Agilent - Breakthroughs in Immunotherapy- 2019 - 10

Breakthroughs in Immunotherapy

bring greater activity and accuracy to CRISPR
editing. These solutions are combined with the
ability to assess and validate the results on
live-cell platforms that deliver relevant, timeresolved functional data.
Although the future remains bright for CRISPR
gene editing, current challenges for wide-spread
therapeutic use include better activity, specificity,
and tunability for different situations. Guide
RNAs that enable targeting of specific genomic
loci can be made using enzymes to copy a DNA
template into an RNA. However, direct chemical
synthesis of guide RNAs such as used for Agilent
SureGuide gRNAs (Agilent Technologies)4, has
distinct advantages. Chemical synthesis provides
a robust method for scalable manufacturing of
highly pure sgRNAs, affords unique opportunities for sgRNA design, and precise installation of
molecular functionality to augment CRISPR-Cas
performance for research, industrial, and therapeutic applications. Figure 1 shows that chemical
modifications added near the ends of guide RNAs
can improve the stability and activity of the guide
RNAs5. In a more recent study, improvements in
CRISPR specificity were achieved by incorporating
chemical modifications in guide RNAs at specific
sites in their DNA recognition sequence and then
systematically evaluating their on-target and
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Figure 1

Figure 1. Chemical synthesis of guide RNAs enables incorporation of combinations of natural, modified
natural, or unnatural nucleotides at defined positions that can impact CRISPR performance. Evaluation
of modified single guide RNAs targeting the CCR5 gene containing three 2′-O-methyl (M), 2′-O-methyl
3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) modified nucleotides at both the 5′ and 3′
ends were evaluated for activity in T cells using Cas9 delivered as mRNA or purified protein. MS and MSP
modified guide RNAs had significantly higher activity than unmodified guide RNAs. Figure adapted from
A. Hendel et al., Nat. Biotechnol. 33(9):985-989 (2015)

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Agilent - Breakthroughs in Immunotherapy- 2019

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