Dear Juxiang, Parwez, and others interested in the multiplex design and application,
I strongly recommend everyone to check back at our website regularly for updates and additional information as we are working constantly to improve it.
Regarding the design, we have supplied detailed sequence information as well as design in the supplementary information of the paper, please refer to Supple Figure S5.
Meantime, I wrote a note below that summarizes and provides information for design of CRISPR constructs, esp. for the CRISPR array design and DR sequence, here in detail:
(1) Chimeric vs. Array design
In our most recent updated design, the full-length longer chimeric RNA design in general has been the most efficient system for introducing genome cleavage, the system and construct maps, reagent links to Addgene are all provided at our website:
http://www.genome-engineering.org/crispr/?page_id=23
(2) Direct Repeat Sequence
Despite the new chimeric design, The CRISPR array design allows for simultaneous cleavage at multiple genomic loci with a single array.
The DR sequence we used for S. pyogenes is: GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAAC
(3) Multiplexing CRISPR Array Design
Therefore, when trying to clone a two-spacer CRISPR array into our backbone vector like PX260, you should first define your two 30-bp protospacer targets, then order a pair of oligos looks like:
5' - AAACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-DR-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGT - 3'
and
3' - NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-DR-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAAAAT - 5'
So the annealed product looks like:
5' - AAACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-DR-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGT - 3'
3' - NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-DR-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAAAAT - 5'
Which can be readily cloned into the target backbone vector PX260 or PX334.
Please let me know if me know if you have any further questions or comments. Hope this helps with your experiments.