公开(公告)号：US10190106B2

公开(公告)日：2019-01-29

申请号：US14976196

申请日：2015-12-21

Applicant: University of Massachusetts

Inventor： Scot Andrew Wolfe ,  Mehmet Fatih Bolukbasi ,  Ankit Gupta ,  Erik J Sontheimer ,  Nadia Amrani

IPC: C12N9/22

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

公开(公告)号：US20250066751A1

公开(公告)日：2025-02-27

申请号：US18899677

申请日：2024-09-27

Applicant: University of Massachusetts

Inventor： Scott WOLFE ,  Mehmet Fatih Bolukbasi ,  Ankit Gupta ,  Erik J. Sontheimer ,  Nadia Amrani

IPC: C12N9/22

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

公开(公告)号：US20210395710A1

公开(公告)日：2021-12-23

申请号：US17313328

申请日：2021-05-06

Applicant: University of Massachusetts

Inventor： Scot A Wolfe ,  Mehmet Fatih Bolukbasi ,  Ankit Gupta ,  Erik J. Sontheimer ,  Nadia Amrani

IPC: C12N9/22

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

公开(公告)号：US11028380B2

公开(公告)日：2021-06-08

申请号：US16226202

申请日：2018-12-19

Applicant: University of Massachusetts

Inventor： Scot Andrew Wolfe ,  Mehmet Fatih Bolukbasi ,  Ankit Gupta ,  Erik J Sontheimer ,  Nadia Amrani

IPC: C12N9/22

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

公开(公告)号：US12098363B2

公开(公告)日：2024-09-24

申请号：US16964461

申请日：2018-07-20

Applicant: THE CHILDREN'S MEDICAL CENTER CORPORATION ,  UNIVERSITY OF MASSACHUSETTS

Inventor： Daniel E. Bauer ,  Scot Wolfe ,  Mehmet Fatih Bolukbasi ,  Benjamin Roscoe ,  Pengpeng Liu ,  Kevin Luk ,  Yuxuan Wu ,  Jing Zeng

IPC: C12N15/01 ,  A61K38/46 ,  C12N9/22 ,  C12N15/113

CPC classification number: C12N15/01 ,  A61K38/465 ,  C12N9/22 ,  C12N15/113 ,  C12N2310/20 ,  C12N2510/00 ,  C12N2800/80

Abstract: Provided herein are methods and compositions for increasing fetal hemoglobin levels in a cell by disrupting BCL11A expression at the genomic level. Also provided herein are methods and compositions relating to the treatment of hemoglobinopathies by reinduction of fetal hemoglobin levels.

公开(公告)号：US20190276810A1

公开(公告)日：2019-09-12

申请号：US16226202

申请日：2018-12-19

Applicant: University of Massachusetts

Inventor： Scot A. Wolfe ,  Mehmet Fatih Bolukbasi ,  Ankit Gupta ,  Erik J. Sontheimer ,  Nadia Amrani

IPC: C12N9/22

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

公开(公告)号：US20160177278A1

公开(公告)日：2016-06-23

申请号：US14976196

申请日：2015-12-21

Applicant: University of Massachusetts

Inventor： Scot Andrew Wolfe ,  Mehmet Fatih Bolukbasi ,  Ankit Gupta ,  Erik J. Sontheimer ,  Nadia Amrani

IPC: C12N9/22

CPC classification number: C12N9/22 ,  C07K2319/80 ,  C12Y301/00

Abstract: The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

Abstract translation: 本发明提供一种用于促进人类基因组内的单位点核酸酶基因编辑精度的Cas9平台。 例如，Cas9核酸酶/ DNA靶向单元（Cas9-DTU）融合蛋白精确地将Cas9 / sgRNA复合物递送到基因组内的特定靶位点，用于随后的相邻靶序列的sgRNA依赖性切割。 或者，使用原始相邻基序（PAM）识别结构域的突变来减弱Cas9结合使Cas9靶位点识别依赖于相关的DTU，同时保留Cas9的sgRNA介导的DNA裂解保真度。 Cas9-DTU融合蛋白具有比标准Cas9系统更高的靶位点结合精度，更大的核酸酶活性和更广泛的序列靶向范围。 现有的Cas9或sgRNA变体（例如截短的sgRNA（tru-gRNA），切口酶和FokI融合物）与这些改进相容以进一步减少脱靶裂解。 公开了一种稳健且广泛应用的策略，以使Cas9基因组编辑系统具有安全，有效临床应用所需的单基因组位点准确度。