Page 9 - PERIODIC Magazine Issue 7

Brown Group containing 2′-O-methyl (2′-OMe) RNA monomers. The primary
motivation was to improve yield and cost-efficiency, since 2′-OMe
Split and click RNA monomers couple more efficiently and are less expensive than
their RNA analogues.
strategy for All the CuAAC-ligated sgRNAs showed good DNA cleavage activity
CRISPR sgRNA in vitro. After one hour, clicked sgRNA containing the unnatural
triazole backbone cleaved roughly half the DNA compared to
Faster, easier and more accurate than control sgRNA (containing only natural backbones). Surprisingly,
previous genome editing methods, the chimeric clicked sgRNA with 2′-OMe performed better than the
CRISPR-Cas9 has revolutionised clicked RNA-only version, with levels of activity similar to that of the
biomedical research. The technology control sgRNA. The Brown group hypothesises that the 2′-OMe
consists of a single-guide (sg) RNA bound modifications may increase folding stability, offsetting destabilising
to a Cas9 enzyme, which acts like a pair effects from the triazole backbone. At longer timepoints (16 hours),
of molecular scissors. When the sgRNA all versions of sgRNA showed almost complete DNA cleavage.
recognises a complementary stretch of Similar results were obtained for experiments performed in cells.
DNA, Cas9 makes a double-stranded
break in the DNA at the specified
location. CRISPR-Cas9 is so powerful
in large part due to this ability to target
editing sites using straightforward
Watson-Crick base-pairing rules.
As researchers worldwide explore the
potential of CRISPR-Cas9, they need
sgRNAs in increasing numbers and
complexity. RNA can be synthesised
enzymatically, but these methods can
be expensive and time-consuming. On
the other hand, solid-phase chemical An sgRNA-Cas9 complex is depicted bound to double-stranded DNA. The variable DNA
synthesis of RNA is generally limited to binding 20-mer region of the sgRNA is shown in red, and the fixed Cas9-binding 79-mer
around 80 nucleotides in length due to is shown in black. The unnatural triazole backbone at the click ligation site is shown in
imperfect coupling. One way to surmount purple and magnified.
this length limit is to ligate chemically Analysis of the cleavage products found no significant difference in
Dr. Lapatrada Taemaitree, Dr.
Arun Shivalingam and Dr. Afaf synthesised oligonucleotides. specificity between clicked and control sgRNA.
El-Sagheer
The Brown group has extensively The split-and-click strategy reported by the Brown group enables
investigated ligation of oligonucleotides efficient, cost-effective production of diverse sgRNA libraries for
via copper(I)-catalysed azide-alkyne CRISPR-Cas9 applications. By taking advantage of untemplated
cycloaddition (CuAAC), in which an azide and an alkyne CuAAC reactions, the ligation method is freed from the need for
group react to form a 5-membered triazole ring. CuAAC complementary splints or self-templating design. Other benefits
is one of the most well-known click reactions, which are of the completely synthetic route include the ability to incorporate
characterised by high yields, fast reaction rates, little to site-specific modifications to enhance monitoring, binding,
no byproducts and mild conditions. Previous work by the activity or in vivo stability. For example, the Brown group reported
group showed that CuAAC and the resulting unnatural synthesis of an sgRNA library labelled with fluorophores. The next
triazole backbone at the ligation site are compatible with step is to use these labelled sgRNAs to image multiple sites in
replication, transcription and other biological processes. fixed or live cells at the same time. By increasing access to sgRNA
In a recent paper published in Nature Communications, libraries, the Brown group will help researchers further explore the
the Brown group demonstrate that CuAAC ligation is vast potential of CRISPR-Cas9 technology.
also compatible with CRISPR-Cas9 gene editing. Jointly
first-authored by Drs. Lapatrada Taemaitree and Arun
Shivalingam, the article describes a “split-and-click”
method to efficiently generate libraries of sgRNAs. Their
strategy consists of splitting the sgRNA into two chemically
synthesised oligonucleotides, a variable DNA-binding
20-mer and a fixed Cas9-binding 79-mer. The shorter
variable section can be produced with high specificity and
purity, while the fixed section can be synthesised cost-
effectively at large scales. The two RNA pieces are then
ligated via untemplated CuAAC to construct the complete
sgRNA.

In addition to the fully RNA version, the Brown group The split-and-click strategy developed by the Brown group enables the generation of
also synthesised the Cas9-binding 79-mer as a chimera diverse sgRNA libraries for CRISPR-Cas9 applications. The libraries can be generated via
individual click reactions or by a pooled one-pot reaction.
Reference: Taemaitree, L.; Shivalingam, A.; El-Sagheer, A.H.; Brown, T.; An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically
modified CRISPR sgRNA, Nat. Commun. 2019, 10, 1610, (doi.org/10.1038/s41467-019-09600-4). 9
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