Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci

Baohui Chen; Jeffrey Hu; Ricardo Almeida; Harrison Liu; Sanjeev Balakrishnan; Christian Covill-Cooke; Wendell A Lim; Bo Huang

doi:10.1093/nar/gkv1533

Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci

Nucleic Acids Res. 2016 May 5;44(8):e75. doi: 10.1093/nar/gkv1533. Epub 2016 Jan 5.

Authors

Baohui Chen¹, Jeffrey Hu¹, Ricardo Almeida², Harrison Liu³, Sanjeev Balakrishnan⁴, Christian Covill-Cooke⁵, Wendell A Lim⁶, Bo Huang⁷

Affiliations

¹ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA.
² Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA.
³ Graduate Program of Bioengineering, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁵ MRC Laboratory of Molecular Cell Biology, University College London, London, WC1E 6BT, UK.
⁶ Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 4143, USA.
⁷ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA [email protected].

Abstract

In order to elucidate the functional organization of the genome, it is vital to directly visualize the interactions between genomic elements in living cells. For this purpose, we engineered the Cas9 protein from Staphylococcus aureus (SaCas9) for the imaging of endogenous genomic loci, which showed a similar robustness and efficiency as previously reported for Streptococcus pyogenes Cas9 (SpCas9). Imaging readouts allowed us to characterize the DNA-binding activity of SaCas9 and to optimize its sgRNA scaffold. Combining SaCas9 and SpCas9, we demonstrated two-color CRISPR imaging with the capability to resolve genomic loci spaced by <300 kb. Combinatorial color-mixing further enabled us to code multiple genomic elements in the same cell. Our results highlight the potential of combining SpCas9 and SaCas9 for multiplexed CRISPR-Cas9 applications, such as imaging and genome engineering.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Associated Proteins / genetics*
CRISPR-Cas Systems / genetics*
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
DNA-Binding Proteins / genetics
Genome / genetics
HEK293 Cells
Humans
Mice
Staphylococcus aureus / enzymology*
Staphylococcus aureus / genetics
Streptococcus pyogenes / enzymology*
Streptococcus pyogenes / genetics

Substances

CRISPR-Associated Proteins
DNA-Binding Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding