Nucleobase editors comprising nucleic acid programmable dna binding proteins

Abstract

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cpf1 or variants thereof, and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a napDNAbp (e.g., CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute) and nucleic acid editing proteins or domains, are provided.

1 .- 207 . (canceled) 
     
     
         208 . A method for producing a ribonucleoprotein (RNP) complex, the method comprising:
 (i) complexing a base editor comprising a (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a cytidine deaminase domain; and (iii) a uracil glycosylase inhibitor (UGI) with an RNA in an aqueous solution, thereby forming a complex comprising the base editor and the RNA in the aqueous solution; and   (ii) contacting the complex of (i) with a cationic lipid.   
     
     
         209 . The method of  claim 208 , wherein the base editor and the RNA of (i) are complexed at a molar ratio from 1:1 to 1:1.5. 
     
     
         210 . The method of  claim 208 , wherein the base editor is in the aqueous solution at a concentration ranging from 10 μM to 100 μM. 
     
     
         211 . The method of  claim 208 , wherein the RNA is a sgRNA. 
     
     
         212 . The method of  claim 211 , wherein the sgRNA is from 10-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. 
     
     
         213 . The method of  claim 212 , wherein the target sequence is a DNA sequence. 
     
     
         214 . The method of  claim 213 , wherein the target sequence is in the genome of an organism. 
     
     
         215 . The method of  claim 211 , wherein the RNA comprises the amino acid sequence of SEQ ID NO: 741, wherein each of the Ts of SEQ ID NO: 741 are uracil (U). 
     
     
         216 . The method of  claim 208 , wherein the complex in the aqueous solution of (i) is contacted with the cationic lipid of (ii) at a volumetric ratio that is from 1:2 to 2:1. 
     
     
         217 . The method of any one of  claim 208 , wherein the cationic lipid is Lipofectamine®. 
     
     
         218 . The method of  claim 217 , wherein the Lipofectamine® is selected from the group consisting of Lipofectamine® 2000, Lipofectamine® 3000, Lipofectamine® MessengerMAX, Lipofectamine® LTX, and Lipofectamine® RNAiMAX. 
     
     
         219 . The method of  claim 208 , wherein the base editor further comprises (iv) a second uracil glycosylase inhibitor (UGI) domain. 
     
     
         220 . The method of  claim 219 , wherein the nucleic acid programmable DNA binding protein (napDNAbp) is a CasX, CasY, Cpf1, Cpf1 nickase, dCpf1, C2c1, C2c2, C2c3, Cas9, dCas9, Cas9 nickase or Argonaute protein. 
     
     
         221 . The method of  claim 220 , wherein the napDNAbp is a dCas9 or Cas9 nickase. 
     
     
         222 . The method of  claim 220 , wherein the napDNAbp comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 34 or 38. 
     
     
         223 . The method of  claim 219 , wherein the cytidine deaminase domain is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family. 
     
     
         224 . The method of  claim 219 , wherein the cytidine deaminase domain comprises an amino acid sequence that is at least 85% identical to an amino acid sequence of SEQ ID NO: 49-84, wherein said at least 85% amino acid sequence identity is based on an alignment against any one of SEQ ID NOs: 49-84 by NCBI Constraint-based Multiple Alignment Tool (COBALT). 
     
     
         225 . The method of  claim 219 , wherein the base editor is a fusion protein that comprises the structure:
 NH 2 -[cytidine deaminase domain]-[napDNAbp]-[first UGI domain]-[second UGI domain]-COOH;   NH 2 -[first UGI domain]-[second UGI domain]-[cytidine deaminase domain]-[napDNAbp]-COOH;   NH 2 -[napDNAbp]-[cytidine deaminase domain]-[first UGI domain]-[second UGI domain]-COOH; or   NH 2 -[first UGI domain]-[second UGI domain]-[napDNAbp]-[cytidine deaminase domain]-COOH;   wherein each instance of “]-[” comprises an optional linker.   
     
     
         226 . The method of  claim 219 , wherein the cytidine deaminase domain and the napDNAbp are linked via a linker comprising the amino acid sequence: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 605) 
                 
                     
                   SGGSSGGSSGSETPGTSESATPESSGGSSGGS. 
                 
             
                
                
               
            
           
         
       
     
     
         227 . A pharmaceutical composition produced by the method of  claim 208 . 
     
     
         228 . A method comprising delivering the pharmaceutical composition of  claim 227  to a subject.