Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated Knockin

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Rezza A, Jacquet C, Le Pillouer A, Lafarguette F, Ruptier C, Billandon M, Isnard Petit P, Trouttet S, Thiam K, Fraichard A, Chérifi Y.
Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.
Sci Rep. 2019 Mar 5.

It’s been almost 6 years now since the gene-editing community was shaken by the transformation of a bacterial adaptative immunity system into a most elegant gene-editing tool, CRISPR. Even non-specialists have heard of this system that allows specifically targeting a DNA sequence for DSB (double-strand break), using a single enzyme and an RNA molecule.

This promising tool has since been extensively used and many labs, including ours, have contributed to its improvements and optimizations. The efficiency of cutting as well as the limitation of off-targets have focused a lot of attention, leading to the development of improved CRISPR enzymes and optimized algorithms for guide RNA design.

Interestingly, its efficiency has not been thoroughly compared to homologous recombination (HR) in cells that allow it, i.e., embryonic stem (ES) cells. More recently, several studies have indicated that using CRISPR may lead to complex, unpredicted modifications at the targeted locus.

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In our new manuscript, freshly published in Scientific Reports, we systematically compared CRISPR/Cas9 (using paired nickases) and classical HR efficiency for the targeted insertion of large DNA fragments (>1.5kb) in mouse ES cells, at 128 loci.

This large-scale study showed that although CRISPR/Cas9 allowed for the detection of more clones, classical HR was eventually more efficient for the generation of validated recombinant clones.

Interestingly, we also identified that CRISPR/Cas9-mediated gene editing repeatedly led to unpredicted on-target genomic rearrangements. It is, to our knowledge, the first large-scale study to include in-depth on-target characterization of CRISPR-edited materials.

Our study, along with our previous work (1), confirms that CRISPR/Cas9 is highly efficient for the production of edited clones, also underlining the capital importance of thoroughly characterizing all materials obtained with this technology for potential off-targets, but also at the targeted locus.

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(1) Our previous CRISPR publications:

Dehairs J, Talebi A, Cherifi Y, Swinnen JV.
CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing.
Sci Rep. 2016 Jul 1.

Renaud JB, Boix C, Charpentier M, De Cian A, Cochennec J, Duvernois-Berthet E, Perrouault L, Tesson L, Edouard J, Thinard R, Cherifi Y, Menoret S, Fontanière S, de Crozé N, Fraichard A, Sohm F, Anegon I, Concordet JP, Giovannangeli C.
Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.
Cell Rep. 2016 Feb 24.

Ménoret S, De Cian A, Tesson L, Remy S, Usal C, Boulé JB, Boix C, Fontanière S, Crénéguy A, Nguyen TH, Brusselle L, Thinard R, Gauguier D, Concordet JP, Cherifi Y, Fraichard A, Giovannangeli C, Anegon I.
Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins.
Sci Rep. 2015 Oct 7.