“…This poses a challenge for ab initio modelling of off-target activity, as biophysical models of off-target binding and cleavage are bound to be of limited accuracy unless they incorporate position-dependent energetic penalties for each base mismatch type and for deletions, as well as position-and basespecific penalties for insertions (Boyle et al, 2021;Jones et al, 2020;Zhang et al, 2020). In addition, as certain off-target sequences that are incompatible with dsDNA cleavage can undergo NTS nicking (Fu et al, 2019;Jones et al, 2020;Murugan et al, 2020;Zeng et al, 2018), future bioinformatic models need to be able to predict off-target nicking activity as well.…”