Moving from in vitro to in vivo CRISPR screens

Clustered regularly interspaced short palindromic repeats (CRISPR) screens emerged as the gold standard technology in genetic screening in recent years. Most CRISPR screens are conducted in vitro, although current technologies fail to completely recapitulate the in vivo physiological environment. Direct in vivo screening - where cells are targeted within their natural niche - is emerging as a powerful approach to unravel biological processes in intact tissues and organs, taking into account complex cellular interactions, immune response, extracellular matrix, and tissue architecture. Several recent studies have demonstrated the capacity of in vivo screens to identify unique genetic dependencies left uncovered by in vitro screens. Together with new single cell readout techniques, in vivo CRISPR screens will continue to fuel progress towards identifying genetic elements controlling development, health, and disease

Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors