Adding to the ever growing potential of genome editing, one of the latest breakthroughs from the lab of Feng Zhang at the Broad Institute of MIT and Harvard puts CRISPR/Cas9 to the test with an interesting research trifecta of:
- in vivo genome editing focused on the brain
- genome editing that targeted DNA methylation machinery including key readers and writers.
- both single and multi-plex genome editing
The team created a novel system for probing gene function in the brain by introducing frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. But then they also added some variables, each perhaps worthy of it’s own paper.
The researchers optimized CRISPR/Cas9 for in vivo targeting using adeno-associated viral (AAV) vectors to deliver the custom molecular payloads, with the two different styles.
Single Gene Genome Editing Approach
- They targeted the key transcriptional regulator: MeCP2.
- The team gave a number of short, neuron-specific promoters a whirl, as well as polyA signals in order to get the best fit in their AAV vectors.
- In their final master design they chose a truncated version of the mouse MeCP2 promoter (235 bp) and a minimal polyA signal (48 bp).
Multiplex Genome Editing:
- The multi-gene approach targeted some writers more classic than Socrates: Dmnt1, Dnmt3a, Dmnt3b.
- They achieved some impressive biallelic modification ratios in the brain and confirmed by Westerns.
- It seems that the observed variation in indel formation between different loci may be due to variations in the “chromatin state and accessibility of target sites”.
The sgRNA vector also contained a GFP fusion protein (driven by the human Synapsin I promoter) for easy identification of transduced neurons. The team then went on the characterize the effects of the genome editing in post mitotic adult neurons using a comprehensive suite of biochemical, genetic, electrophysiological, and behavioural techniques on the adult mice.
Overall, the single vector approach allows for some great confirmations, visualization and sorting, but the multi-gene approach allows for efficient multiplex targeting.
There’s a lot of detail we left out so hustle over to Nature Biotechnology, October 2014 and take this one in.