Poster abstracts
Poster number 129 submitted by Daniel Tener
Synthesis of single guide RNAs for CRISPR/Cas
Arun Kalliat Thazhathveetil (Process Development, Avecia), Daniel L. Tener (Small Scale Oligo Unit, Avecia)
Abstract:
Recent advent in genome-editing technologies has resulted in renewed interest in long oligonucleotides, especially RNA longmers that are used as single guide RNAs (sgRNA) for CRISPR-Cas9 system. These RNA longmers can be synthetically synthesized or produced in vitro or in vivo. Chemically synthesizing sgRNA provides versatility in incorporating modifications that can potentially improve the CRISPR gene editing efficiency. However, even with the recent advances in automated solid-phase synthesis, the production of long RNA oligomers is still difficult and time consuming. Poor coupling efficiency as the chain length increases resulting in poor yields, instability under cleavage and deprotection (C & D) conditions, difficulty in separating the full length product from truncated impurities are some of the main factors that affects the efficiency of longmer RNA manufacture. In this poster, we present the syntheses, C & D and purification studies of a series of RNA hundred-mers. These experiments were carried out as proof of concept experiments, to develop a process for the efficient and economic syntheses of RNA longmers. The information gleaned from these experiments was successfully used in a scale-up manufacturing process at Avecia, Cincinnati.
Keywords: RNA longmer, CRISPR, sgRNA