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. 2016 Jul 27;44(13):6482-92.
doi: 10.1093/nar/gkw524. Epub 2016 Jun 8.

Probing the impact of chromatin conformation on genome editing tools

Xiaoyu Chen  1 , Marrit Rinsma  1 , Josephine M Janssen  1 , Jin Liu  1 , Ignazio Maggio  1 , Manuel A F V Gonçalves  2
Affiliations

Probing the impact of chromatin conformation on genome editing tools

Xiaoyu Chen et al. Nucleic Acids Res. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Experimental systems for tracking designer nuclease-induced indel formation at target sites with alternate epigenetic states. (A) Drug-dependent control over the chromatin conformation of designer nuclease target sites. In this system, the binding of the trans-acting tTR-KRAB fusion protein to cis-acting TetO sequences leads to the recruitment of epigenetics modulators consisting of, amongst others, KAP1 and HP1 proteins. In the presence of doxycycline, tTR-KRAB cannot bind its cognate TetO elements, resulting in the transition of a compact heterochromatic to a relaxed euchromatic conformation. (B) Designer nuclease-induced gain-of-function system (ORF correction). HER.TLRTetO.KRAB reporter cells contain a TetO-flanked TLR allele. Subjecting tTR-KRAB-expressing HER.TLRTetO.KRAB cells to designer nucleases targeting TLR sequences yields ORF-correcting indels generated by NHEJ-mediated DSB repair and the appearance of mCherry-positive cells. (C) ChIP-qPCR analysis on HER.TLRTetO.KRAB cells. ChIP-qPCR signals detected by using antibodies directed against open and closed chromatin marks (H3Ac and H3K9me3, respectively). Six different regions spanning the TLRTetO gene body were probed. The targeted sequences were located in the EF1α promoter (EF1α), the puromycin resistance ORF (PuroR), the spleen focus-forming virus regulatory elements (SFFV), the EGFP ORF (EGFP a and EGFP b) and the mCherry ORF (mCherry). Standard positive and negative controls (Ctrl) are indicated. (D) Designer nuclease-induced loss-of-function system (ORF disruption). HEK.EGFPTetO.KRAB reporter cells harbor a TetO-flanked EGFP target allele. Exposing tTR-KRAB-expressing HEK.EGFPTetO.KRAB cells to designer nucleases targeting EGFP yields ORF-disrupting indels generated by NHEJ DSB repair and the emergence of EGFP-negative cells. (E) Experimental settings. HEK.EGFPTetO.KRAB and HER.TLRTetO.KRAB cells exposed or not to Dox are transfected with designer nuclease-encoding constructs. After the generation of site-specific DSBs and ensuing NHEJ-mediated indel formation in each of the two parallel settings (yellow boxes), target gene expression is activated allowing to quantify the frequencies of NHEJ-based gene editing by flow cytometry.
Figure 2.
Figure 2.
Detailed diagrammatic representation of the experimental designs used in the present study. The tTR-KRAB-expressing reporter cells HEK.EGFPTetO.KRAB(A) and HER.TLRTetO.KRAB (B) were used for tracking and quantifying designer nuclease-induced gene editing events at target sites subjected to different epigenetic states. The TetO-negative and tTR-KRAB-expressing reporter cells HER.TLRKRAB(C) were also generated to provide for negative controls. The HEK.EGFPTetO.KRAB and HER.TLRTetO.KRAB systems are complementary in that they allow for measuring ORF disruption and ORF correction, respectively. The initial high-order chromatin conformation of both model alleles is controlled through Dox-dependent regulation of tTR-KRAB binding. Reporter cells HEK.EGFPTetO.KRAB and HER.TLRTetO.KRAB, containing target sequences in a heterochromatic (–Dox) or euchromatic (+Dox) state, are transiently transfected with different sets of designer nuclease-encoding constructs. DsRed and hrGFP expression plasmids are included in the transfection mixtures to serve as internal controls for transfection efficiency. After the generation of targeted DSBs in each of the two parallel settings (i.e. –Dox and +Dox), target gene expression is activated allowing to quantifying the frequencies of NHEJ-based gene editing by flow cytometry.
Figure 3.
Figure 3.
The impact of distinct chromatin conformations on the frequencies of NHEJ-based gene editing achieved by TALEN and CRISPR/Cas9 nucleases. Reporter cells HER.TLRTetO.KRAB (A and C) and HEK.EGFPTetO.KRAB (B and D), were subjected to the indicated experimental conditions. The negative controls (Ctrl) in panels A and B, involved transfecting cells with expression plasmids encoding Cas9 mixed with a non-targeting gRNA or with an ‘empty’ gRNA construct, respectively. The negative controls (Ctrl) in panel C and in panel D involved exposing target cells exclusively to a single TALEN monomer. Representative flow cytometry dot plots are also presented next to each graph. Ten thousand events, each corresponding to a single viable cell, were measured per sample. Error bars indicated mean ± s.e.m. P values (by two-tailed t-tests) and the number of independent experiments (n) are shown. (E) Control HER.TLRKRAB cells. The chromatin status of TLR sequences in HER.TLRKRAB cells are not controlled by Dox since they lack cis-acting TetO elements for tTR-KRAB binding. (F) Gene editing in HER.TLRKRAB cells. HER.TLRKRAB cells were either exposed or not to Dox and were subsequently transfected with the indicated constructs. Differences between +Dox and –Dox values were not statistically significant as determined by three-way ANOVA (P = 0.151; two independent experiments done in replicate). The ratios between EGFP knockout levels measured in the presence versus those determined in the absence of Dox. gRNANT, Non-targeting gRNA. (G) Representative flow cytometry dot plots corresponding to the experimental settings presented in panel F.
Figure 4.
Figure 4.
Gene editing experiments at EGFP target sequences subjected to alternative chromatin conformations. (A) Targeted mutagenesis induced by TALENs with different numbers of TALE repeats. HEK.EGFPTetOKRAB cells were either incubated or not with Dox and were subsequently transfected with expression plasmids encoding TALENs with 14.5, 16.5 and 18.5 TALE repeats. Negative controls consisted of parallel cultures exposed exclusively to TALEN-GA-L (Ctrl). After the generation of site-specific DSBs in each of the two parallel settings (i.e. -Dox and +Dox), target gene expression was activated in all cultures by adding Dox for determining the frequencies of NHEJ-mediated EGFP knockout by flow cytometry. The ratios between EGFP knockout levels measured in the presence versus those determined in the absence of Dox (open versus closed chromatin, respectively) are indicated. (B) Representative flow cytometry dot plots corresponding to the experimental settings presented in panel A.
Figure 5.
Figure 5.
Testing the impact of chromatin conformation on high-specificity Cas9 variants. (A) Screening of CRISPR/Cas9 complexes with different Cas9 proteins in HEK.EGFPTetOKRAB cells. HEK.EGFPTetOKRAB cells were incubated in the presence or in the absence of Dox and were subsequently transfected with expression plasmids encoding the indicated CRISPR/Cas9 nuclease components. The chromatin impact index was determined by computing the ratios between EGFP knockout levels measured in the presence versus those determined in the absence of Dox. Error bars indicate mean ± s.e.m. corresponding to three independent experiments. Ten thousand events, each corresponding to a single viable cell, were measured per sample. (B) Cumulative chromatin impact indexes. Boxplot of the chromatin impact indexes presented in panel A. Whiskers, minimum and maximum. One-way ANOVA compared the experimental groups with a subsequent comparison between groups being done by Bonferroni analysis (P < 0.05 was considered significant).
Figure 6.
Figure 6.
Cumulative chromatin impact indexes for the TALEN and CRISPR/Cas9 nuclease systems. Boxplot of the ratios between EGFP knockout levels measured in the presence versus those determined in the absence of Dox presented in Figures 3B, D, 4A and 5B (Cas9 data points). Whiskers, minimum and maximum. The data corresponding to the TALEN pair with the shortest DNA-binding domains (i.e. TALEN-26-L/TALEN-26-R) was not computed in this analysis to avoid skewing the data. Ten thousand events, each corresponding to a single viable cell, were measured per sample. The P-value was determined by two-tailed Student's t-test analysis (P < 0.05 was considered significant).
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