Methods, Cells & Organisms

摘要

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.

主权项

1. An in vitro method for modifying a genome at a genomic locus of interest in a mouse ES cell, the method comprising:
contacting the mouse ES cell with:
a Cas9 protein;a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest;a tracrRNA; andan incoming nucleic acid sequence that is flanked by:
(i) a 5′ homology arm that is homologous to a 5′ target sequence at the genomic locus of interest; and(ii) a 3′ homolog arm that is homologous to a 3′ target sequence at the genomic locus of interest; wherein the incoming nucleic acid sequence is at least 10 kb in size; wherein following the contacting step, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising:
deletion of a region of the genomic locus of interest wherein the deletion is at least 20 kb; and/orinsertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 20 kb.