Benchling is a great tool for gRNA design. Simply import the region of interest (Create/Sequence/Import DNA/Select Chromosomal Region) and use CRISPR/Design and analyze guides tool. All you need is the appropriate guide RNA sequence (i.e. 20 bp sequence preceeding xGG). If you can, choose the one with higher on- and off-target score.
pCRCT (Bao et al. 2015)
Contains iCas9 (D147Y, P411T) that has higher gene disruption efficiency. Designed to use multiple gRNA that make up crRNA array while tracrRNA is expressed separately (as is in bacteria). The crRNA is simply synthesized as a gBlock; refer to the original paper for design guidance. We have no experience with this approach yet.
pRS414gT-NLS-dCas9 (Smith et al. 2016)
This plasmid expresses catalitically dead Cas9 (dCas9) and has inducible gRNA. We've made a version of this plasmid expressing catalitically active Cas9 (pDB31), however the success rate of cloning oligos into it is lower than the original (not tested extensively).
To clone a guide into this plasmid, follow this protocol. However we skip the oligo amplification step and add it directly into Gibson reaction to 50 nM final (as per this guide).
Note that the Gibson mix must have 3'->5' exonuclease activity (i.e. use NEBuilder rather than NEB Gibson mix, or make this home-made Gibson yourself).
7.5 µl of 1.33x Gibson mix (home made);
1 µl of 0.5 µM oligo (1:200 from stock);
1.5 µ of ~ 50 ng/µl NotI digested vector.
To anneal primers, assemble the following components at RT into PCR tubes:
water: 4 µl
2x Annealing buffer (20 mM Tris pH 8.0, 0.1 M NaCl): 5 µl
Forward primer (100 µM): 0.5 µl
Reverse primer (100 µM): 0.5 µl
Incubate in PCR machine using program DIMA>ANNEAL. This takes about 2 h.
In advanced mode:
80ºC : 5 min - melting;
80ºC : 10 sec (600 cycles with -0.1ºC/cycle) - annealing.
Set up phosphorylation/ligation reaction:
water up to 10 µl total;
1.5 µl of the diluted 1:200 annealed oligos;
50-100 ng of BsaI cut pDB17/pDB18 vector;
1 µl T4 DNA Ligase buffer;
0.5 µl T4 PNK;
0.5 µl T4 DNA Ligase;
Incubate 2h-o/n at RT, transform into DH5&alpha. Include X-Gal/IPTG for blue/white screening (choose white colonies).
Plate pDB17 onto Kanamycin plates!
Note these are expressing wild type Cas9. More specific mutants are available elsewhere (Slaymaker et al. 2016).