Homologous recombination directed genome editing in eukaryotes

Abstract

Disclosed herein are synthetic nucleic acids comprising a nucleic acid sequence that encodes an ANAGO that is a species-specific to a eukaryote, and compositions comprising ANAGO and donor molecules for use in homologous recombination directed targeted gene editing in the eukaryote.

What is claimed is: 
     
         1 . A method of editing a genome of a human cell comprising:
 introducing into the human cell   (i) a species-specific codon-Adapted Nuclear Argonaute protein (ANAGO) encoded by a first nucleic acid sequence, or an in vitro messenger RNA transcribed by the first nucleic acid sequence; and   (ii) a donor nucleic acid comprising:
 a desired nucleic acid sequence that is a nucleic acid to be introduced into a target sequence, wherein introducing the desired nucleic acid sequence is induced by an ANAGO, 
 a 5′-flanking sequence, and 
 a 3′-flanking sequence, 
   wherein the 5′-flanking sequence and the 3′-flanking sequence are located on opposite sides of the desired nucleic acid sequence and independently comprise at least 10 consecutive nucleotides that are at least 90% identical to a target sequence located in the genome of the human cell;   wherein the ANAGO is a polypeptide capable of editing a target nucleic acid sequence within a human cell in the presence of the donor nucleic acid without a guide nucleic acid, wherein the ANAGO is species-specific to human, wherein the ANAGO is attached to a nuclear localization signal peptide sequence (NLS);   wherein the first nucleic acid sequence is produced by modifying a second nucleic acid sequence of a microbial species, and wherein the second nucleic acid sequence comprises a coding region that is capable of encoding a microbial Argonaute protein that has endonuclease activities in a microorganism;   wherein the modifying comprises replacing microbial preferred codons of the second nucleic acid sequence with codons that have preferential usage in the human cell; and   wherein the first nucleic acid sequence comprises at least 85% identity to the nucleic acid sequence of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; or SEQ ID NO:4.   
     
     
         2 . The method of  claim 1 , wherein the first nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO:1. 
     
     
         3 . The method of  claim 1 , wherein the first nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO:2. 
     
     
         4 . The method of  claim 1 , wherein the first nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO:3. 
     
     
         5 . The method of  claim 1 , wherein the first nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO:4. 
     
     
         6 . The method of  claim 1 , wherein the ANAGO is introduced into the human cell, wherein the ANAGO is encoded by the first nucleic acid sequence. 
     
     
         7 . The method of  claim 1 , wherein the in vitro messenger RNA is introduced into the human cell, wherein the in vitro messenger RNA is transcribed by the first nucleic acid sequence. 
     
     
         8 . The method of  claim 1 , wherein the ANAGO is cloned into a mammalian expression vector before being introduced into the human cell. 
     
     
         9 . The method of  claim 8 , wherein the mammalian expression vector is a plasmid vector, a lentiviral vector, an adeno-associated viral vector, or any viral vector. 
     
     
         10 . The method of  claim 1 , wherein a promoter is operably linked to the first nucleic acid sequence. 
     
     
         11 . The method of  claim 1 , wherein the ANAGO is stably expressed after being introduced into the human cell. 
     
     
         12 . The method of  claim 1 , wherein the donor nucleic acid is a single-strand molecule. 
     
     
         13 . The method of  claim 1 , wherein the donor nucleic acid is a double-strand molecule. 
     
     
         14 . The method of  claim 1 , wherein the 5′-flanking sequence and the 3′-flanking sequence each contain 10 nucleotides to 500 nucleotides. 
     
     
         15 . The method of  claim 1 , wherein each of the 5′-flanking sequence and the 3′-flanking sequence comprise at least 10 nucleotides that are identical to the target sequence. 
     
     
         16 . The method of  claim 1 , wherein the donor nucleic acid is introduced into the human cell to target different sites of the target sequence for multiplex genome editing at the same time. 
     
     
         17 . The method of  claim 1 , further comprising introducing into the human cell one or more donor nucleic acids of  claim 1  at same time to target different sites of the target sequence for multiplex genome editing at the same time. 
     
     
         18 . The method of  claim 1 , wherein the ANAGO is introduced into the human cell via viral vector, electroporation, lipofection, nucleofection, nanoparticle, or microinjection. 
     
     
         19 . The method of  claim 1 , wherein the editing of the genome of the human cell occurs in a homologous sequence-dependent manner. 
     
     
         20 . The method of  claim 1 , wherein genome editing in the human cell results in modification of the target sequence. 
     
     
         21 . The method of  claim 20 , wherein the modification comprises a deletion, an insertion, replacement of one or more nucleotides, or a combination thereof. 
     
     
         22 . The method of  claim 1 , wherein the ANAGO and the donor nucleic acid are present in a pharmaceutical composition comprising one or more of a pharmaceutical acceptable excipient, diluent, additive, or carrier. 
     
     
         23 . The method of  claim 1 , wherein genome editing in the human cell permanently inactivates a PCSK9 gene. 
     
     
         24 . The method of  claim 1 , wherein genome editing in the human cell is for use in gene therapy.