Presented by: Oyeyemi Ajayi, Prashant Kuntala, and Colin Kruse

CRISPR/CAS 9 SYSTEM In response to DSB, two DNA repair pathways: NHEJ and HDR are initiated. HDR competes with NHEJ during the resolution of DSBs. Current approach induces Indels from cellular response to dsDNA breaks

This new approach enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template.

A SIMPLISTIC BROAD OVERVIEW OF A NEW APPROACH TO BASE EDITING

Of Base Editors ( three Generations ) Protein Engineering Of Base Editors ( three Generations )

Base Editor – generation 1 Catalytically dead Cas9 (dCas9) fused with rAPOBEC1 (Cytidine deaminase enzyme) to the N-terminus. Inactive nuclease activity (Asp10Ala mutation, His840Ala) Programmable conversion of C to U in DNA. Activity window is approx. 3 to 6 nt In vitro efficiency : 50 to 80% C to U conversion of substrate strands

Context dependence of BE1 BE1 activity preference APOBEC1 prefer substrates with TC or CC TC > CC > AC > GC Max editing efficiency at position 7

Base Editor – generation 1 Catalytically dead Cas9 (dCas9) fused with rAPOBEC1 (Cytidine deaminase enzyme) to the N-terminus. Inactive nuclease activity (Asp10Ala and His840Ala mutations) Programmable conversion of C to U in DNA. Activity window is approx. 3 to 6 nt In vitro efficiency : 50 to 80% C to U conversion of substrate strands But efficiency of C to T editing in Human cells was about 0.8 to 7.7% of total DNA.

Base Editor- generation 2 Hypothesis: Base excision repair (BER) reverts U:G to C:G pair BE2 Uracil DNA glycosylase inhibitor (UGI) fused to the C–terminus of BE1. Increased efficiency in human cells by 3-fold, 20% of total DNA sequences. Indel formation rates <= 0.1% in both BE1 and BE2

Base Editor – generation 3 To augment the base editing efficiency – manipulate cellular DNA repair further. Hypothesis: By nicking the unedited strand, MMR will preferentially repair the unedited strand (G to A) BE3 Restored the catalytic His residue in Cas9 (BE2) that nicks the non- edited strand, containing a G opposite of edited U .

Outcome of BE3 Nicking the non-edited strand augmented base editing efficiency in human cells 2-6 fold relative to BE2, resulting in up to 37% of total DNA sequences containing the targeted C to T.

The Pros and Cons of BE3

BE3-Mediated Correction of Disease-Relevant Mutations Komor et al. set out to correct the two potent missense mutations that could be corrected by C to T (or G to A) base editing p53 Tyr163Cys mutation: cancer associated. APOE4, Cys158Arg mutation: potent Alzheimer's risk factor

BE3-Mediated Induced Base Correction

The Potential of Base Editing

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