The Year in CRISPRs - 19

through the entire genome to find and bind
to their prescribed target sequence.
"It's crazy that the Cas9 complex manages
to scan the vast space of eukaryotic genomes,"
remarked lead author of the Science paper and
graduate student Spencer Knight. "There is a lot
of off-target binding by Cas9, but we found that
these interactions are very brief-from milliseconds to seconds-before Cas9 moves on."

Spencer Knight video, UC Berkeley

The Year in CRISPRs * CRISPR Triple Checks Its Target before Cutting

The Berkeley team estimated that a few
thousand CRISPR-Cas9 complexes can scour the
entire genome extremely rapidly to find one
targeted stretch of DNA. Additionally, Cas9 must
also recognize a short three-base-pair DNA
sequence immediately following the primer
sequence, or PAM, which occurs roughly 300
million times within the human genome.
"If Cas9 bound for tens of seconds or minutes
at each off-target site, it would never, ever be
able to find a target and cut in a timely manner,"
Knight said.

19

| GENengnews.com

Several hundred Cas9 enzymes (red dots) searching the nucleus of a live mammalian cell for a particular
DNA sequence. They become white when they bind briefly before moving on. The lines show the paths
these enzymes have taken, color coded according to time. The Cas9 tracks show that the enzymes search by
diffusing through the nucleus, and that off-target binding events are predominately short lived. Motion is
slowed to half normal speed.

https://www.youtube.com/watch?v=YjuMDtjElfY https://www.youtube.com/watch?v=YjuMDtjElfY http://www.GENengnews.com

The Year in CRISPRs

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