5mM TCEP) per 5g of cell pellet. The cells are lysed using a cell disruptor (Constant System Limited) and then centrifuged at 20,000 x g for 20 minutes at 4°C to clarify the lysate. 0.2% polyethyleneimine (PEI) is added to the clarified lysate and incubated for 20 minutes at 4°C with constant end-over-end rotation. The lysate is then centrifuged again at 20,000 x g for 10 minutes. The lysate is purified via ion exchange chromatography. After purification, fractions are run on an SDS-PAGE gel, and fractions containing proteins of the appropriate size are pooled and concentrated using 30 kD Amicon Ultra15 Centrifugal Units. The protein is buffer-exchanged into 12.5 mM HEPES pH 7.0, 120 mM NaCl, 0.5 mM TCEP, and 50% glycerol. The concentration is then measured using a Nanodrop™ (ThermoFisher®) and the protein is stored at -20°C.

[0386] RNPs are prepared using a 2:1 ratio of synthetic crRNA (Integrated DNA Technologies) to protein. RNPs are complexed in 1x NEB2 buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) at 37°C for 30 minutes. After complexation, RNPs are diluted using 1x NEB2 as a dilution buffer. Apo reactions (protein without RNA guide) are prepared in the same manner and made up to the volume of crRNA with HO.

[0387] The target dsDNA substrate (Integrated DNA Technologies) was added to the RNP and apo samples at 20 nM. After thorough mixing, the reaction was incubated at 37°C for 1 hour and then quenched with 1 μL of 20 mg / mL proteinase K (ThermoFisher®). The reaction was incubated at 50°C for an additional 15 minutes, and then the entire reaction was run on a 2% agarose E-gel (ThermoFisher®). The gel was visualized with ethidium bromide on a Gel DocEZ Gel Imager (Bio-Rad®).

[0388] The intensity of two types of bands is measured: 1) full-length (uncleaved) DNA band, and 2) one or more downshifted cleaved DNA bands. Inactive RNPs are characterized by a full-length DNA band. Active RNPs result in one or more downshifted cleaved DNA bands. As the concentration of active RNP decreases, the intensity of the full-length band increases and the intensity of the cleaved bands decreases. In comparing the activities of multiple RNPs, RNPs with higher activity than others are characterized by stronger cleaved bands at lower RNP concentrations.

[0389] The method of this example allows for the comparison of the in vitro cleavage activity of wild-type or mutant CRISPR nuclease RNPs (binary complexes) on target DNA.

[0390] Example 5 - In vitro stability assays of mutant polypeptides and mutant binary complexes In this example, the stability of mutant RNPs is assessed.

[0391] For accelerated stability studies, RNP (5 μM) is produced in the same manner as described in Example 4, and samples are then stored at 25° C. for 48 hours.

[0392] In vitro cleavage assays (as described in Example 4) are performed on RNP samples. These results are compared to those in Example 4 to determine the extent to which RNPs stored at 25° C. for 48 hours retain biochemical activity.

[0393] The apo polypeptide (without the RNA guide) is also incubated for 48 hours at 25° C. An RNA EMSA assay is performed on the apo sample using the method described in Example 3. These results are compared to those of Example 3 to determine the extent to which the mutant CRISPR nuclease can form a binary complex with the RNA guide.

[0394] The apo sample incubated at 25°C for 48 hours is also complexed with an RNA guide to form RNPs using the method described in Example 4. An in vitro cleavage assay is then performed according to the method of Example 4. The results of the assay are compared to those of Example 4 to assess the activity level of the mutant RNPs formed with the protein incubated at 25°C.

[0395] The method of this example allows for comparison of the stability of wild-type and mutant polypeptides, and wild-type and mutant RNPs (binary complexes). A CRISPR nuclease polypeptide that demonstrates greater specific binding to an RNA guide than that of another CRISPR nuclease polypeptide is indicative of a more stable polypeptide. A CRISPR nuclease RNP that demonstrates more robust in vitro cleavage of target DNA than that of another CRISPR nuclease polypeptide is indicative of a more stable binary complex.

[0396] Example 6 - DNA electrophoretic mobility shift assay for detection of mutant ternary complexes This example describes the use of DNA EMSA to determine the ability of an RNA guide, a CRISPR nuclease polypeptide (wild-type or mutant polypeptide), and a target DNA substrate to form a ternary complex.

[0397] The CRISPR nuclease vector was transformed into E. coli BL21(DE3) (New England Biolabs®) for expression under the T7 promoter. Transformed cells were first grown overnight in 5 mL of Luria Broth (TEKNOVA) + 50 μg / mL kanamycin, and then inoculated into 1 L of Terrific Broth (TEKNOVA) + 50 μg / mL kanamycin. Cells were grown to an OD of 0.6–0.8. 600 The cells are grown at 37°C until 50% saturation is reached, and then protein expression is induced with 0.5 mM IPTG. The cultures are then grown at 18°C ​​for an additional 14-18 hours. The cultures are harvested, pelleted by centrifugation, and resuspended in 1 mL of extraction buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP) per 5 g of cell pellet. The cells are lysed using a cell disruptor (Constant System Limited) and then centrifuged at 20,000 x g for 20 minutes at 4°C to clarify the lysate. 0.2% polyethyleneimine (PEI) is added to the clarified lysate and incubated for 20 minutes at 4°C with constant end-over-end rotation. The lysate is then centrifuged again at 20,000 x g for 10 minutes. The lysate is purified via ion exchange chromatography. After purification, fractions were run on an SDS-PAGE gel, and fractions containing proteins of the appropriate size were pooled and concentrated using 30 kD Amicon Ultra15 Centrifugal Units. The protein was buffer-exchanged into 12.5 mM HEPES pH 7.0, 120 mM NaCl, 0.5 mM TCEP, and 50% glycerol. The concentration was then measured using a Nanodrop (ThermoFisher®), and the protein was stored at -20°C.

[0398] RNPs are prepared using a 2:1 ratio of synthetic RNA guide (Integrated DNA Technologies) to polypeptide. Targets flanked by the PAM sequences disclosed herein are selected, and the RNA guides are designed using direct repeat sequences as described herein. The RNPs are complexed in 1x NEB2 buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) at 37°C for 30 minutes. After complexation, a 5-point 1:2 serial dilution from 5 μM to 37.5 μM is performed using 1x NEB2 as the dilution buffer. The apo reaction (protein without the RNA guide) is prepared in the same manner and made up to the volume of the RNA guide with HO.

[0399] The dsDNA target substrate is generated by PCR from oligos (Integrated DNA Technologies). Prior to PCR, the 5' end of the forward primer is labeled with IR800 dye as described in Yan et al., 2018. The dsDNA is then amplified with an IR800-labeled forward primer and an unlabeled reverse primer using Amplitaq Gold (ThermoFisher®). The resulting dsDNA is purified using a DNA Clean and Concentrator®-5 Kit (Zymo Research) and quantified using Nanodrop™ (ThermoFisher®).

[0400] The RNP and apo (control) samples were diluted 1:10 in 1x binding buffer (50 mM NaCl, 10 mM Tris-HCl, 1 mM TCEP, 10% glycerol, 2 mM EDTA, pH 8.0) plus 20 nM IR800-labeled target DNA substrate and mixed thoroughly. The reaction was incubated at 37°C for 1 hour. For full-field visualization, bromophenol blue was added to the reaction, and the entire reaction was loaded onto a 6% DNA retardation gel (ThermoFisher®) and run at 80V for 90 minutes. The gel was imaged on a Licor® Odyssey® CLx.

[0401] In this assay, the rate at which DNA migrates through the gel is determined by its size. A DNA-only sample can migrate a specific distance. However, when RNPs bind to DNA, a band representing a larger, slower-migrating DNA complex appears, which is "upshifted" on the gel.

[0402] This example shows how the affinity of a mutant RNP (mutant binary complex) for a DNA target (to generate a ternary complex) can be compared to the affinity of a wild-type RNP (wild-type binary complex) for a DNA target.

[0403] Example 7 - Editing of mammalian targets using 5'-CTG-3' PAM sequences This example describes indel assessment for multiple target sequences flanking a 5'-CTG-3'PAM sequence.

[0404] The CRISPR nuclease of SEQ ID NO: 3 was cloned into the pcda3.1 backbone (Invitrogen™). RNA guides, each containing the direct repeat sequence of SEQ ID NO: 6, were cloned into the pUC19 backbone (New England Biolabs®) under a U6 promoter. The plasmids were then maxi-prepped and diluted. The RNA guides (i.e., crRNA sequences of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, and 23) and target sequences (i.e., SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, and 22) are shown in Table 4.

[0405] [Table 4]

[0406] Approximately 16 hours before transfection, 25,000 HEK293T cells in DMEM / 10% FBS + Pen / Strep were seeded into each well of a 96-well plate. On the day of transfection, cells were 70-90% confluent. For each well to be transfected, a mixture of GeneJuice® Transfection Reagent (Millipore Sigma) and OptiMEM™ (ThermoFisher®) was prepared and then incubated at room temperature for 5 minutes (Solution 1). After incubation, the GeneJuice®:OptiMEM™ mixture was added to a separate solution containing CRISPR nuclease plasmid and guide plasmid diluted in OptiMEM™ (Solution 2). For the negative control, crRNA was not included in Solution 2. The mixture of Solution 1 and Solution 2 was mixed by pipetting up and down and then incubated at room temperature for 5 minutes. After incubation, Solution 1 and Solution 2 were added dropwise to each well of the 96-well plate containing the cells. 72 hours after transfection, the cells were trypsinized by adding TrypLE™ (ThermoFisher®) to the center of each well and incubated for approximately 5 minutes. D10 medium was then added to each well and mixed to resuspend the cells. The cells were transferred to a 96-well PCR plate and centrifuged for 10 minutes. After centrifugation, the supernatant was discarded, and the pellet was resuspended in 20 μL of QuickExtract™ extraction reagent (Biosearch™ Technologies). The resuspended cell solution was incubated in a thermal cycler at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.

[0407] Samples for next-generation sequencing (NGS) were prepared by two rounds of PCR. The first round (PCR1) was used to amplify specific genomic regions depending on the target. The PCR1 product was purified by column purification. Round 2 PCR (PCR2) was performed to add Illumina adapters and indexes. The reactions were then pooled and purified by column purification. Sequencing runs were performed using a 150-cycle NextSeq™ v2.5 mid or high output kit. Edited targets were defined as targets that showed indel levels above background (>0.5% in this assay).

[0408] Figure 1 shows the percentage of NGS reads containing indels (% raw indels). The average indel percentage for eight different targets was 10.6%. This example demonstrates that a CRISPR nuclease of SEQ ID NO: 3 and an RNA guide containing a direct repeat sequence of SEQ ID NO: 6 generate indels in the target sequence adjacent to the 5'-CTG-3' PAM sequence.

[0409] Example 8 - Targeting mammalian genes with CRISPR nuclease mutants This example describes indel assessment against multiple targets using wild-type and mutant effectors (e.g., CRISPR nuclease mutants) introduced into mammalian cells by transient transfection.

[0410] The CRISPR nuclease is cloned into the pcda3.1 backbone (Invitrogen™). The plasmid is then maxi-prepped and diluted to 1 μg / μL. A target flanked by the PAM sequence disclosed herein is selected, and an RNA guide is designed using a direct repeat sequence as described herein. To prepare the RNA guide, a dsDNA fragment encoding the crRNA is derivatized with Ultramer containing the target sequence scaffold and U6 promoter. Ultramer is resuspended in 10 mM Tris·HCl, pH 7.5, to a final stock concentration of 100 μM. The working stock is then diluted to 10 μM, again using 10 mM Tris·HCl, to serve as a template for the PCR reaction. Amplification of crRNA was performed in a 50 μL reaction containing the following components: 0.02 μL of the above template, 2.5 μL of forward primer, 2.5 μL of reverse primer, 25 μL of HiFi polymerase (New England Biolabs®), and 20 μL of water. The cycling conditions were 1× (98°C for 30 seconds), 30× (98°C for 10 seconds, 67°C for 15 seconds), and 1× (72°C for 2 minutes). PCR products were cleaned with 1.8× SPRI treatment and normalized to 25 ng / μL.

[0411] Approximately 16 hours before transfection, 25,000 HEK293T cells were seeded into each well of a 96-well plate in 100 μl of DMEM / 10% FBS + Pen / Strep. On the day of transfection, cells were 70-90% confluent. For each well to be transfected, a mixture of 0.5 μl of Lipofectamine™ 2000 and 9.5 μl of OptiMEM™ was prepared and then incubated at room temperature for 5-20 minutes (Solution 1). After incubation, the Lipofectamine™:OptiMEM™ mixture was added to a separate mixture containing 182 ng of CRISPR nuclease plasmid and 14 ng of crRNA, with water up to 10 μL (Solution 2). The mixture of Solutions 1 and 2 was mixed by pipetting up and down and then incubated at room temperature for 25 minutes. After incubation, 20 μL of the mixture of Solution 1 and Solution 2 is added dropwise to each well of the 96-well plate containing the cells. 72 hours after transfection, the cells are trypsinized by adding 10 μL of TrypLE™ to the center of each well and incubated for approximately 5 minutes. 100 μL of D10 medium is then added to each well and mixed to resuspend the cells. The cells are then centrifuged at 500 g for 10 minutes and the supernatant is discarded. QuickExtract™ buffer is added to 1 / 5 of the original cell suspension volume. The cells are incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.

[0412] Samples for next-generation sequencing were prepared by two rounds of PCR. The first round (PCR1) was used to amplify a specific genomic region depending on the target. The PCR1 product was purified by column purification. Round 2 PCR (PCR2) was performed to add Illumina adapters and indexes. The reactions were then pooled and purified by column purification. Sequencing runs were performed using a 150-cycle NextSeq™ v2.5 mid or high output kit.

[0413] Edited targets are defined as targets that exhibit indel levels above background (>0.5% in this assay). Indels induced by wild-type and mutant CRISPR nucleases across the same target set are compared to identify mutant CRISPR nucleases with higher nuclease activity than the wild-type CRISPR nuclease.

[0414] Example 9 - Mammalian gene targeting with mutant polypeptides This example describes indel evaluation against multiple targets using mutants introduced into mammalian cells by transient transfection.

[0415] Variants of SEQ ID NO:3 (Tables 6-8) were cloned into the pcDNA3.1 backbone (Invitrogen®). RNA guides were cloned into the pUC19 backbone (New England Biolabs®). Plasmids were then maxi-prepped and diluted. The crRNA, target, and PAM sequences are listed in Table 5.

[0416] [Table 5]

[0417] Approximately 16 hours before transfection, 25,000 HEK293T cells in DMEM / 10% FBS + Pen / Strep (D10 medium) were seeded into each well of a 96-well plate. On the day of transfection, cells were 70-90% confluent. For each well to be transfected, a mixture of Lipofectamine™ 2000 (Invitrogen®) and Opti-MEM™ (Gibco™) was prepared and incubated at room temperature for 5 minutes (Solution 1). After incubation, the Lipofectamine 2000™:Opti-MEM™ mixture was added to a separate mixture containing the nuclease plasmid, RNA guide plasmid, and Opti-MEM™ (Solution 2). For the negative control, the RNA guide plasmid was not included in Solution 2. Solution 1 and Solution 2 were mixed by pipetting up and down and then incubated at room temperature for 25 minutes. After incubation, the mixture of Solution 1 and Solution 2 was added dropwise to each well of the 96-well plate containing the cells. Approximately 72 hours after transfection, the cells were trypsinized by adding TrypLE™ (Gibco™) to the center of each well and incubated at 37°C for approximately 5 minutes. D10 medium was then added to each well and mixed to resuspend the cells. The resuspended cells were centrifuged at 500g for 10 minutes to obtain a pellet, and the supernatant was discarded. The cell pellet was then resuspended in QuickExtract™ buffer (Lucigen®), and the cells were incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.

[0418] Samples for next-generation sequencing were prepared by two rounds of PCR. The first round (PCR1) was used to amplify specific genomic regions depending on the target. Round 2 PCR (PCR2) was performed to add Illumina adapters and indexes. Reactions were then pooled and purified by column purification. Sequencing runs were performed using a 150-cycle NextSeq 500 / 550 Mid or High Output v2.5 kit (Illumina®).

[0419] [Table 6]

[0420] [Table 7-1]

[0421] [Table 7-2]

[0422] [Table 7-3]

[0423] [Table 7-4]

[0424] [Table 7-5]

[0425] [Table 8-1]

[0426] [Table 8-2]

[0427] As shown in Tables 6-8, multiple mutants containing a single amino acid substitution, such as D509R, or two to five amino acid substitutions, such as L580G and L385G, resulted in at least a two-fold increase in indel activity.

[0428] Example 10 - Mammalian gene targeting with mutant polypeptides This example describes indel assessment against multiple targets using mutant polypeptides introduced into mammalian cells by transient transfection.

[0429] Additional combinatorial mutants of SEQ ID NO: 3 were analyzed using the assay and three targets described in Example 9. Combination mutations that result in at least a 2-fold increase in indel activity, such as K136G, N220R, and M380R, are shown in Table 9.

[0430] [Table 9]

[0431] Enumeration of Embodiments The following enumerated embodiments are provided, the numbering of which should not be construed as designating a level of importance.

[0432] Embodiment 1 provides a variant polypeptide, or a composition comprising the variant polypeptide, wherein the variant polypeptide comprises a modification relative to a parent polypeptide, wherein the parent polypeptide comprises SEQ ID NO: 3, wherein the variant polypeptide is capable of binding to an RNA guide and a target nucleic acid, and wherein the variant polypeptide or a complex comprising the variant polypeptide exhibits increased enzymatic activity, increased binding activity, increased binding specificity, and / or increased stability compared to the parent polypeptide or a complex comprising the parent polypeptide.

[0433] Embodiment 2 pro...

Claims

1. The amino acid sequence of SEQ ID NO: 3 includes modifications, such as D509R, S511R, I521R, D535G, Q514G, L516R, L516G, E198R, S527R, D509K, D509G, L580G, V359R, D535K, Y381R, R354G, M380K, M3 80R, V383R, L580R, E367R, I521K, E367G, E507R, I521G, W350R, D535R, R528K, S5 27K, L385G, W350G, L516K, Y381G, H532R, D129R, P615R, G578R, M380G, V595G, R5 31G, D129G, R531K, D158G, N476G, G578K, A512R, P618R, V595R, V595K, V383G, A597G, Y381K, P618G, E198K, T288R, E507K, L385R, C538G, P615G, D386R, S511K, C371G, K136G, N220R, S78K, K141G, K240R, D277R, T165R or K374R, and which comprises at least 90% identity to the amino acid sequence of SEQ ID NO:3 or differs from the amino acid sequence of SEQ ID NO:3 by 1 to 20 amino acid residues.

2. The modification is i) D509R, ii) S511R, iii) I521R, iv) D535G, and v) Q514G 2. The mutant polypeptide of claim 1, selected from the group consisting of:

3. The variant polypeptide of claim 1 further comprising a second modification to the amino acid sequence of SEQ ID NO:

3.

4. i) the modification comprises L580G and the second modification comprises L385G ii) the modification comprises D509R and the second modification comprises M380R; iii) the modification comprises L580G and the second modification comprises A512R; iv) the modification comprises S527R and the second modification comprises C538G; v) the modification comprises D129R and the second modification comprises P618R; vi) the modification comprises Q514G and the second modification comprises A512R; or vii) the second modification comprises an insertion of a polypeptide domain, the insertion being at the N-terminus, C-terminus or internally of the sequence of SEQ ID NO: 3; The mutant polypeptide of claim 3.

5. The variant polypeptide of claim 3 further comprising a third modification to the amino acid sequence of SEQ ID NO:

3.

6. i) the modification comprises D535G, the second modification comprises L516R, and the third modification comprises I521R; ii) the modification comprises L516R, the second modification comprises Q514G, and the third modification comprises I521R; iii) the modification comprises D509R, the second modification comprises L516R, and the third modification comprises I521R; iv) the modification comprises D535G, the second modification comprises L516R, and the third modification comprises Q514G; v) the modification comprises D535G, the second modification comprises Q514G, and the third modification comprises I521R; vi) the modification comprises K136G, the second modification comprises N220R, and the third modification comprises M380R; vii) the modification comprises S78K, the second modification comprises E198R, and the third modification comprises R354G; viii) the modification comprises K141G, the second modification comprises N220R, and the third modification comprises M380R; ix) the modification comprises K141G, the second modification comprises K240R, and the third modification comprises M380R; or x) the modification comprises K141G, the second modification comprises D277R, and the third modification comprises M380R; The mutant polypeptide of claim 5 .

7. The mutant polypeptide of claim 5 further comprising a fourth modification.

8. 8. The mutant polypeptide of claim 7, wherein the modification comprises Q514G, the second modification comprises I521R, the third modification comprises L580G, and the fourth modification comprises E198R.

9. The mutant polypeptide of claim 7 further comprising a fifth modification.

10. 10. The mutant polypeptide of claim 9, wherein the modification comprises D509R, the second modification comprises D535G, the third modification comprises L516R, the fourth modification comprises Q514G, and the fifth modification comprises I521R.

11. 10. The variant polypeptide of claim 9, further comprising a sixth modification, and optionally further comprising a seventh, eighth, ninth and tenth modification.

12. 10. The mutant polypeptide of claim 9, wherein the second modification, the third modification, the fourth modification, or the fifth modification each independently comprises a substitution, an insertion, or a deletion. (i) the mutant polypeptide or a complex comprising the mutant polypeptide exhibits enhanced enzymatic activity and / or enhanced stability compared to the polypeptide of SEQ ID NO: 3; or (ii) The variant polypeptide of claim 1, wherein the variant polypeptide exhibits enhanced binding or specificity for an RNA guide compared to the polypeptide of SEQ ID NO:

3.

14. (i) the enhancement of the enzyme activity is enhancement of nuclease activity; or (ii) the variant polypeptide or a complex comprising the variant polypeptide comprises at least a 1.5-fold, 2-fold, 2.5-fold or 3-fold increase in enzymatic activity compared to the polypeptide of SEQ ID NO: 3, e.g., as measured by indel activity, e.g., as described in Examples 9 and 10; The mutant polypeptide of claim 13. (i) comprising a RuvC domain or a split RuvC domain; (ii) contains one or more catalytic residues (e.g., aspartic acid or glutamic acid); (iii) comprises one or more catalytic residues, said one or more catalytic residues comprising D345 and E506; (iv) a polypeptide with reduced or no nuclease activity; (v) further comprising a peptide tag, a fluorescent protein, a base editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcriptional modulator, a photogating factor, a chemically inducible factor, or a chromatin visualization factor; and / or (vi) capable of binding to an RNA guide; The mutant polypeptide of claim 1.

16. 1. An RNA guide or a nucleic acid encoding said RNA guide, comprising a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 4-6, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and wherein the nucleotide immediately 3' to the direct repeat sequence is selected from C, T, or G.

17. 10. A system comprising the mutant polypeptide of claim 1 or a first nucleic acid encoding said mutant polypeptide, and an RNA guide or a second nucleic acid encoding said RNA guide, wherein said RNA guide comprises a direct repeat sequence and a spacer sequence.

18. (i) the direct repeat sequence comprises a nucleotide sequence having at least 90% or 95% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6; (ii) the spacer sequence comprises 15 to 35 nucleotides in length; and / or (iii) the first nucleic acid is located in a first vector and the second nucleic acid encoding the RNA guide is located in a vector (e.g., the first vector or a second vector), and optionally the first and / or second vector is a viral vector; 20. The system of claim 17.

19. The target nucleic acid is (i) comprising a sequence complementary to a nucleotide sequence in the spacer sequence; (ii) adjacent to a protospacer adjacent motif (PAM) sequence, said PAM sequence comprising a nucleotide sequence designated as 5'-NTN-3', 5'-HTN-3', or 5'-TNA-3', where N is any nucleotide and H is A, C, or T; (iii) the target nucleic acid is double-stranded DNA; and / or (iv) the PAM sequence comprises a nucleotide sequence designated as 5'-CTG-3' or 5'-CTC-3'; 20. The system of claim 17.

20. A nucleic acid encoding the mutant polypeptide of claim 1.

21. The nucleic acid, (i) is codon-optimized for expression in cells; (ii) further comprising a sequence encoding a guide RNA; (iii) operably linked to a promoter; and / or (iv) The nucleic acid of claim 20, comprising RNA (e.g., mRNA).

22. 21. The nucleic acid of claim 20 in a vector.

23. 23. The vector of claim 22, wherein the vector comprises a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector, and optionally, the viral vector is an adeno-associated viral (AAV) vector.

24. 24. A composition comprising a mutant polypeptide according to any one of claims 1 to 15, a system according to any one of claims 17 to 19, a nucleic acid according to any one of claims 20 to 22, or a vector according to claim 23, wherein the composition is present in a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

25. A cell comprising the mutant polypeptide of any one of claims 1 to 15, the system of any one of claims 17 to 19, the nucleic acid of any one of claims 20 to 22, or the vector of claim 23.

26. The cell described in claim 25, wherein the cell is a eukaryotic cell, optionally the cell is a mammalian cell or a plant cell, and further optionally the cell is a human cell.

27. 16. A method for producing a mutant polypeptide according to any one of claims 1 to 15, said method comprising: (A) (i) generating a nucleic acid sequence encoding the variant polypeptide, (ii) introducing the nucleic acid sequence into a suitable host cell capable of expressing the nucleic acid sequence, and (iii) enabling the host cell to express the variant polypeptide; or (B) A method comprising: (i) introducing one or more nucleotide substitutions into a nucleic acid comprising SEQ ID NO:1 or SEQ ID NO:2 to generate a mutant nucleic acid encoding the mutant polypeptide; and (ii) expressing the mutant polypeptide from the mutant nucleic acid.

28. 16. A composition comprising a mutant polypeptide of any one of claims 1 to 15 or a nucleic acid encoding said mutant polypeptide, for use in a method of delivering said mutant polypeptide to a cell, said method comprising introducing said mutant polypeptide or a nucleic acid encoding said mutant polypeptide into said cell, and optionally introducing an RNA guide or a nucleic acid encoding said RNA guide, wherein said introducing optionally comprises introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof.

29. A composition for use in a method for modifying a target DNA molecule in a cell, comprising a mutant polypeptide according to any one of claims 1 to 15 or a nucleic acid encoding said mutant polypeptide, said method comprising the steps of introducing said mutant polypeptide or a nucleic acid encoding said mutant polypeptide into said cell, and introducing an RNA guide or a nucleic acid encoding said RNA guide, said introducing step optionally comprising introducing a nanoparticle, a liposome, an exosome, a microvesicle, a viral vector, or any combination thereof.

30. The method comprising: (i) the step of introducing into the cell comprises transfecting or transducing the cell; (ii) the step of introducing into the cell comprises the use of electroporation, injection, a gene gun, or any combination thereof; and / or (iii) the nucleic acid encoding the variant polypeptide comprises RNA (e.g., mRNA); 30. The composition of claim 28, further comprising:

31. The method comprising: (i) the step of introducing into the cell comprises transfecting or transducing the cell; (ii) the step of introducing into the cell comprises the use of electroporation, injection, a gene gun, or any combination thereof; and / or (iii) the nucleic acid encoding the variant polypeptide comprises RNA (e.g., mRNA); 30. The composition of claim 29, further comprising:

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