Methods, cells & organisms

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....

AI Technical Summary

Benefits of technology

The patent describes a way to add or remove specific parts of DNA from a genome. This is done by creating breaks in the DNA and using a specific repair process that reduces the chance of errors. This technique is more efficient than previous methods and can be used in cells or organisms.

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 nuclease has been shown to generate precise predefined mutations but the efficiency at which this occurs in both alleles is low and mutation is seen in one of the strands of the dsDNA target (Wang et al).

While it may be possible to modify a desired sequence in one strand of dsDNA, the sequence in the other strand is often mutated through undesirable NHEJ.

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