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© Research
Publication : Science (New York, N.Y.)

Dynamics of CRISPR-Cas9 genome interrogation in living cells

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Science (New York, N.Y.) - 13 Nov 2015

Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, Zhang ET, El Beheiry M, Masson JB, Dahan M, Liu Z, Doudna JA, Tjian R

Link to Pubmed [PMID] – 26564855

Science 2015 Nov;350(6262):823-6

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

https://www.ncbi.nlm.nih.gov/pubmed/26564855