公开(公告)号：US20230235364A1

公开(公告)日：2023-07-27

申请号：US18046066

申请日：2022-10-12

Applicant: TECHNISCHE UNIVERSITÄT DRESDEN

Inventor： Frank BUCHHOLZ ,  Martin SCHNEIDER ,  Felix LANSING

IPC: C12N15/90 ,  C12N9/22 ,  C12N15/10 ,  C12Q1/6869

CPC classification number: C12N15/907 ,  C12N9/22 ,  C12N15/1058 ,  C12Q1/6869

Abstract: The invention provides methods and means for specifically altering the DNA sequence in a genome, in particular for genome editing by deleting or replacing a sequence of interest. Advantageously, the invention uses two non-identical sequences naturally occurring in a genome as target sites two which DNA-recombining enzymes are generated. The invention is in particular useful for medicine, in particular to repair a mutation in a genome or to delete predefined genetic material from cells or tissue and to cure diseases. An advantage of the invention is that it allows precise site directed altering of DNA without engaging host DNA repair pathways and thereby works without inducing random insertions and deletions (in-dels).

公开(公告)号：US20230183713A1

公开(公告)日：2023-06-15

申请号：US18055545

申请日：2022-11-15

Applicant: TECHNISCHE UNIVERSITÄT DRESDEN

Inventor： Jenna HOERSTEN ,  Felix LANSING ,  Frank BUCHHOLZ

IPC: C12N15/52 ,  C12N15/63

CPC classification number: C12N15/52 ,  C12N15/63

Abstract: The invention relates to the field of genome editing and provides a method of generating DNA recombinases or provides DNA recombinases, which efficiently and specifically recombine genomic target sequences as obligate DNA recombinase enzymes. The invention provides a genetically engineered DNA recombining enzyme comprising a complex of a first and a second recombinase enzyme, wherein said first recombinase enzyme and said second recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and/or a downstream target site of a DNA recombinase, wherein said first recombinase enzyme and said second recombinase enzyme each comprises at least one mutation in its catalytic site, wherein said first recombinase enzyme and said second recombinase enzyme carrying said at last one mutation in their catalytic site, when expressed in isolation, do not show the catalytic activity of a DNA recombinase, and wherein said first DNA recombinase enzyme and said second DNA recombinase enzyme carrying said at least one mutation in their catalytic site when co-expressed and forming a complex show the catalytic activity of a DNA recombinase. The invention further relates to nucleic acid molecules encoding said genetically engineered DNA recombinases and complexes, as well as to pharmaceutical compositions comprising the same. The invention further provides the use of said genetically engineered DNA recombinases, complexes, nucleic acid molecules and pharmaceutical compositions in medicine and specifically for treating genetic disorders such as hemophilia A.

公开(公告)号：US20210147878A1

公开(公告)日：2021-05-20

申请号：US16622937

申请日：2018-06-14

Applicant: TECHNISCHE UNIVERSITÄT DRESDEN

Inventor： Frank BUCHHOLZ ,  Martin SCHNEIDER ,  Felix LANSING

IPC: C12N15/90 ,  C12Q1/6869 ,  C12N15/10 ,  C12N9/22

Abstract: The invention provides methods and means for specifically altering the DNA sequence in a genome, in particular for genome editing by deleting or replacing a sequence of interest. Advantageously, the invention uses two non-identical sequences naturally occurring in a genome as target sites two which DNA-recombining enzymes are generated. The invention is in particular useful for medicine, in particular to repair a mutation in a genome or to delete predefined genetic material from cells or tissue and to cure diseases. An advantage of the invention is that it allows precise site directed altering of DNA without engaging host DNA repair pathways and thereby works without inducing random insertions and deletions (indels).

公开(公告)号：US20170306356A1

公开(公告)日：2017-10-26

申请号：US15634949

申请日：2017-06-27

Applicant: Technische Universität Dresden

Inventor： Frank BUCHHOLZ ,  Madina KARIMOVA

IPC: C12N15/90 ,  C12N9/12 ,  C12N15/52 ,  C12N15/85

CPC classification number: C12N15/907 ,  C12N9/12 ,  C12N9/1241 ,  C12N15/52 ,  C12N15/85 ,  C12N15/902 ,  C12N2320/33 ,  C12Y207/07

Abstract: The invention relates to the use of a protein with recombinase activity to catalyze a site-specific DNA recombination and a method for producing a site-specific DNA recombination. The invention is applicable alone or in combination with other recombinase systems for genetic manipulation, for example in medical research. The objective of the invention is solved by the use of a protein with recombinase activity to catalyze a site-specific DNA recombination at, preferably at two, recognition sites that are identical or reverse complementary to each other. The invention also includes a method for producing a site-specific DNA recombination comprising the steps of a) providing a cell comprising at least two recognition sites that are identical or reverse complementary to each other; and b) contacting a protein with recombinase activity with the recognition sites, thereby producing the site-specific DNA-recombination.

公开(公告)号：US20250145973A1

公开(公告)日：2025-05-08

申请号：US18916979

申请日：2024-10-16

Applicant: Technische Universität Dresden Körperschaft des öffentlichen Rechts

Inventor： Liliya MUKHAMETZYANOVA ,  Frank BUCHHOLZ ,  Julia TORRES RIVERA

IPC: C12N9/12 ,  A61K38/00 ,  C12N15/10

Abstract: The present invention provides a method of identifying an amino acid position of a DNA modifying enzyme for insertion of a heterologous DNA binding domain (DBD), the method comprising the steps of providing a library of DNA modifying enzymes, wherein the members of the library comprise heterologous amino acid sequence insertions throughout the DNA modifying enzyme; identifying those DNA modifying enzymes of the library that have DNA modifying activity; and identifying the position of the insertion in those DNA modifying enzymes identified in the previous step. The present invention further pertains to methods of producing DNA modifying enzymes comprising an inserted heterologous DBD, to methods for modifying a nucleic acid sequence in a cell, and to methods for evolving a DNA binding domain on desired target sequences. Further provided are nucleic acid sequences encoding such DNA modifying enzymes and DNA binding domains, respective vectors and host cells.

公开(公告)号：US20170016029A1

公开(公告)日：2017-01-19

申请号：US15216215

申请日：2016-07-21

Applicant: Technische Universität Dresden

Inventor： Frank BUCHHOLZ ,  Madina KARIMOVA

IPC: C12N15/90 ,  C12N9/12

CPC classification number: C12N15/907 ,  C12N9/12 ,  C12N9/1241 ,  C12N15/52 ,  C12N15/85 ,  C12N15/902 ,  C12N2320/33 ,  C12Y207/07

Abstract: The invention relates to the use of a protein with recombinase activity to catalyze a site-specific DNA recombination and a method for producing a site-specific DNA recombination. The invention is applicable alone or in combination with other recombinase systems for genetic manipulation, for example in medical research. The objective of the invention is solved by the use of a protein with recombinase activity to catalyze a site-specific DNA recombination at, preferably at two, recognition sites that are identical or reverse complementary to each other. The invention also includes a method for producing a site-specific DNA recombination comprising the steps of a) providing a cell comprising at least two recognition sites that are identical or reverse complementary to each other; and b) contacting a protein with recombinase activity with the recognition sites, thereby producing the site-specific DNA-recombination.

Abstract translation: 本发明涉及具有重组酶活性的蛋白质用于催化位点特异性DNA重组的用途和用于产生位点特异性DNA重组的方法。 本发明可单独应用或与用于遗传操作的其他重组酶系统组合应用，例如在医学研究中。 本发明的目的是通过使用具有重组酶活性的蛋白质来催化位点特异性DNA重组，优选在两个彼此相同或相互互补的识别位点。 本发明还包括用于产生位点特异性DNA重组的方法，包括以下步骤：a）提供包含彼此相同或相互互补的至少两个识别位点的细胞; 和b）使蛋白质与重组酶活性与识别位点接触，从而产生位点特异性DNA重组。