Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.

ABSTRACT: The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the 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 sgRN

SUBMITTER: Hoy A 

PROVIDER: S-EPMC9805849 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature