Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs
Nucleic Acids Research ,
Jan 2018
Ryan, Daniel E , Taussig, David , Steinfeld, Israel , Phadnis, Smruti M , Lunstad, Benjamin D , Singh, Madhurima , Vuong, Xuan , Okochi, Kenji D , McCaffrey, Ryan , Olesiak, Magdalena , et al.
Ryan, Daniel E
Taussig, David
Steinfeld, Israel
Phadnis, Smruti M
Lunstad, Benjamin D
Singh, Madhurima
Vuong, Xuan
Okochi, Kenji D
McCaffrey, Ryan
Olesiak, Magdalena
et al.
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
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Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs
Nucleic Acids Research
Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs
Daniel E. Ryan 2
David Taussig 2
Israel Steinfeld 2
Smruti M. Phadnis 2
Benjamin
D. Lunstad 1
Madhurima Singh 2
Xuan Vuong 2
Kenji D. Okochi 1
Ryan McCaffrey 1
Magdalena Olesiak 0
Subhadeep Roy 1
Chong Wing Yung 2
Bo Curry 2
Jeffrey
R. Sampson 2
Laurakay Bruhn 2
Douglas J. Dellinger 1
0 University of Colorado , Boulder, CO , USA
1 Agilent Research Laboratories , Boulder, CO , USA
2 Agilent Research Laboratories , Santa Clara, CA , USA
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2 -O-methyl-3 -phosphonoacetate, or 'MP') incorporated at select sites in the ribosephosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
INTRODUCTION
CRISPR–Cas systems evolved to function on relatively
small genomes in bacteria, but the recent proliferation of
applications in larger genomes has created a growing need
for higher specificity (
1–5
), especially for applications such
as therapeutics where off-target activities have the potential
to introduce deleterious effects (
6,7
). Various approaches
have been reported to increase the specificity of CRISPR–
Cas9 to minimize off-target events (8). These include
truncations (
9
) and extensions (
10
) at the 5 ends of gRNAs,
colocalization of paired nickase mutants of Cas9 (
11
), fusion
of catalytically inactive dCas9 to dimerization-dependent
Fok1 nuclease (
12,13
), and engineered higher-fidelity
versions of Cas9 protein (
14–16
). Other approaches control the
duration of CRISPR activity in eukaryotic cells, for
example by transient delivery of Cas9 and gRNA as a
ribonucleoprotein complex (gRNP) via cationic lipids or
electroporation (
17,18
), by inducible temporal control (
19,20
), or
by timed addition of a CRISPR–Cas9 inhibitor (21) and
other strategies. Among these options, transient delivery of
gRNP complex has gained widespread favor and the other
methods have yet to be broadly adopted.
Here, we explore an alternative means of modulating the
specificity of CRISPR–Cas systems by incorporating
chemical modifications in the 20-nucleotide (nt) guide sequence
at the 5 end of a gRNA which mediates the specificity of
Cas9 in recognizing and hybridizing complementary
protospacers. We have developed robust synthetic chemistry
methods to produce single-guide RNAs (sgRNAs) with
high fidelity and purity (
22
) and have reported that certain
chemical modifications in the first and last three terminal
nucleotides of sgRNAs can substantially boost CRISPR–
Cas9 indel rates and homology-directed repair (HDR)
editing events (
23
). The terminal modifications are thought to
provide resistance to exonucleases, which is especially
helpful in primary cells. Other studies exploring the potential
advantages of chemical modifications in gRNAs employed
crRNA and tracrRNA strands in dual-guide RNA
systems and showed that chemical modifications in the crRNA
strand can boost indel frequencies in transfected human
cells (
24–26
), can enable conjugation of donor DNA for
template-directed repair (
27
), and reportedly can improve
the targeting specificity of truncated crRNAs by
extensive modification of their ribose-phosphate backbones with
2 -O-methine-4 bridges, phosphorothioates, and 2 -fluoro
substituents in specific combinations (
24
).
In the present work, we chemically synthesized sgRNAs
in which we systematically walked individual MP
modifications across the 20-nt guide sequence of sgRNAs
targeting four different genes, and each sgRNA was complexed
with Streptococcus pyogenes Cas9 protein to evaluate the
gene editing specificity of the Cas9 sgRNP. Importantly,
we identified specific positions in sgRNA guide sequences
where MP modification improves cleavage specificity across
a variety of target loci and cell types, in several instances
improving specificity by an order-of-magnitude or greater
while maintaining high on-target activity.
MATERIALS AND METHODS
sgRNA synthesis
RNA oligomers were synthesized on a Dr Oligo 48
synthesizer (Biolytic Lab Performance, Inc. (...truncated)
This is a preview of a remote PDF: https://academic.oup.com/nar/article-pdf/46/2/792/23595387/gkx1199.pdf
Ryan, Daniel E, Taussig, David, Steinfeld, Israel, Phadnis, Smruti M, Lunstad, Benjamin D, Singh, Madhurima, Vuong, Xuan, Okochi, Kenji D, McCaffrey, Ryan, Olesiak, Magdalena, Roy, Subhadeep, Yung, Chong Wing, Curry, Bo, Sampson, Jeffrey R, Bruhn, Laurakay, Dellinger, Douglas J.
Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs ,
Nucleic Acids Research,
2018, pp. 792-803, Volume 46, Issue 2, DOI: 10.1093/nar/gkx1199