Bio-orthogonal chemistry-based conjugation strategy facilitates investigation of impacts of s2U, s4U, m1A and m6A guide RNA modifications on CRISPR activity.
Hoy, A.; Zheng, Y. Y.; Sheng, j.; Royzen, M.
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The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential towards treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on incorporation of modifications into the single guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as m1A, m6A, s2U and s4U. The impacts of these RNA modifications on overall CRISPR activity was investigated in vitro and in Cas9-expressing HEK293T cells.
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