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Repair template linkage to endonucleases for genome engineering

A nuclease and genome technology, used in genetic engineering, plant genetic improvement, applications, etc., can solve the problems of discovering and using site-specific nucleases and repair templates that have not yet been exploited, the process is error-prone, and the efficiency is low.

Pending Publication Date: 2019-11-19
KWS SAAT SE & CO KGAA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It thus provides high fidelity, however less efficient
In contrast, NHEJ is an efficient and direct pathway that rejoins 2 ends independent of significant homology, this efficiency is accompanied by the disadvantage that the process is error-prone and may be associated with insertions or deletions
[0026] So far, these discoveries regarding the ability of biotinylated molecules and their cognate binding partners or other high-affinity molecular binding pairs (such as antibodies or single-chain variable fragments and their cognate partners) have not been exploited for the use of positional Targeted genome engineering of site-specific nucleases and repair templates

Method used

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  • Repair template linkage to endonucleases for genome engineering
  • Repair template linkage to endonucleases for genome engineering
  • Repair template linkage to endonucleases for genome engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0289] Embodiment 1: be suitable for being combined with Cas or Cpf1 or Argonaute polypeptide as the hybrid nucleic acid sequence of RTDD / RT pair

[0290] In one experiment, tailed sgRNAs or sgDNAs were hybridized via complementary base pairing and RNA-DNA or DNA-DNA ligated single-strand repair templates. For covalent conjugation, synthetic DNA oligonucleotides are manipulated with the manufacturer's ssRNA ligase to covalently join the 3' ends of RNA / DNA oligonucleotides. For non-covalent conjugation, RNA / DNA and DNA oligonucleotides with partially complementary sequences were mixed and allowed to complex via Watson Crick base pairing. Successful hybridization can be determined in a gel shift assay. Treatment of hybrid nucleic acid aliquots with RNase and DNase enzymes prior to gel shift assays revealed that, for those experiments using sgRNA, some hybrid nucleic acids consisted of RNA and some consisted of DNA. The nucleic acid hybrid is then complexed with a recombinant C...

Embodiment 2

[0291] Example 2: In vitro cleavage of DNA targets by complexes of Cas9 protein and hybrid RNA-DNA nucleic acids

[0292] In one experiment, the Cas9 protein was tested for its function as a site-specific endonuclease when used with the described nucleic acid hybridization technology. A linearized plasmid containing at least one target site for the sgRNA is mixed with the Cas9-sgRNA-RT complex as described in the present invention. After incubation under conditions suitable for nuclease activity, including correct pH, temperature, and cofactors, etc. known to those skilled in the art for a variety of CRISPR nucleases and their variants, the DNA target plasmids are run on an agarose gel And observe the band size indicating the expected target site for cleavage. In vitro cleavage of target DNA demonstrated that RT associated with sgRNA as "cargo" did not interfere with the function of the Cas9 complex as a site-specific endonuclease.

Embodiment 3

[0293] Example 3: In vivo editing by Cas9 protein complexed with hybrid RNA-DNA nucleic acid

[0294] To demonstrate that target genes can be edited in vivo by delivering a complex containing the Cas9 protein and hybrid RNA-DNA nucleic acid, the non-functional tdTomato gene contained within the transformed plasmid was repaired by exchanging a single nucleotide to restore the fluorescent signal from the tdTomato gene . To determine optimal use for editing by providing ssDNA repair templates that are complementary to the target or non-target strand, complexes carrying either strand repair templates were compared.

[0295] The hybrid nucleic acid RNA / DNA-Cas polypeptide complex obtained in Example 1 was used to repair the episomal plasmid target encoding the tdTomato gene with a single point mutation from A to T at codon position 51 Generate an early termination signal. This plasmid was introduced into maize protoplasts by PEG- or electroporation-mediated delivery of an editing...

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Abstract

The present invention relates to artificial molecular complexes comprising at least one site-specific nuclease and directly interacting therewith at least one repair template docking domain, the repair template docking domain interacting with at least one repair template nucleic acid sequence. The artificial complex can further comprise at least one interaction domain. The artificial molecular complexes are configured to mediate repair of a DNA target sequence in a prokaryotic or eukaryotic organism with high precision in a targeted way and can thus be used for genome engineering in a prokaryotic or a eukaryotic cell or organism, or editing of a viral genome. Further provided are methods of modifying at least one DNA target sequence in a prokaryotic or eukaryotic cell or a viral genome, e.g., for trait development, or for treating a disease. Additionally, there is provided a method for manufacturing a plant, plant cell, a plant material, or a derivative, or a progeny thereof comprisingor edited by at least one artificial molecular complex.

Description

technical field [0001] The present invention relates to an artificial molecular complex comprising at least one site-specific nuclease and at least one repair template docking domain interacting directly with it, said repair template docking domain interacting with at least one repair template nucleic acid sequence. The artificial complex may further comprise at least one interaction domain. Artificial molecular complexes are configured to mediate the repair of DNA target sequences in prokaryotic or eukaryotic or viral organisms or genomes in a targeted manner with high precision, and thus can be used for genome engineering of prokaryotic or eukaryotic cells or organisms or in In vivo or in vitro for genome engineering in prokaryotic, eukaryotic or viral genomes. Also provided are methods of modifying at least one DNA target sequence in the genome of a prokaryotic or eukaryotic cell or virus, for example for trait development or for treatment of disease. In addition, methods...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/10C12N15/11C12N15/90C12N15/55
CPCC12N9/22C12N15/102C12N15/11C12N15/111C12N15/8213C12N15/902C12N2310/20C12N2310/3519A61P35/00A61P37/06A61P43/00
Inventor M·拉布斯
Owner KWS SAAT SE & CO KGAA
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