CRISPR enzymes and systems
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorsFeng Zhang, David Arthur Scott, Winston Xia Yan, Sourav Choudhury, Matthias Heidenreich
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Abstract
In one aspect, embodiments disclosed herein are directed to engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein that enhances binding of the of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type. In certain example embodiments, the CRISPR-Cas effector proteins a Type II effector protein. In certain other example embodiments, the Type V effector protein is Cas9 or an orthologs or engineered variant thereof. Example Cas9 proteins suitable for use in the embodiments disclosed herein are discussed in further detail below.
Claims14
An allogeneic modified isolated T cell or progeny thereof, obtained by a method comprising:
- selecting a set of candidate target sequences for one or more genes selected from the group consisting of CEP290, CFTR, DMD, G6PC, HBB, IDUA, IL2RG, PCSK9, SERPINA1, TTR, VEGFA, BCL11A, SLC2A2, GLUT2, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, and PFKM;
- wherein off-target candidates have 1 to 3 mismatches between an off-target site and the target sequences;
- preparing a set of CRISPR-Cas9 systems based on the final target sequences set,
- treating a first unmodified T cell ex vivo with the set of CRISPR-Cas9 systems to obtain the modified isolated T cell comprising modifications to one or more genes, or regions which regulate production of a gene product of the one or more genes, selected from the group consisting of CEP290, CFTR, DMD, G6PC, HBB, IDUA, IL2RG, PCSK9, PDCD1, SERPINA1, TTR, VEGFA, BCL11A, SLC2A2, GLUT2, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, and PFKM;
- and
- wherein the CRISPR-Cas9 system comprises a Cas9 protein and one or more Cas9 guides wherein the guides are based on the final target sequences set, and is capable of binding and cleaving one or more of the final target sequences set, and generating one or more insertions, deletions, or substitutions in one or more of the target sequences sets compared to a second unmodified T cell;
- wherein the modified isolated T cell or progeny thereof is capable of allogeneic adoptive cell therapy due to altered expression of B2M, CTLA4, TRAC, PDCD1, and CD52, compared to the second unmodified T cell;
- and
- wherein the degree of complementarity between one or more Cas9 guides and its target sequence is greater than 94.5%.
An allogenic modified isolated T cell or progeny thereof for treating a glycogen storage disease, obtained by a method comprising:
- selecting a set of candidate target sequences for one or more genes selected from the group consisting of SLC2A2, GLUT2, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, and PFKM;
- wherein off-target candidates have 1 to 3 mismatches between an off-target site and the target sequences;
- preparing a set of CRISPR-Cas9 systems based on the final target sequences set,
- treating a first unmodified T cell ex vivo with the set of CRISPR-Cas9 systems to obtain the modified isolated T cell comprising modifications to one or more genes, or regions which regulate production of a gene product of the one or more genes, selected from the group consisting of SLC2A2, GLUT2, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, and PFKM;
- and
- wherein the CRISPR-Cas9 system comprises a Cas9 protein and one or more Cas9 guides wherein the guides are based on the final target sequences set, and is capable of binding and cleaving one or more of the final target sequences set, and generating one or more insertions, deletions, or substitutions in one or more of the target sequences sets compared to a second unmodified T cell;
- wherein the modified isolated T cell or progeny thereof is capable of allogeneic adoptive cell therapy for the treatment of glycogen storage diseases due to altered expression of B2M, CTLA4, TRAC, PDCD1, and CD52, compared to the second unmodified T cell;
- and
- wherein the degree of complementarity between one or more Cas9 guides and its target sequence is greater than 94.5%.
Dependencies are inferred from the claim text.