Methods, cells & organisms

a technology of cells and organisms, applied in the field of methods, cells & organisms, can solve the problems of low efficiency at which this occurs in both alleles, time and cost of developing such engineered proteins, and high undesirable effects, and achieve the effect of large and precise nucleotide sequence deletions or insertions

Inactive Publication Date: 2015-03-19
KIMAB LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0024]In aspects of the configurations of the invention there is provided a method of sequential endonuclease-mediated homology directed recombination (sEHDR) comprising carrying out the method of any preceding configuration a first time and carrying out the method of any preceding configuration a second time. In this way, the invention enables serial nucleic acid modifications, e.g., genome modifications, to be carried out, which may comprise p

Problems solved by technology

These have been successfully used in various eukaryotic hosts but they all suffer from important limitations notably off-target mutagenesis leading to nuclease-related toxicity and also the time and cost of developing such engineered proteins.
This is highly undesirable when precise nucleic acid or genome editing is required.
This approach with Cas9 nuc

Method used

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  • Methods, cells & organisms
  • Methods, cells & organisms
  • Methods, cells & organisms

Examples

Experimental program
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Effect test

example 1

Precise DNA Modifications

[0174](a) Use of Nickase for HDR

[0175]It has been reported that the Cas9 nuclease can be converted into a nickase through the substitution of an aspartate to alanine (D10A) in the RuvCl domain of SpCas9 (Cong et al). It is noteworthy that DNA single-stranded breaks are preferentially repaired through the HDR pathway. The Cas9 D10A nickase, when in a complex with mature crRNA:tracrRNA, can specifically induce DNA nicking at a precise location. With this in mind, we propose extending the application of the CRISPR / Cas system by creating a nick in a given location in a genome using Cas9 D10A nickase and then exploiting the HDR pathway for inserting a single-stranded DNA fragment (endogenous or exogenous) which will contain DNA homology flanking the nick. Typically for recombineering 50 by is enough for efficient recombination) flanking the nicked DNA junction to bring in and insert a given DNA in a precision location; similar size homology will be used with the ...

example 2

Recycling PAM for Sequential Insertions or Deletions

[0177]In certain settings it may be useful to edit a genome by chromosome walking. Using any of the three examples outlined above, it could be possible to carry out sequential genome editing in a stepwise fashion whereby the PAM sequence used in a previous round of CRISPR / Cas mediated genome editing, can be re-used to carry out multiple rounds of genome editing such as deletions, insertions or the simultaneous deletion and insertion. An example of sequential deletion whereby the PAM sequence from the previous genome editing step is recycled is shown in FIG. 4. Using the PAM recycling approach, it is possible to carry out sequential insertions as well as sequential simultaneous deletion and insertion.

example 3

Rapid Insertion of Lox Sites Using CRISPR / Cas System

[0178]Targeting efficiency using conventional homologous recombination methods in ES cells is low. In a different setting, the CRISPR / Cas system can be used to rapidly and efficiently introduce lox sites or other recombinase recognition sequence such as Frt in a defined location to act as a landing pad for genome editing using recombinase mediated cassette exchange (RMCE) (Qiao J, Oumard A, Wegloehner W, Bode J: Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties. J Mol Biol 2009, 390(4):579-594; and Oumard A, Qiao J, Jostock T, Li J, Bode J: Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology. Cytotechnology 2006, 50(1-3):93-108). Once the lox sites are introduced into the genome, inversion, deletion or cassette exchange to delete and introduce DNA fragment varying in size at this site can be e...

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PUM

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Abstract

The invention relates to an approach for introducing one or more desired insertions and/or deletions of known sizes into one or more predefined locations in a nucleic acid (eg, in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location. The technique is based on the observation that DNA single-stranded breaks are preferentially repaired through the HDR pathway, and this reduces the chances of indels (eg, produced by NHEJ) in the present invention and thus is more efficient than prior art techniques. The invention also provides sequential insertion and/or deletions using single- or double-stranded DNA cutting.

Description

[0001]This application claims the benefit of Great Britian application number 1321210.5, filed Dec. 2, 2013, and Great Britian application number 1316560.0, filed Sep. 18, 2013, the disclosures of which are herein incorporated by reference in their entireties.[0002]The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 13581—001—999 SequenceListing.txt, date recorded: Sep. 18, 2014, size: 28 kilobytes).[0003]The inventors have devised an approach for introducing one or more desired insertions and / or deletions of known sizes into one or more predefined locations in a nucleic acid (eg, in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location....

Claims

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Application Information

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IPC IPC(8): C12N15/90
CPCC12N15/907C12N15/102C12N2800/80A01K67/0278A01K2207/15A01K2217/072A01K2227/105A01K2267/01C07K16/00C07K2317/14C07K2317/24C07K2317/52C07K2317/56C12N5/0635C12N2510/04C07K2317/20A01K2217/052
Inventor BRADLEY, ALLANALI, HANIFLEE, E-CHIANG
Owner KIMAB LTD
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