Nucleosomes impede Cas9 access to DNA in vivo and in vitro
- Howard Hughes Medical Institute, University of California, San Francisco, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
Abstract
The prokaryotic CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.
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© 2016, Horlbeck et al.
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Further reading
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- Biochemistry and Chemical Biology
- Chromosomes and Gene Expression
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 allow Cas9 to effectively act on chromatin in vivo.
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- Chromosomes and Gene Expression
- Evolutionary Biology
Repression of retrotransposition is crucial for the successful fitness of a mammalian organism. The domesticated transposon protein L1TD1, derived from LINE-1 (L1) ORF1p, is an RNA-binding protein that is expressed only in some cancers and early embryogenesis. In human embryonic stem cells, it is found to be essential for maintaining pluripotency. In cancer, L1TD1 expression is highly correlative with malignancy progression and as such considered a potential prognostic factor for tumors. However, its molecular role in cancer remains largely unknown. Our findings reveal that DNA hypomethylation induces the expression of L1TD1 in HAP1 human tumor cells. L1TD1 depletion significantly modulates both the proteome and transcriptome and thereby reduces cell viability. Notably, L1TD1 associates with L1 transcripts and interacts with L1 ORF1p protein, thereby facilitating L1 retrotransposition. Our data suggest that L1TD1 collaborates with its ancestral L1 ORF1p as an RNA chaperone, ensuring the efficient retrotransposition of L1 retrotransposons, rather than directly impacting the abundance of L1TD1 targets. In this way, L1TD1 might have an important role not only during early development but also in tumorigenesis.
