We have made a money-saving update to the CRISPR protocol, based on a publication from Alex Scheir's lab (Gagnon et al., 2014). Instead of purchasing a 119 bp "guide-oligo" (as in Hruscha et al., 2013), you can cut this sequence in half, and buy two 60 bp oligos with overlap and use PCR to sew the two pieces together. This saves money in two ways: 1) One of the two oligos which encodes the scaffold sequence is reusable, and only needs to be purchased once. 2) Because both oligos are much shorter, they can each be ordered at a smaller synthesis scale resulting in a much lower overall cost.

The short oligo method drops the price of each gRNA enough that I'm now designing two gRNA at once for each target gene, providing a backup for mutagenesis.

 I generated tyrosinase gRNA (Jao et al., 2013) using all three methods, and found similar mutagenesis rates. With this in mind, I recommend using this "short oligo" method for gRNA synthesis, because it is the least expensive and fastest method to make gRNA.

Our lab has tested the short oligo method on 14 targets, and found good mutagenesis for 4 of these CRISPRs; this is about half the success rate seen for the two methods in our publication ("synthetic" and "cloning based"). This may be statistical fluctuation, and we are testing the protocol further.

The "short oligo" method for gRNA synthesis is described on page 13 of our updated "CRISPR3.2" method.