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High throughput pegRNA screening platform using lentiviral delivery

D Satyapertiwi(1) J H Haldrup(1) K M Miah(1) S C Hyde(1) D R Gill(1)

1:University of Oxford

Prime Editing (PE) has good flexibility in the scope for targeting pathogenic mutations, but selecting the most appropriate prime editing guide RNA (pegRNA) to correct any target remains a significant challenge. This challenge is mainly due to the large number of possible permutations in the 3’ extension region of the pegRNA. To address this, a platform was developed utilising a HEK293T cell line expressing PEMax-eGFP for pooled lentiviral screening of pegRNA candidates, containing a unique barcode and synthetic DNA target sequence for high throughput interrogation of pegRNA performance. The HEK293T-PEMax-eGFP cell line was generated by lentiviral transduction and single cell clones (n=6) were expanded to ensure homogenous editing performance throughout. Plasmid transfection of enhanced pegRNA targeting the endogenous HEK3 locus identified one HEK293T-PEMax-eGFP clone that demonstrated 33% of the intended edit. The integrity of this approach was confirmed by lentiviral transduction of one library element targeting the HEK3 locus, which resulted in the desired insertion of CTT in HEK3 loci in the synthetic target (22.5%-35%) and in the endogenous loci (50%-75%). As a clinically relevant exemplar, a synthetic library was generated targeting the common F508del mutation in the CFTR locus (comprising 445 pegRNAs with 9 additional pegRNAs as positive and negative controls). Assembly of the library was confirmed with 100% incorporation and an average of 300x coverage for each library element. A series of whole library transductions has been performed and data analysis is underway to identify candidate CFTR F508del pegRNAs that could have therapeutic potential.

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