Nucleosome breathing and remodeling constrain CRISPR‐Cas9 function

  1. R Stefan Isaac
  2. Fuguo Jiang
  3. Jennifer A Doudna
  4. Wendell A Lim
  5. Geeta J Narlikar  Is a corresponding author
  6. Ricardo AB Almeida
  1. University of California, San Francisco, United States
  2. University of California, Berkeley, United States
  3. Howard Hughes Medical Institute, University of California, Berkeley, United States
  4. Howard Hughes Medical Institute, United States

Abstract

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allows Cas9 to effectively act on chromatin in vivo.

Article and author information

Author details

  1. R Stefan Isaac

    Department of Biochemistry and Biophysics and Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  2. Fuguo Jiang

    Department of Molecular and Cell Biology, California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Jennifer A Doudna

    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    Jennifer A Doudna, Co‐founder of Caribou Biosciences; Editas Medicine; Intellia Therapeutics.
  4. Wendell A Lim

    Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, San Francisco, United States
    Competing interests
    Wendell A Lim, Founder of Cell Design Labs, and member of its scientific advisory board.
  5. Geeta J Narlikar

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    Geeta.Narlikar@ucsf.edu
    Competing interests
    No competing interests declared.
  6. Ricardo AB Almeida

    Department of Cellular and Molecular Pharmacology, Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.

Reviewing Editor

  1. Karen Adelman, National Institute of Environmental Health Sciences, United States

Publication history

  1. Received: December 2, 2015
  2. Accepted: April 16, 2016
  3. Accepted Manuscript published: April 28, 2016 (version 1)
  4. Version of Record published: May 25, 2016 (version 2)

Copyright

© 2016, Isaac et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 8,723
    Page views
  • 2,260
    Downloads
  • 131
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. R Stefan Isaac
  2. Fuguo Jiang
  3. Jennifer A Doudna
  4. Wendell A Lim
  5. Geeta J Narlikar
  6. Ricardo AB Almeida
(2016)
Nucleosome breathing and remodeling constrain CRISPR‐Cas9 function
eLife 5:e13450.
https://doi.org/10.7554/eLife.13450
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    Ziyue Wang, Michael Mülleder ... Markus Ralser
    Research Article

    The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Jinli Geng, Yingjun Tang ... Xiaodong Liu
    Research Article Updated

    Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2–3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.