Paper of the month October 2018

Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.

CRISPR-Cas9 is a novel gene editing technique with promising applications in clinical settings (https://www.youtube.com/watch?v=2pp17E4E-O8). This technology allows to introduce a double-strand break (DBS) in the DNA in a specific genomic location (target site). This brake results in the activation of cell repair mechanisms, that can be exploit to obtain targeted genomic modifications. One of the most appealing application would, for example consist in correcting disease-causing genetic mutations. It is believed that on-target CRISPR-Cas9 mediated-DSBs most commonly result in small insertions or deletions (indels). Therefore, the prevalent method to assess repair outcomes of a Cas9 induced-DSBs relay focus on detecting genetic alterations in the immediate vicinity of the target site using mostly short-range PCR and Sanger sequencing. It is important to be able to distinguish between all the possible genetic events derived from Cas9-mediated genome editing, especially considering the possible clinical implications. The authors of this paper speculate that the current assessment methods may overlook a substantial part of possible outcomes that results from on-target Cas9 cutting and repair. Indeed, it is here reported that Cas9 activity not only results in indels formation but can also lead to on-target extensive deletions and complex rearrangements.

In this study Cas9 and several gRNAs targeting exonic and intronic region of PigA X-linked locus were introduced in mouse male embryonic stem cells. Afterwards the region surrounding the target site in PigA deficient cells has been analysed, using long-read sequencing and long-range PCR. This analysis revealed a large variety of mutation between large deletions, ranging up to 9.5 Kb, and complex rearrangements, involving contiguous and non-contiguous regions. To further corroborate this results, similar experiments have been carried out in different cell types (human female pigment epithelial cell line and mouse hematopoietic progenitors). All together the editing of actively transcribed loci in normal embryonic stem cells and progenitor cells, as well as in immortalized differentiated human cells, shows that extensive on-target genomic damage is a common outcome of Cas9-mediated DSB introduction.

This paper call the attention to the need of a thorough comprehensive genomic analysis of Cas9 outcomes to identify this class of unwanted alterations that could be unnoticed using the common short-range analysis. Especially looking forward a translation into clinical setting, this results, represent an important mile stone in improving Cas9 safety and specificity.

Further information regarding the methods mentioned above can be find in the following link.

PCR: https://simple.wikipedia.org/wiki/Polymerase_chain_reaction
Sequencing: https://en.wikipedia.org/wiki/DNA_sequencing