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

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application under 35 U.S.C. §120 of co-pending U.S. application Ser. No. 14 / 490,549, filed Sep. 18, 2014, which claims the benefit of Great Britain application number 1321210.5, filed Dec. 2, 2013, and Great Britain application number 1316560.0, filed Sep. 18, 2013, the disclosures of which are herein incorporated by reference in their entireties.SEQUENCE LISTING[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: Sequence_Listing_14490549.txt, created on Mar. 7, 2016, size: 27,892 bytes).FIELD OF THE INVENTION[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 sequ...

AI Technical Summary

Benefits of technology

These methods enable serial nucleic acid modifications, allowing for large and precise deletions or insertions, improving the fidelity and efficiency of genome editing by reducing indel formation and enhancing the precision of nucleic acid modifications compared to previous techniques.

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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