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Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

Ruben Schep, Eva Brinkman, Christ Leemans, Xabier Vergara, Ben Morris, Tom van Schaik, Stefano G. Manzo, Daniel Peric-Hupkes, Jeroen van den Berg, Roderick L. Beijersbergen, Rene H. Medema, Bas van Steensel

Posted on: 11 June 2020

Article now published in Molecular Cell at http://dx.doi.org/10.1016/j.molcel.2021.03.032

How does the chromatin context of a Cas9 double-strand break influence repair choice? Read this pre-print to learn more

Selected by Katie Weiner

Background:

The chromatin context in which a DNA double strand break (DSB) occurs influences the choice of repair pathway. Previous studies have primarily investigated the chromatin contexts that favor repair by the canonical repair pathways homologous recombination (HR) and non-homologous end-joining (NHEJ). However, there are additional repair pathways that cells can use to repair DSBs. One such pathway is micro-homology mediated end-joining (MMEJ), in which short homologous sequences near the DSB are recombined. Further, in the context of genome editing in which a single-stranded oligodeoxynucleotide (ssODN) donor is provided to direct specific mutations or indels, the single-stranded template repair (SSTR) is used. It remains unclear how chromatin environments influence these lesser characterized repair pathways, namely MMEJ and SSTR.

Main Method:

The authors creatively combine techniques to generate a multiplexed reporter assay that allows them to induce and monitor DNA double strand breaks of a single DNA sequence randomly integrated at different chromatin environments. The system is comprised of (1) the reporter sequence, which is a previously characterized DNA sequence with distinguishable DNA repair products for NHEJ, MMEJ, and SSTR repair pathways (Brinkman et al., 2018), (2) an inducible double strand break catalyzed by Cas9 and a gRNA targeting the reporter sequence, (3) the random integration of the reporter sequence into the human genome using the PiggyBac transposon method, (4) unique barcoding of the reporter sequence such that each integration event can be independently followed and quantified, (5) and a cell line that has been extensively epigenetically characterized to understand the genomic integration site of the reporter sequence (K562 cells). Combined, these elements allow the authors to question how chromatin context, and not simply DNA sequence, impacts repair decisions as well as monitor the repair kinetics of a single double strand break.

Key Findings: 

Following validation of their novel methodology (described above), the paper makes several claims regarding the DNA repair of Cas9-induced DSBs:

  • NHEJ correlates with euchromatic regions (for example H3K4me1, H3K4me2, or H3K27ac) and MMEJ with heterochromatin regions (for example H3K27me3, H3K9me2, or lamina-associated domains). In the presence of a ssODN repair template, SSTR can be activated and weakly correlates with heterochromatin.
  • Heterochromatin features may redundantly favor MMEJ repair as reduction of H3K9me2 by treatment with a G9a-inhibitor, global loss of H3K27me3 by treatment with a EZH2-inhibitor, or knock-down of either LaminA/C or Lamin B Receptor had minor effects on repair pathway usage.
  • Kinetically, MMEJ has a very slow activation in all chromatin contexts. In contrast, NHEJ is rapidly activated, especially in euchromatin. This kinetic difference may explain why euchromatic regions have a stronger NHEJ repair preference. SSTR has intermediate kinetics, although this rate was only measured while NHEJ was inhibited.
  • Heterochromatin marked by the triple combination of H3K9me2, lamina-association, and late-replication timing behaves markedly differently than other heterochromatin types. It most strongly favors MMEJ, has the slowest indel accumulation, and has the slowest repair kinetics.
  • MMEJ and SSTR appear to be competing pathways that both require end resection by CtIP. Kinetically, MMEJ is activated first in triple marked heterochromatin, while SSTR is activated first in H3K27me3 heterochromatin and euchromatin.

Importance:

This study details an interesting new technique that has the potential to greatly increase our understanding of how DSBs are repaired in different disease contexts. Many studies are limited to studying how chromatin context in combination with the underlying DNA sequence affects repair. However, as all of the DNA sequences are the same in this method, this pitfall is overcome and the authors can look at just the contribution of chromatin environment on repair pathway choice. Using this method, they observe the repair preference for more than ~1,000 genomic locations and follow the kinetics of repair for a few of these locations. Their observations have implications for genome editing, as they show the preferred genomic context for the SSTR pathway which is used for Cas9-mediated repair template guided editing. Further, the methodology described is amenable to robotic, large-scale culture growth. As the authors point out, it is easy to imagine using a system like this to screen different drugs or mutants to better understand how they affect DSB repair across the genome.

Questions for the authors:

  • It seems that there are certain genomic regions that are resistant to reporter integration. Is there any thinking as to why these regions may be resistant and/or is there any expectations that this resistance may relate to altered DSB repair behavior?
  • The SSTR kinetics were investigated in the presence of NHEJ inhibitors. Do the authors have an understanding of the SSTR vs. NHEJ kinetics in the absence of such inhibitors, especially in euchromatin where both are preferred over MMEJ? On a related note, do the authors have any intuition as to whether the efficiency of Cas9-genome editing via SSTR would improve in the presence of NHEJ inhibitors?
  • An interesting future experiment could repeat some of these experiments in synchronized cultures. It would be interesting to see if different cell cycle stages effect either repair pathway presence or pathway kinetics?
  • The authors mention that this system is amenable to larger scale screens. In the authors opinions, what screens would be the most interesting to start with?

Tags: cas9, dna break repair, mmej, nhej, sstr

doi: https://doi.org/10.1242/prelights.21919

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Author's response

The author team shared

  • It seems that there are certain genomic regions that are resistant to reporter integration. Is there any thinking as to why these regions may be resistant and/or is there any expectations that this resistance may relate to altered DSB repair behavior?

It is true that there is a slight preference of PiggyBac transposition into more accessible chromatin. Previously, Huang et al. (Mol. Ther. 2010) showed that PiggyBac has a preference to integrate near transcription start sites (TSS) and the same was observed in our previous work with ~ 3-fold preference towards TSS. (Akhtar, de Jong, Pindyurin et al, Cell, 2013). But this would not explain the blank regions, as you might have noticed in figure 2a. These regions are, in majority, regions packed with repeats, in which we cannot uniquely map our integrated reporters by inverse PCR. They include centromeres, telomeres and other repeats throughout the genome. Other than these repetitive regions it is fair to consider that some regions are just resistant to reporter integration. Note, that we map here ~1000 reporter integrations in K562 cells, which means that is also comes down to a bit of chance. To determine PB resistance regions we could consider other performed TRIP experiments (e.g. Akhtar, de Jong, Pindyurin et al., Cell, 2013; Bruckner et al., Epigenetics Chromatin, 2016; Leemans et al., Cell, 2019; Gisler et al., Commun 2019). If such regions can resist PB transposition, it is reasonable to think that repair pathway balance may also be altered, and we would guess towards MMEJ.

  • The SSTR kinetics were investigated in the presence of NHEJ inhibitors. Do the authors have an understanding of the SSTR vs. NHEJ kinetics in the absence of such inhibitors, especially in euchromatin where both are preferred over MMEJ? On a related note, do the authors have any intuition as to whether the efficiency of Cas9-genome editing via SSTR would improve in the presence of NHEJ inhibitors?

We did not run a full time series with ssODN and without a NHEJ inhibitor, because our data on the pool (which were done with and without inhibitor) indicated that we might not have enough coverage of the MMEJ & SSTR pathways in absence of a DNAPKcs inhibitor. As you can see in figure 7a, without inhibitor only ~7% of all indels are repaired with SSTR at t = 64h.

We did check at an intermediate timepoint (t = 16h) and the interaction of SSTR vs. NHEJ is quite constant over 3 time points (16h, 64h, 88h), with or without DNAPKcs inhibitor, in both euchromatin and heterochromatin.

As to whether the efficiency of SSTR mediated genome editing would increase with NHEJ inhibition, we certainly do support that statement, and it’s also visible in figure 7a with the addition of NU7441. We would however recommend M3814, as S. Reisenberg et al. (NAR, 2019) showed (it works in our system as well), it has an even stronger bias towards SSTR, for an unknown reason.

  • An interesting future experiment could repeat some of these experiments in synchronized cultures. It would be interesting to see if different cell cycle stages effect either repair pathway presence or pathway kinetics?

We completely agree. It is thought that homology-based repair pathways are more active in S/G2 compared to G1 and it might affect the repair pathway balance (e.g. Hustedt & Durocher, Nature cell biology, 2017). It has been in our mind since we started this project. However, it is rather tricky to get a perfect synchronization of K562 cells. Any deviation from the synchronization will induce noise into the system. If we can improve this, it would be a very good experiment to increase our understanding of the impact of cell cycle and chromatin combined, especially with a time series setup.

  • The authors mention that this system is amenable to larger scale screens. In the authors opinions, what screens would be the most interesting to start with?

The strength of this system lies in its ability to dissociate chromatin effects from DNA sequence effects. Targeting chromatin factors would therefore be the obvious choice by using either CRISPR or epigenetic drugs for example. Then the relative activity of three repair pathways can be easily revealed under these conditions by determining the frequencies of the three specific indels.

The time series also bring a huge amount of possibilities to the table in terms of large data sets. We could try blocking Cas9 during the process or perturbing the cell cycle/checkpoints and then measure the effects on repair pathway balance over time at multiple sites in the genome. Blocking Cas9 will allow us to dissect precise repair kinetics (without re-cutting a perfectly repaired sequence), cell cycle and checkpoint changes might help us understand the interplay between cell cycle timing, chromatin and repair pathways.

 

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