Inhibition of unintended mutations in gene editing

Abstract

Provided are fusion proteins and related molecules useful for conducting base editing with reduced or no off-target mutations. The fusion protein may include a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitor, and a protease cleavage site between the first fragment and the second fragment. Also provided are improved prime editing systems, including prime editing guide RNA with improved stability.

1 . A pair of helper guide RNA/guide RNA for editing a human PCSK9 nucleic acid sequence, wherein the guide RNA specifically targets a first site on the PCSK9 nucleic acid to enable base editing to convert a non-stop codon to a stop codon, and wherein the helper guide RNA specifically targets a second site on the PCSK9 nucleic acid that is 20 to 100 bases from the first site. 
     
     
         2 . The pair of helper guide RNA/guide RNA of  claim 1 , wherein the non-stop codon is CAG, CAA, or CGA. 
     
     
         3 . The pair of helper guide RNA/guide RNA of  claim 1 , wherein the helper guide RNA specifically binds to a sequence that is 7-23 nucleotides in length. 
     
     
         4 . The pair of helper guide RNA/guide RNA of  claim 1 , wherein the helper guide RNA specifically binds to a sequence that is 8-15 nucleotides in length. 
     
     
         5 . The pair of helper guide RNA/guide RNA of  claim 1 , wherein the helper guide RNA specifically binds to a sequence within the first site, and the guide RNA specifically binds to a sequence within the second site. 
     
     
         6 . The pair of helper guide RNA/guide RNA of  claim 5 , wherein the second site comprises the sequence of any one of SEQ ID NO:166-180. 
     
     
         7 . The pair of helper guide RNA/guide RNA of  claim 5 , wherein the second site and the first site, respectively, comprises the sequences of:
 SEQ ID NO:166 and 181;   SEQ ID NO:167 and 182;   SEQ ID NO:168 and 183;   SEQ ID NO:169 and 184;   SEQ ID NO:170 and 185;   SEQ ID NO:171 and 186;   SEQ ID NO:172 and 187;   SEQ ID NO:173 and 188;   SEQ ID NO:174 and 189;   SEQ ID NO:175 and 190;   SEQ ID NO:176 and 191;   SEQ ID NO:177 and 192;   SEQ ID NO:178 and 193;   SEQ ID NO:179 and 194; or   SEQ ID NO:180 and 195.   
     
     
         8 . One or more polynucleotide sequences encoding the helper guide RNA and guide RNA of  claim 1 . 
     
     
         9 . A method of inactivating a PCSK9 gene in a cell, comprising contacting the cell with a pair of helper guide RNA and guide RNA of  claim 1 , a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein, and a nucleobase deaminase.