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

2. 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

3. 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.

4. A SIMPLISTIC BROAD OVERVIEW OF A NEW APPROACH TO BASE EDITING

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

6. 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

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

8. 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.

9. 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

10. 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 .

11. 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.

12. The Pros and Cons of BE3

13. 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

14. BE3-Mediated Induced Base Correction

15. The Potential of Base Editing

16. Questions?