Search

CN-121975771-A - Compositions and methods for targeting proteins to specific loci in the genome

CN121975771ACN 121975771 ACN121975771 ACN 121975771ACN-121975771-A

Abstract

Compositions and methods for targeting proteins to specific loci in the genome and uses thereof are disclosed. In one aspect, the disclosed methods allow targeting of a protein to a specific locus in the genome of an organism, including the step of providing a fusion protein comprising a DNA localization component and an effector molecule. Preferred embodiments of the present disclosure include, but are not limited to, fusion proteins of dSaCas9-Clo051, dCAS9-Clo051, xanthomonas-TALE-Clo 051, and Ralstonia-TALE-Clo 051.

Inventors

  • E. oester
  • T. leaf history
  • X.LI

Assignees

  • 波赛达治疗公司

Dates

Publication Date
20260505
Application Date
20160616
Priority Date
20150617

Claims (10)

  1. 1. A fusion protein comprising a DNA localization component and an effector molecule.
  2. 2. The fusion protein of claim 1, wherein the DNA localization component comprises at least one guide RNA (gRNA).
  3. 3. The fusion protein of claim 2, wherein the DNA localization component comprises two guide RNAs (grnas), wherein a first gRNA specifically binds to a first strand of a double-stranded DNA target sequence and a second gRNA specifically binds to a second strand of the double-stranded DNA target sequence.
  4. 4. The fusion protein of claim 1, wherein the DNA localization component comprises a DNA binding domain of a transcriptional activator-like effector nuclease (TALEN).
  5. 5. The fusion protein of any one of the preceding claims, wherein the effector molecule comprises a homodimer.
  6. 6. The fusion protein of any one of the preceding claims, wherein the effector molecule comprises a heterodimer.
  7. 7. The fusion protein of any one of the preceding claims, wherein the effector molecule comprises a nuclease or endonuclease.
  8. 8. The fusion protein of claim 7, wherein the effector molecule comprises a type IIS endonuclease.
  9. 9. The fusion protein of claim 7 or 8, wherein the effector molecule comprises AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I or Clo051.
  10. 10. The fusion protein of any one of claims 1, 2, 3, 5, 6, 7, or 8, wherein the effector molecule comprises Cas9, a Cas9 nuclease domain, or a fragment thereof.

Description

Compositions and methods for targeting proteins to specific loci in the genome The application is a divisional application of the application application of which the application date is 2016, 6 and 16, china national application number is 201680047154.3, and the application name is 'composition and method for targeting proteins to specific loci in genome'. Technical Field The present invention relates to compositions and methods for targeted genetic modification. Background There are many situations where it is desirable to locate a protein at a specific locus in the genome of an organism in order for the protein to perform a specific function. One example of a desire to localize a protein to a specific location in the genome is in the case of gene editing. In an example of such a gene editing tool, the DNA binding domain is fused to the nuclease domain via a peptide bond through a covalent bond. The present disclosure provides compositions and methods for fusion proteins for gene editing with excellent efficacy. Disclosure of Invention The present disclosure provides compositions and methods for targeting proteins to one or more specific loci in the genome of an organism. After contacting the genome with the compositions or polypeptides of the present disclosure, one or more strands of double-stranded DNA may be cleaved. If cleavage is performed in the presence of one or more DNA repair pathways or components thereof, gene expression may be interrupted or genomic sequence modifications provided by insertions, deletions or substitutions of one or more base pairs. The compositions and methods of the present disclosure provide excellent and unexpectedly effective nuclease activity at one or more target loci in the genome. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule. In certain embodiments of the fusion proteins of the present disclosure, the DNA localization component may comprise, consist essentially of, or consist of at least one guide RNA (gRNA). In certain aspects of these embodiments, the DNA localization component may comprise, consist essentially of, or consist of two guide RNAs (grnas), wherein a first gRNA specifically binds to a first strand of a double-stranded DNA target sequence and a second gRNA specifically binds to a second strand of the double-stranded DNA target sequence. In certain embodiments of the present disclosure, the DNA localization component may comprise, consist essentially of, or consist of at least one guide RNA (gRNA), and the effector molecule may comprise, consist essentially of, or consist of a Cas9, cas9 nuclease domain, or fragment thereof. In certain embodiments of the present disclosure, the DNA localization component may comprise, consist essentially of, or consist of at least one guide RNA (gRNA), and the effector molecule may comprise, consist essentially of, or consist of an inactivated Cas9 (dCas 9) or an inactivated nuclease domain. In certain embodiments of the present disclosure, the DNA localization component may comprise, consist essentially of, or consist of at least one guide RNA (gRNA), and the effector molecule may comprise, consist essentially of, or consist of inactive small Cas9 (dscas 9). In each of these embodiments, the effector molecule can comprise, consist essentially of, or consist of Cas9, dCas9, dscas 9, or a nuclease domain thereof, and a second endonuclease. The second endonuclease may comprise, consist essentially of, or consist of a type IIS endonuclease. The second endonuclease may comprise, consist essentially of, or consist of a type IIS endonuclease, including but not limited to AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments, the effector molecule may comprise, consist essentially of, or consist of dCas9 or a nuclease domain thereof and a type IIS endonuclease. The second endonuclease may comprise, consist essentially of, or consist of a type IIS endonuclease, including but not limited to AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I or Clo051. In certain embodiments, the effector molecule may comprise, consist essentially of, or consist of dCas9 or a nuclease domain thereof, and does not comprise, consist essentially of, or consist of fokl. In certain embodiments, the effector molecule may comprise, consist essentially of, or consist of a h