US 12,331,291 B2
Split complementary base editing systems based on bimolecular deaminases and uses thereof
Jianfeng Li, Guangdong (CN); Xiangyu Xiong, Guangdong (CN); Kehui Liu, Guangdong (CN); and Xionglei He, Guangdong (CN)
Assigned to SUN YAT-SEN UNIVERSITY, Guangzhou (CN)
Filed by SUN YAT-SEN UNIVERSITY, Guangdong (CN)
Filed on Jul. 19, 2024, as Appl. No. 18/777,597.
Application 18/777,597 is a continuation of application No. PCT/CN2022/106402, filed on Jul. 19, 2022.
Claims priority of application No. 202210503831.5 (CN), filed on May 10, 2022.
Prior Publication US 2024/0368590 A1, Nov. 7, 2024
Int. Cl. C12N 15/11 (2006.01); C12N 9/22 (2006.01); C12N 9/78 (2006.01); C12N 15/81 (2006.01); C12N 15/82 (2006.01); C12N 15/85 (2006.01)
CPC C12N 15/111 (2013.01) [C12N 9/22 (2013.01); C12N 9/78 (2013.01); C12N 15/81 (2013.01); C12N 15/82 (2013.01); C12N 15/85 (2013.01); C12N 2310/20 (2017.05)] 3 Claims
 
1. A complementary base editing system based on bimolecular deaminases, comprising any one selected from the group consisting of the following (1) to (5):
(1) a base editing fusion protein A, a base editing fusion protein B, and a guide RNA (gRNA);
(2) an expression construct comprising nucleotide sequences encoding the base editing fusion protein A and the base editing fusion protein B, and the gRNA;
(3) the base editing fusion protein A, the base editing fusion protein B, and an expression construct comprising the nucleotide sequence encoding the gRNA;
(4) the expression construct comprising the nucleotide sequences encoding the base editing fusion protein A and the base editing fusion protein B, and the expression construct comprising the nucleotide sequence encoding the gRNA; and
(5) an expression construct comprising the nucleotide sequences encoding the base editing fusion protein A and the base editing fusion protein B and the nucleotide sequence encoding the gRNA,
wherein the base editing fusion protein A comprises a first nCas9 polypeptide fragment, a flexible peptide linker, and a first nucleobase deaminase polypeptide fragment sequentially from N-terminus to C-terminus; the base editing fusion protein B comprises a second nucleobase deaminase polypeptide fragment, the flexible peptide linker, and a second nCas9 polypeptide fragment sequentially from N-terminus to C-terminus; the first nucleobase deaminase polypeptide fragment and the second nucleobase deaminase polypeptide fragment are derived from the same nucleobase deaminase; and
a combination of the base editing fusion protein A and the base editing fusion protein B in the base editing system is any one selected from the group consisting of the following (a) to (g):
(a) the base editing fusion protein A having an amino acid sequence shown in SEQ ID NO: 15 (Split-AID10-N), and the base editing fusion protein B having an amino acid sequence shown in SEQ ID NO: 16 (Split-AID10-C);
(b) the base editing fusion protein A having an amino acid sequence shown in SEQ ID NO: 17 (Split-AID10-N5), and the base editing fusion protein B having an amino acid sequence shown in SEQ ID NO: 18 (Split-AID10-C4);
(c) the base editing fusion protein A having an amino acid sequence shown in SEQ ID NO: 19 (Split-BE3-N), and the base editing fusion protein B having an amino acid sequence shown in SEQ ID NO: 20 (Split-BE3-C);
(d) the base editing fusion protein A having an amino acid sequence shown in SEQ ID NO: 21 (Split-A3A-N), and the base editing fusion protein B having an amino acid sequence shown in SEQ ID NO: 22 (Split-A3A-C);
(e) the base editing fusion protein A having an amino acid sequence shown in SEQ ID NO: 23 (Split-A3B-N), and the base editing fusion protein B having an amino acid sequence shown in SEQ ID NO: 24 (Split-A3B-C);
(f) the base editing fusion protein A having an amino acid sequence shown in SEQ ID NO: 29 (Split-ABE8e-N), and the base editing fusion protein B having an amino acid sequence shown in SEQ ID NO: 30 (Split-ABE8e-C); and
(g) the base editing fusion protein A having an amino acid sequence shown in SEQ ID NO: 31 (Split-ABE8e-N7), and the base editing fusion protein B having an amino acid sequence shown in SEQ ID NO: 32 (Split-ABE8e-C2).