The Year in CRISPRs - 11

The Year in CRISPRs * Improving CRISPR Specificity

they found that while Cas9 previously
showed full activity with most gRNAs
that are truncated by up to three
nucleotides, truRFNs induce efficient
mutagenesis with the gRNAs truncated
by only one nucleotide.

"Here we show that the on-target
activities of RFNs can be directed by
tru-gRNAs and that the activities of
these tru-RFNs can be comparable to
the activities of RFNs directed by fulllength gRNAs," the scientists explain.
"Additionally, the use of tru-gRNAs can
reduce the residual undesired mutagenic activity of monomeric RFNs. Our
findings suggest the utility of tru-RFNs
for applications requiring the highest
possible genome editing specificity."

Additionally the tru-RFN system
was able to minimize the off-target
mutation rates to levels significantly
lower than what has been observed
for CRISPR-Cas9 previously. This led the
researchers to the conclusion that "our
data show that tru-RFNs provide a
useful and further improved tool for
high-precision genome editing
applications in human cells."

The Boston-based researchers were
able to identify the gRNA truncation
length for highly specific genome
editing within human cells. Interestingly,

11

| GENengnews.com

A comprehensive review of the
CRISPR-Cas9 technology can also be
found in the July issue of Human Gene
Therapy in an article entitled "The
Bacterial Origins of the CRISPR
Genome-Editing Revolution." n

Human Gene Therapy

specificity of CRISPR-Cas9 nucleases,
but the researchers questioned if
the fidelity could be amplified by
combining the two technologies
together into what they have
named tru-RFN.

Schematic of a dimeric tru-RFN binding to DNA. Two FokI-dCas9 proteins
are targeted to DNA half-site sequences by two tru-gRNAs (red) enabling
FokI dimerization and cleavage of the intervening spacer sequence.

http://online.liebertpub.com/doi/full/10.1089/hum.2015.091 http://online.liebertpub.com/doi/full/10.1089/hum.2015.091 http://www.GENengnews.com

The Year in CRISPRs

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