Abstract
The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9’s efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.
Highlights
Reverse genetics, the study of phenotypes due to the targeted mutation of a gene, has been and still is largely used to decipher genes or genetic elements function
A great deal of attention has focused on the risk of off-targets inherent to CRISPR/Cas[9] technology, and many optimizations are being established to minimize this risk and make this technology safe for medical applications[22]
To try and compare Cas9n and classical homologous recombination (cHR) efficiency in mouse Embryonic Stem (ES) cells, rather than evaluating their performance in parallel assays at the same locus, we chose to look at experiments performed at different loci all together
Summary
The study of phenotypes due to the targeted mutation of a gene, has been and still is largely used to decipher genes or genetic elements function. This greater efficiency of cHR over Cas9n was observed in all tested conditions, except for inserts of more than 8 kb where both technologies proved equivalent These observations were made for the targeting of the Rosa[26] locus, but were validated over all tested loci, suggesting that CRISPR/Cas9-mediated KI in mouse ES cells is more efficient than cHR for the detection of recombined clones, but that cHR is more robust and efficient for error-free insertions. For Cas9n-mediated KI, we observed a significant increase in the number of experiments and clones showing bands of unexpected sizes when using an external probe, i.e. insertion at the targeted locus This observation suggests the presence of undesired rearrangements at the targeted loci when using CRISPR/Cas[9] technology, rearrangements that never occur when using cHR. This study show that all materials obtained with CRISPR/Cas[9] technology must be carefully characterized by SB and/or long-range sequencing at the targeted locus before further use
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