This paper nails programmable cleavage in vitro. As an outsider this always confused me: why was off-target activity such a big deal later if the guide is so specific? What changes between a clean tube and a live cell that makes specificity hard?
Accepted answer
Several things: chromatin accessibility, much higher effective enzyme/guide concentrations and dwell times in cells, and tolerance for mismatches (especially distal to the PAM). In a tube you control all of that; in a cell you don't.
That makes sense — so 2012 is 'it's programmable', and a decade of work was 'make it programmable and precise under messy in-cell conditions'.
And the assays matter: early off-target claims swung a lot depending on whether detection was homology-biased or genome-wide unbiased. Methodology drove the narrative as much as the biology.