CRISPR-aid using catalytically inactive RNA-guided endonuclease
The present invention relates to nucleobase editing complexes comprising a) a catalytically inactive RNA-guided endonuclease having a sequence identity of at least 60% to SEQ ID NO: 126 or SEQ ID NO: 155 and b) a nucleobase editing domain, as well as polynucleotides encoding said nucleobase editing complexes, nucleic acid constructs and expression vectors comprising said polynucleotides, host cells comprising said nucleobase editing complexes and/or polynucleotides, and methods for preparing and using said nucleobase editing complexes.
1 . A nucleobase editing complex comprising:
a) a catalytically inactive RNA-guided endonuclease having a sequence identity of at least 90% to SEQ ID NO: 126; and
b) a nucleobase editing domain;
wherein the catalytically inactive RNA-guided endonuclease and the nucleobase editing domain are fused end-to-end or connected via a linker polypeptide.
2 . The nucleobase editing complex according to claim 1 , wherein the catalytically inactive RNA-guided endonuclease comprises an alteration of an amino acid at a position corresponding to position 877 of SEQ ID NO: 126.
3 . The nucleobase editing complex according to claim 1 , wherein the amino acid at a position corresponding to position 877 of SEQ ID NO: 126 is Ala.
4 . The nucleobase editing domain according to claim 1 , wherein the catalytically inactive RNA-guided endonuclease comprises SEQ ID NO: 126.
5 . The nucleobase editing complex according to claim 1 , wherein the nucleobase editing domain is a cytosine base editor (CBE).
6 . The nucleobase editing complex according to claim 5 , wherein the nucleobase editing domain is a cytosine base editor of the APOBEC1/AID family.
7 . The nucleobase editing complex according to claim 5 , wherein the nucleobase editing domain is PmCDA1.
8 . The nucleobase editing complex according to claim 5 , wherein the nucleobase editing domain comprises or consists of a polypeptide having a sequence identity of at least 90% to SEQ ID NO: 128.
9 . The nucleobase editing complex according to claim 5 , wherein the nuclease editing domain comprises SEQ ID NO: 128.
10 . The nucleobase editing complex according to claim 5 , which further comprises an uracil DNA glycosylase inhibitor (UGI).
11 . The nucleobase editing complex according to claim 10 , wherein the uracil DNA glycosylase inhibitor has a sequence identity of at least 90% to SEQ ID NO: 132.
12 . The nucleobase editing complex according to claim 10 , the uracil DNA glycosylase inhibitor comprises SEQ ID NO: 132.
13 . The nucleobase editing complex according to claim 1 , wherein the nucleobase editing domain is an adenine base editor (ABE).
14 . The nucleobase editing complex according to claim 1 , wherein the nucleobase editing domain is selected from the group consisting of TadA, TadA*, TadA homodimer, and TadA-TadA* heterodimer.
15 . The nucleobase editing complex according to claim 1 , wherein the nucleobase editing domain is a TadA-TadA* heterodimer.
16 . The nucleobase editing complex according to claim 1 , wherein the catalytically inactive RNA-guided endonuclease, the linker polypeptide, and the nucleobase editing domain are encoded in frame and are expressed as a single polypeptide.
17 . The nucleobase editing complex according to claim 16 , wherein the linker polypeptide comprises at least 10 amino acid residues.
18 . The nucleobase editing complex according to claim 16 , wherein the linker polypeptide comprises at least 50 amino acid residues.
19 . The nucleobase editing complex according to claim 16 , wherein the linker polypeptide has a sequence identity at least 90% to SEQ ID NO: 130.
20 . The nucleobase editing complex according to claim 16 , wherein the linker polypeptide comprises SEQ ID NO: 130.
21 . A polynucleotide encoding a nucleobase editing complex according to claim 1 .
22 . A host cell comprising the polynucleotide according to claim 21 .
23 . The host cell according to claim 22 , which is a bacterial host cell.
24 . The host cell according to claim 23 , wherein the bacterial host cell is Bacillus licheniformis.
25 . The host cell according to claim 22 , which is a filamentous fungal host cell.
26 . The host cell according to claim 25 , wherein the filamentous fungal host cell is a Aspergillus niger, Aspergillus oryzae , or Trichoderma reesei cell.
27 . The host cell according to claim 22 , which is a yeast host cell.
28 . The host cell according to claim 27 , wherein the yeast host cell is Pichia pastoris.
29 . The host cell according to claim 22 , which is a mammalian host cell.
30 . A method for modifying at least one nucleobase in a DNA target sequence, the method comprising:
a) providing a nucleobase editing complex according to claim 1 complexed with a gRNA that is complementary to and capable of hybridizing to the DNA target sequence; and
b) contacting the nucleobase editing complex with the DNA target sequence;
wherein at least one nucleobase in the DNA target sequence is converted to a different nucleobase without introducing a double-strand break in the DNA sequence of interest.
31 . The nucleobase editing complex according to claim 1 , wherein the catalytically inactive RNA-guided endonuclease has a sequence identity of at least 95% to SEQ ID NO: 126.
32 . The nucleobase editing complex according to claim 5 , wherein the nucleobase editing domain comprises or consists of a polypeptide having a sequence identity of at least 95% to SEQ ID NO: 128.
33 . The nucleobase editing complex according to claim 10 , wherein the uracil DNA glycosylase inhibitor has a sequence identity of at least 95% to SEQ ID NO: 132.
34 . The nucleobase editing complex according to claim 16 , wherein the linker polypeptide has a sequence identity of at least 95% to SEQ ID NO: 130.