Chromatin’s Impact on Editing

Chen et al (2016) report in Nucleic Acids Research results of a systematic analysis of chromatin conformation on various genome editing tools and observe effects that are not equally shared among the most popular systems.

https://upload.wikimedia.org/wikipedia/en/8/80/Sha-Boyer-Fig1-CCBy3.0.jpg

Open and compact chromatin. Image credit: Sha, K. and Boyer, L. A. The chromatin signature of pluripotent cells (May 31, 2009), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.45.1.

The authors focus on two gene-editing systems, TALENS and CRISPR/Cas9 and were motivated by absence of any quantitative analysis on the effects of chromatin conformation on isogenic sites. In addition, the authors wanted to assess the relative performance of TALENS and CRISPR/Cas9 at target sites that have different epigenetic modifications.

Basics of Cas9 Activity

Basics of Cas9 Activity

Chen et al. make use of two human cell lines (human embryonic kidney – HEK; human embryonic retinoblasts – HER), each with a report gene system that was enabled the monitoring of double strand break activity. In one system DSB and NHEJ would result in a gain of function of the reporter gene (DsRed), while in the other a loss of reporter gene function (EGFP).

What was particularly interesting about these editing targets is that they were in a transgene construct that allowed the authors to transition the chromatin state from compact to relaxed with the use of a small molecular (doxycycline). The details of the reporter system are nicely described and illustrated in the paper.

With these two FACS-based assays in place the authors did a number of experiments and made a couple of observations.

First, compact chromatin states reduce editing activity regardless of the system

Second, the authors found that TALENs seem to more sensitive than Cas9

Finally, Cas9 and some of the popular variants that are currently in use show different sensitivities to chromatin structure. The high specificity variant – SpCas9-HF1 showed significantly more sensitivity to chromatin structure compared to Cas9 and another high specificity variant eSpCas9(1.1).

While the data reported by Chen et al. are convincing and the impact of chromatin on editing systems is not surprising it remains to be seen whether there is anything a potential user of editing systems can do about these limitations of these systems.

It seems that from a practical perspective users are advised to design good targeting molecules and hedge those efforts by designing alternative targeting molecules to different regions of the target sequence, since local chromatin structure among other things may limit the effectiveness of any particular molecule.

Xiaoyu Chen, Marrit Rinsma, Josephine M. Janssen, Jin Liu, Ignazio Maggio, and Manuel A.F.V. Gonçalves,  2016.  Probing the impact of chromatin conformation on genome editing tools Nucl. Acids Res. first published online June 8, 2016 doi:10.1093/nar/gkw524

 

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