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1. 发明申请

US20210395710A1 CAS9-CAS9 FUSION PROTEINS 有权
公开(公告)号：US20210395710A1
公开(公告)日：2021-12-23
申请号：US17313328
申请日：2021-05-06
申请人： University of Massachusetts
发明人： Scot A Wolfe , Mehmet Fatih Bolukbasi , Ankit Gupta , Erik J. Sontheimer , Nadia Amrani
IPC分类号： C12N9/22
摘要： 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.

2. 发明申请

US20230132569A1 DNase H Activity of Neisseria Meningitidis Cas9 有权
公开(公告)号：US20230132569A1
公开(公告)日：2023-05-04
申请号：US17870336
申请日：2022-07-21
申请人： UNIVERSITY OF MASSACHUSETTS
发明人： Erik J. Sontheimer , Yan Zhang
IPC分类号： C12N9/22 , C12N15/11
摘要： Many strains of the human pathogen Neisseria meningitidis carry a compact Cas9 (NmeCas9) that can serve to limit genetic exchange via natural transformation. Cas9 orthologues (including NmeCas9) have recently been adopted for RNA-guided genome engineering and DNA binding, adding to the need to define better their activities and properties. The present invention examines DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 is found able to cleave single-stranded DNA (ssDNA) targets in a manner that is RNA-guided but both PAM- and tracrRNA-independent. Beyond the requirement for guide-target pairing, this activity has no apparent sequence requirements, and the cleavage sites are measured from the 5′ end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA domains are not strictly required for enzymatic activation of NmeCas9, and expand the list of targeting activities exhibited by these revolutionary RNA-guided nucleases.

3. 发明申请

US20190382741A1 ANTI-CRISPR COMPOUNDS AND METHODS OF USE 审中-公开
公开(公告)号：US20190382741A1
公开(公告)日：2019-12-19
申请号：US16084397
申请日：2017-03-13
申请人： UNIVERSITY OF MASSACHUSETTS
发明人： Erik J. Sontheimer , Alan Davidson , Karen Maxwell
IPC分类号： C12N9/22 , C12N15/90
摘要： The present invention is related to the field of CRISPR-Cas9 gene editing platforms. In particular, the present invention has identified Type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity. Co-administration of such Acr inhibitors may provide an advantageous adjunct in permitting safe and practical biological therapeutics through spatial or temporal control of Cas9 activity; controlling Cas9-based gene drives in wild populations to reduce the ecological consequences of such forced inheritance schemes; and contributing to general research into various biotechnological, agricultural, and medical applications of gene editing technologies.

4. 发明申请

US20190338308A1 Targeted CRISPR Delivery Platforms 审中-公开
公开(公告)号：US20190338308A1
公开(公告)日：2019-11-07
申请号：US16186352
申请日：2018-11-09
申请人： University of Massachusetts
发明人： Erik J. Sontheimer , Raed Ibraheim , Wen Xue , Aamir Mir , Alireza Edraki , Ildar Gainetdinov
IPC分类号： C12N15/86 , C12N15/11 , A61K48/00
摘要： The present invention is related to compositions and methods for gene therapy. Several approaches described herein utilize the Neisseria meningitidis Cas9 system that provides a hyperaccurate CRISPR gene editing platform. Furthermore, the invention incorporates full length and truncated single guide RNA sequences that permit a complete sgRNA-Nme1Cas9 vector to be inserted into an adeno-associated viral plasmid that is compatible for in vivo administration. Furthermore, Type II-C Cas9 orthologs have been identified that target protospacer adjacent motif sequences limited to between one-four required nucleotides.

5. 发明申请

US20220290113A1 PROGRAMMABLE DNA BASE EDITING BY NME2CAS9-DEAMINASE FUSION PROTEINS 有权
公开(公告)号：US20220290113A1
公开(公告)日：2022-09-15
申请号：US17285440
申请日：2019-10-15
申请人： UNIVERSITY OF MASSACHUSETTS
发明人： Erik J. Sontheimer , Xin Gao , Aamir Mir , Alireza Edraki , Scot A. Wolfe , Pengpeng Liu
IPC分类号： C12N9/22 , C12N9/78 , C12N15/11 , C12N15/62 , C12N15/86 , A61P3/00
摘要： The present invention is related to the field of gene editing. In particular, the gene editing is directed toward single nucleotide base editing. For example, such single nucleotide base editing results in a conversion of a OG base pair to a T*A base pair. The high accuracy and precision of the presently disclosed single nucleotide base gene editor is accomplished by an NmeCas9 nuclease that is fused to a nucleotide deaminase protein. The compact nature of the NmeCas9 coupled with a larger number of compatible protospacer adjacent motifs provide the Cas9 fusion constructs contemplated herein to have a gene editing window that can edit sites that are not targetable by other conventional SpyCas9 base editor platforms.

6. 发明授权

US11530394B2 Anti-CRISPR compounds and methods of use 有权
公开(公告)号：US11530394B2
公开(公告)日：2022-12-20
申请号：US16084397
申请日：2017-03-13
申请人： UNIVERSITY OF MASSACHUSETTS , THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO
发明人： Erik J. Sontheimer , Alan Davidson , Karen Maxwell , April Pawluk , Yan Zhang , Jooyoung Lee , Nadia Amrani
IPC分类号： C12N9/22 , C12N15/90
摘要： The present invention is related to the field of CRISPR-Cas9 gene editing platforms. In particular, the present invention has identified Type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity. Co-administration of such Acr inhibitors may provide an advantageous adjunct in permitting safe and practical biological therapeutics through spatial or temporal control of Cas9 activity; controlling Cas9-based gene drives in wild populations to reduce the ecological consequences of such forced inheritance schemes; and contributing to general research into various biotechnological, agricultural, and medical applications of gene editing technologies.

7. 发明申请

US20220389447A9 Targeted CRISPR Delivery Platforms 有权
公开(公告)号：US20220389447A9
公开(公告)日：2022-12-08
申请号：US16186352
申请日：2018-11-09
申请人： University of Massachusetts
发明人： Erik J. Sontheimer , Raed Ibraheim , Wen Xue , Aamir Mir , Alireza Edraki , Ildar Gainetdinov
IPC分类号： C12N15/86 , C12N15/11 , A61K48/00
摘要： The present invention is related to compositions and methods for gene therapy. Several approaches described herein utilize the Neisseria meningitidis Cas9 system that provides a hyperaccurate CRISPR gene editing platform. Furthermore, the invention incorporates full length and truncated single guide RNA sequences that permit a complete sgRNA-Nme1Cas9 vector to be inserted into an adeno-associated viral plasmid that is compatible for in vivo administration. Furthermore, Type II-C Cas9 orthologs have been identified that target protospacer adjacent motif sequences limited to between one-four required nucleotides.

8. 发明授权

US11453864B2 DNase H activity of Neisseria meningitidis Cas9 有权
公开(公告)号：US11453864B2
公开(公告)日：2022-09-27
申请号：US15758394
申请日：2016-09-06
申请人： UNIVERSITY OF MASSACHUSETTS
发明人： Erik J. Sontheimer , Yan Zhang
IPC分类号： C12N9/22 , C12N15/11 , A61K38/00
摘要： Many strains of the human pathogen Neisseria meningitidis carry a compact Cas9 (NmeCas9) that can serve to limit genetic exchange via natural transformation. Cas9 orthologues (including NmeCas9) have recently been adopted for RNA-guided genome engineering and DNA binding, adding to the need to define better their activities and properties. The present invention examines DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 is found able to cleave single-stranded DNA (ssDNA) targets in a manner that is RNA-guided but both PAM- and tracrRNA-independent. Beyond the requirement for guide-target pairing, this activity has no apparent sequence requirements, and the cleavage sites are measured from the 5′ end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA domains are not strictly required for enzymatic activation of NmeCas9, and expand the list of targeting activities exhibited by these revolutionary RNA-guided nucleases.

9. 发明申请

US20190276810A1 CAS9-CAS9 FUSION PROTEINS 审中-公开
公开(公告)号：US20190276810A1
公开(公告)日：2019-09-12
申请号：US16226202
申请日：2018-12-19
申请人： University of Massachusetts
发明人： Scot A. Wolfe , Mehmet Fatih Bolukbasi , Ankit Gupta , Erik J. Sontheimer , Nadia Amrani
IPC分类号： C12N9/22
摘要： 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.

10. 发明申请

US20160177278A1 Cas9-DNA Targeting Unit Chimeras 审中-公开
标题翻译： Cas9-DNA靶向单位嵌合体
公开(公告)号：US20160177278A1
公开(公告)日：2016-06-23
申请号：US14976196
申请日：2015-12-21
申请人： University of Massachusetts
发明人： Scot Andrew Wolfe , Mehmet Fatih Bolukbasi , Ankit Gupta , Erik J. Sontheimer , Nadia Amrani
IPC分类号： C12N9/22
CPC分类号： C12N9/22 , C07K2319/80 , C12Y301/00
摘要： 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.
摘要翻译：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。

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