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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.
                            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

              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, (         9
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