Agilent Integrated Solutions for Design, Synthesis and Quality Control of Guide RNA for CRISPR-Cas9 Genome Editing Workflows

The CRISPR-Cas9 system has rapidly been adopted for many genome engineering
applications including creating gene knock-outs/knock-ins, genome mutagenesis,
and gene activation and inhibition studies. This Application Note demonstrates
how several Agilent products can be used in a CRISPR-Cas9 workflow, starting
with the design of a guide RNA (gRNA) against a specific target, synthesis of
the gRNA, quality control methods to ensure the integrity of the gRNA, and a
Cas9 nuclease activity assay to test the efficacy of the generated gRNA. gRNAs
were designed and synthesized against two target genes: ZFP42 and GUSB. The
synthesized gRNAs were assessed for their overall quality and cleavage efficacy
using the Agilent 2100 Bioanalyzer system.
Genome engineering technologies
are indispensable tools in several
biotechnology applications that range
from functional studies to developing
better or novel biological systems
One such tool, the clustered regularly
interspaced short palindromic repeat
(CRISPR) and CRISPR-associated (Cas)
protein system, is gaining interest due
to the ease and versatility in editing
the genome. The system consists of an
RNA-guided nuclease (Cas9), and a guide
RNA (gRNA) against a target sequence.
The RNA-guided nuclease introduces
a double-stranded break at the target
sequence site if a protospacer adaptor
motif (PAM) site is present adjacent to
the target sequence. The double-stranded
break can then be exploited to introduce
gene knockdowns or point mutations.
These RNA-guided nucleases that the
CRISPR-Cas systems are based on
depend on simple complementary base
pairing of a RNA-DNA duplex rather
than complex protein-DNA interactions
as in zinc finger nucleases (ZFN) or
transcription activator-like effector
nucleases (TALENs) techniques. The
target specificity of the Cas9 nuclease
can be modified by designing gRNAs to
target different genes.

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