EXPRESSION AND PURIFICATION OF CAS ENZYMES

1 . A method for expressing and purifying a Cas13a protein, the method comprising:

(a) inserting a nucleotide sequence encoding a polypeptide having the polypeptide sequence of SEQ ID NO: 2 or 4 into an expression plasmid;

(b) transforming one or more cells with the expression plasmid;

(c) inducing expression of the transformed plasmid;

(d) isolating the cells;

(e) extracting the Cas13a protein; and

(f) purifying the protein using affinity purification and ion exchange purification.

2 . The method of claim 1 , wherein the cell comprises E. coli BL21(DE3).

3 . The method of claim 1 , wherein the expression plasmid comprises pET28 or pET28-MBP-TEV plasmids.

4 . The method of claim 1 , wherein the nucleotide sequence is inserted into the expression plasmid using isothermal assembly.

6 . The method of claim 1 , wherein the affinity purification comprises a nickel or a maltose affinity media.

7 . The method of claim 1 , wherein the affinity purification comprises affinity chromatography comprising:

(f)(1) equilibrating a nickel affinity column with a binding buffer and loading the extracted Cas13a protein;

(f)(2) washing the nickel affinity column with a wash buffer; and

(f)(3) eluting the affinity purified Cas13a protein from the nickel affinity column using an elution buffer.

8 . The method of claim 1 , wherein the affinity purification comprises affinity chromatography comprising:

(f)(1) equilibrating a maltose affinity column with a binding buffer and loading the extracted Cas13a protein;

(f)(2) washing the maltose affinity column with a wash buffer; and

(f)(3) eluting the affinity purified Cas13a protein from the maltose affinity column using an elution buffer.

9 . The method of claim 1 , wherein the ion exchange purification comprises a cation exchange media.

10 . The method of claim 1 , wherein the ion exchange purification comprises cation exchange chromatography comprising:

(f)(1) equilibrating a cation exchange column with a binding buffer and loading the extracted Cas13a protein;

(f)(2) washing the cation exchange column with a wash buffer; and

(f)(3) eluting the cation exchange purified Cas13a protein from the cation exchange column using an elution buffer.

11 . The method of claim 1 , further comprising concentrating the purified Cas13a protein to approximately 10 mg/mL.

12 . The method of claim 11 , further comprising dialyzing the concentrated purified Cas13a protein.

13 . A method for purifying a recombinant Cas13a protein, the method comprising:

(a) providing an expressed recombinant Cas13a protein having the polypeptide sequence of SEQ ID NO: 2 or 4;

(b) performing an affinity purification comprising a nickel affinity media or a maltose affinity media;

(c) performing an ion exchange purification comprising a cation exchange media; and

(d) collecting the purified Cas13a protein.

14 . The method of claim 13 , further comprising concentrating the purified Cas13a protein to approximately 10 mg/mL.

15 . The method of claim 14 , further comprising dialyzing the concentrated purified Cas13a protein against three rounds of dialysis buffer.