2016
DOI: 10.1093/nar/gkv1533
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Expanding the CRISPR imaging toolset withStaphylococcus aureusCas9 for simultaneous imaging of multiple genomic loci

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.… Show more

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Cited by 167 publications
(162 citation statements)
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“…Because the S. pyogenes CRIPSR-Cas9 system has the simplest PAM sequence among all the CRIPSR-Cas9 systems adopted for DNA labeling910, one potential advantage of our method over previous two-color CRIPSR labeling strategies78 is that our method may allow easier selection of targeting sequences around certain genomic loci of interest. One could also potentially add more colors by incorporating more orthogonal RNA aptamers, such as the com system12.…”
Section: Discussionmentioning
confidence: 99%
“…Because the S. pyogenes CRIPSR-Cas9 system has the simplest PAM sequence among all the CRIPSR-Cas9 systems adopted for DNA labeling910, one potential advantage of our method over previous two-color CRIPSR labeling strategies78 is that our method may allow easier selection of targeting sequences around certain genomic loci of interest. One could also potentially add more colors by incorporating more orthogonal RNA aptamers, such as the com system12.…”
Section: Discussionmentioning
confidence: 99%
“…For this, dCas9 is fused with a fluorescent protein, either as a part of a fusion protein (Chen et al, 2013; Anton et al, 2014; Ma et al, 2015; Anton et al, 2016; Chen et al, 2016) or through an RNA scaffold (McDonald et al, 2016; Shao et al, 2016) (Fig. 1E).…”
Section: Cas9-mediated Chromatin Visualizationmentioning
confidence: 99%
“…The human alpha-satellite proteome from K562 cells has been analyzed by the PICh-related protocol HyCCAPP ( hy bridization c apture of c hromatin- a ssociated p roteins for p roteomics) 15 , again enabling comparison. We first confirmed dCas9-mCherry-APEX2 inducible expression, specific centromere targeting 16 , and biotinylation (Supplementary Fig. 8b–d), and then used SILAC to identify 1,268 proteins (Supplementary Table 4) from each of two biological replicates.…”
mentioning
confidence: 91%