Probing the impact of chromatin conformation on genome editing tools.
Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions.
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands.
Xiaoyu
X
Chen
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands.
Marrit
M
Rinsma
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands.
Josephine M
JM
Janssen
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands.
Jin
J
Liu
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands.
Ignazio
I
Maggio
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands M.Goncalves@lumc.nl.
Manuel A F V
MA
Gonçalves
D064113:CRISPR-Cas Systems / Q000235:genetics*; D002843:Chromatin / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics; D000072669:Gene Editing; D005818:Genetic Engineering; D005838:Genotype; D006801:Humans; D008968:Molecular Conformation; D013297:Streptococcus pyogenes / Q000235:genetics; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics*
D002843:Chromatin; D000069896:Transcription Activator-Like Effector Nucleases