CRISPR-Cas-Assisted Multiplexing (CAM): Simple Same-Day Multi-Locus Engineering in Yeast.

Demands on the industrial and academic yeast strain engineer have increased significantly in the era of synthetic biology. Installing complex biosynthetic pathways and combining point mutations are tedious and time-consuming using traditional methods. With multiplex engineering tools, these tasks can be completed in a single step, typically achieving up to sixfold compression in strain engineering timelines. To capitalize on this potential, a variety of yeast CRISPR-Cas methods have been developed, differing largely in how the guide RNA (gRNA) reagents that direct the Cas9 nuclease are delivered. However, in nearly all reported protocols, the time savings of multiplexing is offset by multiple days of cloning to prepare the required reagents. Here, we discuss the advantages and opportunities of CRISPR-Cas-assisted multiplexing (CAM), a same-day, cloning-free method for multi-locus engineering in yeast. J. Cell. Physiol. 231: 2563-2569, 2016.