Chemical-inducible genome engineering technology

Patent metadata

Field	Value
Publication number	US-11261435-B2
Application number	US-201615773995-A
Country	US
Kind code	B2
Filing date	Nov 7, 2016
Priority date	Nov 5, 2015
Publication date	Mar 1, 2022
Grant date	Mar 1, 2022

Abstract

The present disclosure refers to an endonuclease-based gene editing construct, wherein the construct comprises a CRISPR-associated endonuclease (such as Cas9 or Cpf1) or a derivative thereof and at least one or more hormone binding domains of the estrogen receptor (ERT2) or derivatives thereof. The present disclosure also describes a method of editing a genome of a host cell using the construct as disclosed herein, the method comprising transfecting the host cell with the nucleic acid sequence as defined herein and incubating the cell with an inducing agent.

First claim

What is claimed is:

1. An endonuclease-based gene editing fusion protein construct, wherein the fusion protein construct comprises the following components:
(a) a CRISPR-associated endonuclease or a derivative thereof; and
(b) at least two hormone binding domain of the estrogen receptor (ERT2) comprising SEQ ID NO: 4 or derivatives thereof capable of ligand binding; and
(c) two or more selected from the group consisting of a localization sequence, a binding tag, a self-cleaving peptide, and a selectable marker;
wherein said construct comprises formula (I):

wherein A is absent or is the hormone binding domain of the estrogen receptor (ERT2) or the derivatives thereof, or a binding tag;
wherein B is a localization sequence or derivatives thereof, or a binding tag, or absent;
wherein both C 1 and C 2 are present or only C 1 is present; wherein C 1 and C 2 are each independently selected from the group consisting of a localization sequence, derivatives thereof of the localization sequence, and the hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivatives thereof;
wherein X is a CRISPR-associated endonuclease or a derivative thereof;
wherein D is selected from the group consisting of the hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivatives thereof, a localization sequence and derivatives of the localization sequence;
wherein E is absent or is selected from the group consisting of the hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivatives thereof and a self-cleaving peptide;
wherein F is absent or is selected from the group consisting of a self-cleaving peptide and a selectable marker;
wherein G is absent or is a selectable marker;
wherein L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 and L 8 are linker sequences and at least one of the linker sequences is present and each of the linkers sequences is independently between 1 to 25 amino acids long;
wherein, if any one or more of the linker sequences of L 1 to L 8 is absent, the neighbouring substituents are bound by a peptide bond;
wherein L 1 is selected from the group consisting of PR, TG, TGPGPGGS (SEQ ID NO: 370), TGPGPGGSAGDTTGPGTGPG (SEQ ID NO: 371) and TGGGS (SEQ ID NO: 372);
wherein L 2 is selected from the group consisting of PRGGS (SEQ ID NO: 373), GGSPRGGS (SEQ ID NO: 374), PR, and TPGGPRGGS (SEQ ID NO: 375);
wherein L 3 is selected from the group consisting of PG, SGSEGA (SEQ ID NO: 376), GASGSKTPG (SEQ ID NO: 377), SGSETPGTSESAGA (SEQ ID NO: 378), SGSETPGTGPGGA (SEQ ID NO: 379), SESATPESGA (SEQ ID NO: 380), GTSESATPESGGA (SEQ ID NO: 381), GGSGGSGA (SEQ ID NO: 382), GA, GGGS (SEQ ID NO: 383), TPESGA (SEQ ID NO: 384), SGSETPGTGA (SEQ ID NO: 385), SGSETPGTSEGA SEQ ID NO: 386), PAG, PAGGGS (SEQ ID NO: 387), SGSETPGTPGGA (SEQ ID NO: 388), TPESGPGGA (SEQ ID NO: 389) and GASGS (SEQ ID NO: 390);
wherein L 4 is GGGS (SEQ ID NO: 383);
wherein L 5 is PAG or PAGGGS (SEQ ID NO: 387);
wherein L 6 is GA;
wherein L 7 and L 8 are independently selected from the linkers as disclosed in any of L 1 to L 6 .

2. The construct of claim 1 , wherein
i) D and E are each one hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivatives thereof;
ii) A is a mutated hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivative thereof, B is a binding tag, C 1 is a localization sequence and C 2 is absent, X is a CRISPR-associated endonuclease or derivative thereof, D is a localization sequence, E is a hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivative thereof, F is a self-cleaving peptide and G is a selectable marker;
iii) wherein A is a binding tag, B is a localization sequence, C 1 is the hormone binding domain of the estrogen receptor (ERT2) of (b) of the derivative thereof and C 2 is absent, X is the CRISPR-associated endonuclease or derivative thereof, D is the localization sequence, E is the hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivative thereof, F is a self-cleaving peptide and G is a selectable marker.

3. An endonuclease-based gene editing fusion protein construct, wherein the fusion protein construct comprises the following components:
(a) a CRISPR-associated endonuclease or a derivative thereof; and
(b) at least two hormone binding domain of the estrogen receptor (ERT2) comprising SEQ ID NO: 4 or derivatives thereof capable of ligand binding; and
(c) two or more selected from the group consisting of a localization sequence, a binding tag, a self-cleaving peptide, and a selectable marker;
wherein said construct comprises formula (II):

wherein B is a localization sequence or derivatives thereof, or a binding tag;
wherein both C 1 and C 2 are present or only C 1 is present; wherein C 1 and C 2 are each independently selected from the group consisting of a localization sequence, derivatives thereof of the localization sequence, and the hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivatives thereof;
wherein X is a CRISPR-associated endonuclease or a derivative thereof,
wherein D is selected from the group consisting of the hormone binding domain of the estrogen receptor (ERT2) or the derivatives thereof, a localization sequence and derivatives of the localization sequence;
wherein E is absent or is selected from the group consisting of the hormone binding domain of the estrogen receptor (ERT2) or the derivatives thereof and a self-cleaving peptide;
wherein F is absent or is selected from the group consisting of a self-cleaving peptide and a selectable marker;
wherein G is absent or is a selectable marker;
wherein L 1 , L 2 , L 3 , L 4 , L 5 , L 7 and L 8 are linker sequences; wherein at least one of the linker sequences is present and each of the linkers sequences is independently between 1 to 25 amino acids long;
wherein, if any one or more of the linker sequences of L 1 to L 8 is absent, the neighbouring substituents are bound by a peptide bond;
wherein L 1 is selected from the group consisting of PR, TG, TGPGPGGS (SEQ ID NO: 370), TGPGPGGSAGDTTGPGTGPG (SEQ ID NO: 371) and TGGGS (SEQ ID NO: 372);
wherein L 2 is selected from the group consisting of PRGGS (SEQ ID NO: 373), GGSPRGGS (SEQ ID NO: 374), PR, and TPGGPRGGS (SEQ ID NO: 375);
wherein L 3 is selected from the group consisting of PG, SGSEGA (SEQ ID NO: 376), GASGSKTPG (SEQ ID NO: 377), SGSETPGTSESAGA (SEQ ID NO: 378), SGSETPGTGPGGA (SEQ ID NO: 379), SESATPESGA (SEQ ID NO: 380), GTSESATPESGGA (SEQ ID NO: 381), GGSGGSGA (SEQ ID NO: 382), GA, GGGS (SEQ ID NO: 383), TPESGA (SEQ ID NO: 384), SGSETPGTGA (SEQ ID NO: 385), SGSETPGTSEGA SEQ ID NO: 386), PAG, PAGGGS (SEQ ID NO: 387), SGSETPGTPGGA (SEQ ID NO: 388), TPESGPGGA (SEQ ID NO: 389) and GASGS (SEQ ID NO: 390);
wherein L 4 is GGGS (SEQ ID NO: 383);
wherein L 5 and L 7 are independently PAG, SGS or PAGGGS (SEQ ID NO: 387);
wherein L 8 is selected from the linkers as disclosed in any of L 1 to L 5 and L 7 .

4. The construct of claim 3 , wherein
i) D is a localization sequence and E and F are each a mutated hormone binding domain of the estrogen receptor (ERT2) of (b) or the derivatives thereof;
ii) A is absent, B is localization sequence, C 1 is the hormone binding domain of the estrogen receptor (ERT2) of (b) of the derivative thereof, C 2 is absent, X is a CRI

Assignees

• Agency Science Tech & Res
• Univ Nanyang Tech

Inventors

• Tan Meng How

Classifications

• C07K14/70567

  Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title