Download Programmable Editing of a Target Base in Genomic DNA oa pk ck

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
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 nonedited 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
Questions?