Journal Article

Efficient single-copy HDR by 5’ modified long dsDNA donors

Kellner, Tanja, Tsingos, Erika, Gutierrez-Triana, Jose Arturo, Thomas, Isabelle, Anlas, Kerim, Wittbrodt, Joachim, Tavhelidse, Tinatini, Thumberger, Thomas, Wittbrodt, Beate

2018

Overview

CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging. CRISPR/Cas9 technology has revolutionized the ability of researchers to edit the DNA of any organism whose genome has already been sequenced. In the editing process, a section of RNA acts as a guide to match up to the location of the target DNA. The enzyme Cas9 then makes a cut in both strands of the DNA at this specific location. New segments of DNA can be introduced to the cell, incorporated into DNA ‘templates’. The cell uses the template to help it to heal the double-strand break, and in doing so adds the new DNA segment into the organism’s genome. A drawback of CRISPR/Cas9 is that it often introduces multiple copies of the new DNA segment into the genome because the templates can bind to each other before being pasted into place. In addition, some parts of the new DNA segment can be missed off during the editing process. However, most applications of CRISPR/Cas9 – for example, to replace a defective gene with a working version – require exactly one whole copy of the desired DNA to be inserted into the genome. In order to achieve more accurate CRISPR/Cas9 genome editing, Gutierrez-Triana, Tavhelidse, Thumberger et al. attached additional molecules to the end of the DNA template to shield the DNA from mistakes during editing. The modified template was used to couple a stem cell gene to a reporter that produces a green fluorescent protein into the genome of fish embryos. The fluorescent proteins made it easy to identify when the coupling was successful. Gutierrez-Triana et al. found that the additional molecules prevented multiple templates from joining together end to end, and ensured the full DNA segment was inserted into the genome. Furthermore, the results of the experiments showed that only one copy of the template was inserted into the DNA of the fish. In the future, the new template will allow DNA to be edited in a more controlled way both in basic research and in therapeutic applications.

• Publisher: eLife Science Publications, Ltd,eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Animals / Annealing / Biochemistry / Cell cycle / Cloning / Conformation / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / Developmental Biology / DNA / DNA - genetics / DNA - metabolism / DNA End-Joining Repair / DNA repair / Embryo, Nonmammalian - metabolism / endogenous gene tagging / Enzymes / Fish Proteins - genetics / Fish Proteins - metabolism / Fluorescence / Gene Editing - methods / Genes / Genetic research / Genetic testing / Genetics and Genomics / Genome editing / Genomes / Genomics / Green Fluorescent Proteins - genetics / Green Fluorescent Proteins - metabolism / homologous recombination / Homology / Models, Genetic / Mutation / Oryzias / oryzias latipes / Plasmids / precision genome editing / Proteins / Recombinational DNA Repair / RNA / Stem cells / Technology / Tools and Resources