For all the focus on making CRISPR more accurate, effective, compact, and versatile, one key feature has remained elusive: the two-for-one special. In a way, it’s ironic that CRISPR multiplexing – i.e., expressing multiple sgRNAs from the same promoter to target multiple DNA sites – is not more straightforward.
After all, natural CRISPR arrays are very compact, with all RNA spacers expressed from the same promoter. Cutting them apart requires dedicated processing enzymes, however, and most synthetic applications have sacrificed this processing ability to reduce the number of necessary genes. That means if you want to target multiple sequences, you must sandwich each sgRNA between its own promoter and terminator. Not only does that make multiplex CRISPR arrays big, their repetition makes them hard to synthesize.
Fortunately, a pair of studies recently brought CRISPR into the retail age, with a space-saving, express-one-get-one (or more!) -free special. The strategy separates sgRNA spacers with tRNA sequences, which the host cell’s tRNA processing machinery naturally cleaves, neatly releasing the sgRNAs to join up with Cas9. Kabin Xie, Bastian Minkenberg, and Yinong Yang of Penn State first demonstrated this strategy in rice plants, and then Fillip Port and Simon Bullock, of the MRC Lab of Molecular Biology in Cambridge, extended it to flies.
In 2015, Xie, Minkenberg, and Yang found that tRNA processing very efficiently released processed, battle-ready gRNAs in both rice protoplasts and intact plants, and the authors developed a handy, Golden Gate-based method to assemble up to 8x multiplex arrays. Not only could their multiplex arrays knock out multiple genes at once, but ganging up on a single gene with multiple gRNAs increased the knock-out efficiency.
Inspired by this rice demonstration, Port and Bullock showed the same gRNA-tRNA array strategy also works in whole flies, with similar improved knock-out efficiency and multiplexing ability. Additionally, the method worked much better for cell type-specific gene disruption, a previously difficult feat. Interestingly, CpfI did not work as well as Cas9 in flies, but CpfI did get a later start in genome editing, so it may well catch up.