Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance
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 WeinerCategories: bioinformatics, genomics, molecular biology
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?
doi: https://doi.org/10.1242/prelights.21919
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the bioinformatics category:
Deep learning-based predictions of gene perturbation effects do not yet outperform simple linear methods
Benjamin Dominik Maier
Functional Diversity of Memory CD8 T Cells is Spatiotemporally Imprinted
Marina Schernthanner
Enhancer-driven cell type comparison reveals similarities between the mammalian and bird pallium
Rodrigo Senovilla-Ganzo
Also in the genomics category:
A fine kinetic balance of interactions directs transcription factor hubs to genes
Deevitha Balasubramanian
Modular control of time and space during vertebrate axis segmentation
AND
Natural genetic variation quantitatively regulates heart rate and dimension
Girish Kale, Jennifer Ann Black
Enhancer cooperativity can compensate for loss of activity over large genomic distances
Milan Antonovic
Also in the molecular biology category:
Platelet-derived LPA16:0 inhibits adult neurogenesis and stress resilience in anxiety disorder
Harvey Roweth
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor
Jonathan Townson
preListsbioinformatics category:
in the‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
Single Cell Biology 2020
A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.
List by | Alex Eve |
Antimicrobials: Discovery, clinical use, and development of resistance
Preprints that describe the discovery of new antimicrobials and any improvements made regarding their clinical use. Includes preprints that detail the factors affecting antimicrobial selection and the development of antimicrobial resistance.
List by | Zhang-He Goh |
Also in the genomics category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University
This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.
List by | Nándor Lipták |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
List by | Nándor Lipták |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
List by | Jesus Victorino |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
List by | Shikha Nayar |
Also in the molecular biology category:
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
List by | Nathalie Krauth |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |