- Context: this was a talk I attended in-person at the Innovative Genomics Institute Building (IGIB)
- DOI: 10.1038/s41587-024-02240-0
- Single Nucleotide Variant (SNV)
- 96% of human genetic variation is due to SNVs
- Each person has 4-5M genetic variants (small % of genome - 3.2B bp)
- Most of which are in nc regions
- e.g. T → A in HBB, causes SCD
- Most variants (2/3) identified are unclassified, and almost all are from individuals w/ European backgrounds (inequity in precision medicine)
- Functional genomics: methods to study variants in lab settings are needed
- Traditional genome editing using DSBs
- DSB repair (2 pathways are in competition - no way to get 100% one pathway yet)
- EJ → indels
- HDR (+ DNA template) → precise DNA manipulation
- “Nontraditional” genome editing tools: don’t use DSBs; do chemistry on single nt bases instead
- Base editing
- nCas9 + ssDNA cytidine deaminase + gRNA
- CBE: cytidine → uridine (C·G → U·G → T·A conversion)
- ABE: A·T → I·T → G·C conversion
- ABE0.1
couldn’t edit DNA 😢 edits RNA at UACG motifs + edits DNA at TACG motifs (low but detectable editing activity)
- How important are each of the nts in the TACG motif?
- Method: GFP reporter links DNA editing w/ fluorescence (higher sensitivity)
- G: favored but not critical for ABE0.1 editing
- T is critical for ABE0.1 editing; later evolved ones tolerate all bases but prefer T, slight aversion for A
- C and A are favored but not critical for ABE0.1 editing; evolved ABEs tolerate all bases at this position
- ABEs were evolved from TadA (tRNA deaminase enzyme)
- Directed evolution selection system in E. coli
- How important are each of the 14 mutations in ABE7.0?
- Method: Single nt reversion analysis
- Mutations cause host system-dependent impacts on activity
- Reversion combinations increase on-target editing activity
- ABE + CBE covers ~63% of human point mutations
- Summary: insights into ABE evolution
- Dev of new BEs will req engineering/evolving new nucleic acid modifying enzymes
- TadA was good starting point for ABEs due to its substrate promiscuity
- Host system-specific selection pressures may have resulted in mutations not optimal in other systems (bacteria vs. mammalian cells)
- Reversion analyses identified mutations in ABEs that increase on-target editing activity
- Multiplexing w/ BEs
- ex. of treatment involving ABE multiplexing: https://www.ukri.org/news/world-first-use-of-base-edited-cells-to-treat-incurable-leukaemia/
- gRNA crosstalk if multiplex ABE and CBE together - get mixtures of genotypes and undesired genome editing byproducts
- MOBEs avoid these byproducts by recruiting deaminases directly to gRNA
- Engineering aptamer BE systems (Quinn Cowan)
- Recruitment of deaminases directly to gRNA via aptamer-coat protein interactions
- Result: MOBEs (Multiplexed Orthogonal Base Editors)
- Increasing MOBE editing activity
- SNV combos in polygenic disease
- tool: OLIDA (oligogenic diseases database)
- digenic and monogenic diseases
- decided to start w/ digenic diseases: modeling digenic diseases w/ MOBEs