The problem. Jinek’s 2012 work showed Cas9 could be programmed with a guide RNA to cut DNA in vitro. The open question was whether that would work inside a living mammalian cell, where chromatin, repair machinery, and the nucleus all change the picture. If it did, could you edit more than one gene at a time?

The idea. The authors engineered Cas9 from two bacteria to work in human and mouse cells, and drove it with short guide RNAs expressed in the cell. It cut at the programmed sites, and the cell’s own repair sealed the breaks with small insertions or deletions that knock a gene out. Supplying several guides at once let them edit several loci in the same cell, which is where the “multiplex” in the title comes from. They also used a nickase version that cuts one strand to lower off-target damage.

Why it matters. This is the step that made CRISPR a general lab tool rather than a bacterial curiosity. Multiplexing matters for synthetic biology, where you often need to change a pathway, not a single gene. It also sets up the screen papers later in this batch, where thousands of guides act at once across a cell population.

Verdict. A landmark method paper, co-founding mammalian CRISPR editing with Mali. Read it together with Mali 2013 as the pair that opened the door.