I saw this comment on Pubpeer and found it very interesting:
"SpRNase III was not necessary for cleavage of the protospacer (Fig. 1D), and the 89-nt tracrRNA is processed in its absence (fig. S2C). Similarly, maturation of pre-crRNA does not require RNase III (Fig. 1D and fig. S4), suggesting that there may be endogenous mammalian RNases that assist in pre-crRNA maturation (24–26)"
This paper was the very first paper demonstrating the effectiveness of Cas9 in mammalian cells. If this is true, wouldn't it be easier to clone the 2 guides in the direct repeat rather than to clone the 2 U6 system when multiplex is needed?