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Anti-crispr compounds and methods of use

A composition and compound technology, applied in the direction of biochemical equipment and methods, microorganisms, drug combinations, etc., can solve the problems of lack of predictable control and robust inhibition

Active Publication Date: 2019-01-04
UNIV OF MASSACHUSETTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although several engineered systems allow controlled activation of CRISPR-Cas9 for increased precision, all of these systems still lack the ability to provide predictable control and robust repression

Method used

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  • Anti-crispr compounds and methods of use
  • Anti-crispr compounds and methods of use
  • Anti-crispr compounds and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0346] Construction of Acr vector

[0347] Sequences found to encode the Acr protein can be ordered from companies that sell custom DNA sequence samples, or they can be PCR amplified from appropriate biological DNA samples. They can then be introduced into vectors (including but not limited to plasmid and viral vectors) by standard recombinant DNA cloning protocols (eg, restriction endonuclease digestion and religation, or Gibson assembly). The vector typically includes a promoter sequence suitable for expression in the cell of interest. They also contain other sequences that facilitate gene expression and control [eg, 5' and 3' untranslated regions, poly(A) signals, and transcription terminators]. These vectors can then be introduced into target cells by using standard delivery methods well known in the art.

Embodiment II

[0349] Screening of Acr protein candidates

[0350] Many other acr gene candidates can be identified by their genetic association with aca genes or putative aca genes. The Acr open reading frame can be cloned directly from the species of origin, or alternatively, the gene sequence can be synthesized by a company. There are several approaches to screen for Acr activity against type II systems. 1) a test for the inhibition of NmeCas9 activity in its native context, namely in Neisseria meningitidis, by using the assay previously described to measure the transformation efficiency of DNA targeted by CRISPR-Cas9; 2) for Test of inhibition of NmeCas9 activity in a heterologous E. coli system (wherein NmeCas9 is programmed to target coliphage (eg, lambda phage)); 3) by using the T7E1 assay described herein, with respect to Test for inhibition of NmeCas9 activity in cell culture.

Embodiment III

[0352] Acr administration for controlled gene drive spread

[0353]Cas9 is currently being considered for use in gene drives, engineered genetic elements that can spread rapidly throughout natural populations. If that natural population is an undesired population, such as insects that transmit pathogens such as malaria parasites or dengue virus, the gene drive may include elements that induce death or sterility for any insects that acquire the gene drive. While the potential of this method for controlling insect-borne diseases is widely recognized, there are many concerns about its safety and potential for undesired adverse consequences (predictable or unpredictable) following release into the natural environment. worry. Part of that concern arises from the possibility that, once released, the spread of a gene drive may be difficult or impossible to stop. It is conceivable to imagine a gene drive that uses a type II-C Cas9 enzyme, such as NmeCas9, to support its propagatio...

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Abstract

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 Cas9activity; 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.

Description

field of invention [0001] The present invention relates to the field of CRISPR-Cas9 gene editing platform. In particular, the present invention identifies type II-C Cas9 anti-CRISPR (Acr) inhibitors that control the gene editing activity of Cas9. Co-administration of such Acr inhibitors can provide advantageous assistance in: allowing safe and practical biological therapeutics by spatially or temporally controlling Cas9 activity; controlling Cas9-based gene drives in wild populations ( gene drive) to mitigate the ecological consequences of such forced genetic programs; and to contribute to general research into various biotechnological, agricultural and medical applications of gene editing technologies. Background of the invention [0002] CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-associated system) involves the bacterial immune system that recognizes and destroys foreign nucleic acids. The development of type II CRISPR-Cas9 systems as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N1/20A61K31/7088A61P31/00
CPCA61K31/7088C12N15/102C12N2795/00043A61P31/00C12N9/22C12N15/907
Inventor E·J·松特海默尔A·戴维森K·马克斯韦尔
Owner UNIV OF MASSACHUSETTS
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