High-fidelity cas9 variants and applications thereof

A technology of S487 and A488, applied in the field of Cas9 variants, can solve problems such as defective on-target activity

Pending Publication Date: 2019-11-29
THE UNIV OF TORONTO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of these approaches are off-target free and are often deficient in on-target activity due to their inherent molecular complexity

Method used

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  • High-fidelity cas9 variants and applications thereof
  • High-fidelity cas9 variants and applications thereof
  • High-fidelity cas9 variants and applications thereof

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Embodiment Construction

[0017] The present invention describes isolated Cas9 molecules with increased specificity obtained by random mutagenesis of the REC1-II domain of Cas9 and screened using a yeast-based assay to simultaneously assess the neutrality of each resulting variant. On-target and off-target activity. Selected hits were further refined by screening in mammalian systems.

[0018] In a first aspect of the invention, the Cas9 variant comprises at least one mutation at an amino acid residue position selected from the group consisting of K526, K377, E387, D397, R400, D406, A421, L423, R424, Q426, Y430, K442, P449, V452, A456, R457, W464, M465, K468, E470, T474, P475, W476, F478, K484, S487, A488, T496, F498, L502, N504, K506, P509, F518, N522, E523, L540, S541, I548, D550, F553, V561, K562, E573, A589, L598, D605, L607, N609, N612, E617, D618, D628, R629, R635, K637, L651, K652, R654, T657, G658, L666, K673, S675, I679, L680, L683, N690, R691, N692, F693, S701, F704, Q712, G715, Q716, H723,...

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Abstract

To address the limitations deriving from the unspecific genomic cleavages of the Streptococcus pyogenes Cas9 (SpCas9) and to identify variants with higher cleavage fidelity, the present invention describes a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets. The screening of SpCas9 variants obtained by random mutagenesis of the Red-II domain allowed the identification of hits with increased on / off ratios. The best performing nuclease, evoCas9, was isolated through the combination of the identified mutations within asingle variant. Side by side analyses with previously reported rationally designed variants demonstrated a significant improvement in fidelity of evoCas9 of the present invention.

Description

[0001] field of invention [0002] The present invention is in the field of enzymes and nucleic acid binding proteins, in particular Cas9 variants. [0003] Background of the invention [0004] The number of biotechnology applications involving the CRISPR-Cas9 system has seen a dramatic increase over the past few years, driven by the flexibility and efficacy of this new genome editing tool. Typically, Cas9 recognizes a target site via a so-called "guide RNA" (gRNA) sequence that is complementary to a target nucleic acid comprising a protospacer sequence. Target recognition also requires the presence of a short adjacent PAM (protospacer adjacent motif) sequence. The target nucleic acid is usually DNA, but can also be RNA in some cases. Guide RNAs can be formed from one or more small RNAs. Genome editing via the CRISPR-Cas9 approach has been successfully applied to a variety of cell types and species, clearly demonstrating the need to characterize the effectiveness and robustn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/11
CPCC07K2319/71C07K2319/80C12N9/22C12Y301/00A61K38/00C12N2800/80
Inventor A.切雷塞托A.卡西尼G.彼得里斯A.因加M.奥利维利
Owner THE UNIV OF TORONTO
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