February 2020

Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

Xiaoyu Chen, Francesca Tasca, Qian Wang, Jin Liu, Josephine M. Janssen, Marcella D. Brescia, Milena Bellin, Karoly Szuhai, Josefin Kenrick, Richard L. Frock and Manuel A.F.V. Goncalves

In this paper the authors address one of the most discussed aspects regarding the application of Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted genome editing. The use of CRISPR-Cas9 is based on the introduction of a DNA break and relay on endogenous cellular repair pathways to obtain the desired modification. This strategy can be applied to correct genetic mutations causing disease such as B-Thalassemia or Duchenne Muscular Dystrophy. Although this technology has been proved to be easy, cheap and highly versatile, in some specific situation the “classic” CRISPR-Cas9 based genome editing approach has proved limitative. One of the main concerns is the precision of this molecular scissors and the safety of the translation of this tool towards therapeutic applications. For example, it is known that introducing a DNA break into induced pluripotent stem cells (iPSCs) is damaging and results in very few surviving cells . Further, some regions of the DNA have  been classified as impossible to edit with CRISPR-Cas9 because of the presence of repetitive regions that could induce this molecule to introduce a cut in an undesired location. For this reasons it’s important to investigate a way to tackle this sensitive situations with a more delicate approach. 

A cutting-free strategy called In-trans paired nicking has been used here to bypass these limitations. This strategy relays on the use of nickases, a modified version of Cas9 (Cas9^D10A), that allow it to cut only one of the DNA strands, generating a nick rather than a break. The specificity and efficiency of this strategy have been explored on a previous paper targeting intronic regions (Chen et al., 2017). Here, in addition, the strategy have been applied on sensitive cells, i.e. iPSCs, to target repetitive and/or essential DNA region, i.e. Oct4. The data presented showed how this strategy not only preserve cells viability and pluripotency but also results in a precise modification of the targeted regions that couldn’t be efficiently edited with a standard strategy. Most interestingly a method to detect genome wide off-target activity have been applied, underlying how the introduction of nicks instead of breaks it’s in itself less mutagenic. All together this strategy allows to target previously unteachable DNA regions, boosting CRISPR-Cas9 versatility.

About In-trans paired nicking