Conjugation and Evaluation of Triazole-Linked Single Guide RNA for CRISPR-Cas9 Gene Editing

The CRISPR‐Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site‐specific double‐stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne–azide cycloaddition to generate a triazole‐linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified sgRNA constructs. Editing efficiency: A triazole‐linked single guide RNA (sgRNA) was constructed by using click chemistry. The sgRNA showed efficient CRISPR‐Cas9 genome editing activity comparable to that of natural dual RNA and unmodified sgRNA constructs. These findings open new avenues for generating long sgRNA libraries in array or plate formats.