公开(公告)号：US12098381B2

公开(公告)日：2024-09-24

申请号：US17200398

申请日：2021-03-12

Applicant: University of Massachusetts ,  University of Central Florida Research Foundation, Inc.

Inventor： Thoru Pederson ,  Scot Andrew Wolfe ,  Hanhui Ma ,  Metewo Selase Kosi Enuameh ,  Nicola Anne Kearns ,  Ryan Michael Jude Genga ,  Rene Maehr ,  Shaojie Zhang ,  Ardalan Naseri ,  Manuel Garber

IPC: C12N15/85 ,  C12N5/0735 ,  C12N5/074 ,  C12N9/22 ,  C12N15/63 ,  C12N15/86 ,  C12N15/90 ,  C12Q1/6841

CPC classification number: C12N15/86 ,  C12N5/0606 ,  C12N5/0696 ,  C12N9/22 ,  C12N15/63 ,  C12N15/907 ,  C12Q1/6841 ,  C12N2501/65 ,  C12N2501/70 ,  C12N2510/00 ,  C12N2740/15043

Abstract: The present disclosure relates to methods of and systems for modifying the transcriptional regulation of stem or progenitor cells to promote their differentiation or reprogramming of somatic cells. Further, the labeling and editing of human genomic loci in live cells with three orthogonal CRISPR/Cas9 components allow multicolor detection of genomic loci with high spatial resolution, which provides an avenue for barcoding elements of the human genome in the living state.

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。

公开(公告)号：US20150191744A1

公开(公告)日：2015-07-09

申请号：US14571818

申请日：2014-12-16

Applicant: University of Massachusetts

Inventor： Scot Andrew Wolfe ,  Hanhui Ma ,  Thoru Pederson ,  Metewo Selase Kosi Enuameh ,  Nicola Anne Kearns ,  Ryan Michael Jude Genga ,  Rene Maehr ,  Shaojie Zhang ,  Ardalan Naseri ,  Manuel Garber

IPC: C12N15/86 ,  C12Q1/68 ,  C12N5/073 ,  C12N5/074

CPC classification number: C12N15/86 ,  C12N5/0606 ,  C12N5/0696 ,  C12N9/22 ,  C12N15/63 ,  C12N15/907 ,  C12N2501/65 ,  C12N2501/70 ,  C12N2510/00 ,  C12N2740/15043 ,  C12Q1/6841

Abstract: The present disclosure relates to methods of and systems for modifying the transcriptional regulation of stem or progenitor cells to promote their differentiation or reprogramming of somatic cells. Further, the labeling and editing of human genomic loci in live cells with three orthogonal CRISPR/Cas9 components allow multicolor detection of genomic loci with high spatial resolution, which provides an avenue for barcoding elements of the human genome in the living state.

Abstract translation: 本公开涉及用于修饰干细胞或祖细胞的转录调节以促进其体细胞分化或重编程的方法和系统。 此外，用三个正交的CRISPR / Cas9组分在活细胞中标记和编辑人类基因组座位允许以高空间分辨率对基因组基因座进行多色检测，为生命状态中人类基因组的条形码元素提供了途径。

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：US20240271112A1

公开(公告)日：2024-08-15

申请号：US18566367

申请日：2022-05-31

Applicant: UNIVERSITY OF MASSACHUSETTS

Inventor： Scot Andrew Wolfe ,  Ester Mintzer ,  Jeremy Luban ,  Jason Shohet

IPC: C12N9/22 ,  C12N15/11 ,  C12N15/90

CPC classification number: C12N9/22 ,  C12N15/11 ,  C12N15/907 ,  C12N2310/20 ,  C12N2800/80

Abstract: The present invention utilizes a Cas9 nickase which nicks a flanking target sequence to a duplicated gene sequence (e.g., a retroviral LTR). This nicking causes a genomic collapse of the sequence between the nick and the LTR, thereby deleting the sequence from the genome. Because the nickase does not introduce mutations at the target site, this method can be repeated maximize the efficiency (e.g., 100% of retroviral genome excision. For example, this method is useful to delete all PERVs within a pig genome intended for human transplantation. Further, such PERV-free cells can then be used to clone PERV-free pigs. Furthermore, this method is useful to remove amplified gene repeats in cancer cells.

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.