Multiplex using Cas9 direct repeat

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)"

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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?

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Pink Pixiealmost 4 years ago

That's a really interesting idea, and it's further supported by the fact that in that paper (Cong and Ran et al 2013), crRNAs also look like they're more efficient than sgRNAs in some cases. However, in a paper later that year (Hsu and Scott et al 2013--, they showed that if you extend the tracrRNA element to nearly full-length in the sgRNA (+85 rather than +48), then sgRNAs are more efficient than crRNAs/tracrRNAs expressed separately.

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So you could definitely multiplex the crRNAs and have the tracrRNA expressed separately, but it might be less efficient than just using two sgRNAs driven by separate promoters.

Mint Lichalmost 4 years ago

This is a great point. Explained my confusion. In this paper (Hsu and Scott et al 2013), they also showed that longer sgRNA lead to greater transcription efficiency. Wonder why crRNA doesn't have similar KO efficiency ( crRNA~102nt, so transcription should not be a problem). Probably mammalian cells have less efficiency in processing the guide array.

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