I've had "radio silence on this blog for a while. Time to start it back up!

I've been reading and responding to a lot of questions about the 2015 protocol, we published and I hope to soon start posting these responses to the blog.

The most common question has been "what are low-cost alternatives to HRMA?"

In our paper, we indicated that a "standard" qPCR machine could be used to do HRMA-like analysis. A recent paper by D'Agostino et al demonstrated that this is indeed true.

If you're interested, take a look at this short paper: 
A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening.

Upside: Less upfront costs, fast
Downside: not very good at detecting small indels.
While this method can identify F0 carriers, it seems best-suited for F1 analysis.



A somewhat older paper provides another alternative method to HRMA:
An Efficient Genotyping Method for Genome-modified Animals and Human Cells Generated with CRISPR/Cas9 System

In this paper, Zhu et al. 2014, the authors show that acrylamide gels do an excellent job of identifying heteroduplex DNA, as is seen in F1 CRISPR injected embryos.
Simply do a PCR, and then run out the products on a 15% PAGE gel.

According to personal communications from Rich Londraville, this method also does a good job of identifying heterozygotes in F0

Upside: Very low cost, fast
Downside: May be less capable of distinguishing alleles from one another than HRMA.

I hope that the blog's readers find these methods helpful.

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.

Recently, Sharon Amacher and I wrote a protocol for doing CRISPR mutagenesis in zebrafish. The methods we described have been very useful to our lab, and has been the stuff of good conversation. In addition, we have shared the protocol with several other labs, who also found it helpful. We hope to see it published soon, and I would like to move the conversation to an open forum, in case the readers of that publication care to chime in.

With this in mind, I've set up this "zCRISPR" blog. I tried to model this "blog" after Chi-Bin Chien's Tol2kit blog; in which each entry was a fairly brief announcement, and the comments sections became a forum for conversations as new techniques developed. I suspect that this blog wont be as popular as Chi-Bin's, but he laid out a good model to work with.

 Last night, I posted a few conversation starters about the protocol we wrote- and invite any readers  to add comments, questions, and suggestions to those posts.  At the outset, I hope to improve and clarify the methods we've already written up- last night's posts have this goal in mind. As the protocol evolves, we'll post the major updates in new blog entries, which may themselves become fodder for further conversation.

In addition, I hope to use this blog as a place to discuss new  CRISPR techniques as they come out; including things not covered in the protocol Sharon and I wrote, and hopefully including things we've never dreamed of. CRISPR has opened up an exciting world of possibilities. I would be shocked and dissapointed if we have already imagined even the outlines of all the applications and implications of CRISPR/Cas.

Sincerely,
Jared Talbot

Emerging techniques

In this thread we invite discussion about emerging techniques in zebrafish CRISPR, including techniques that were not included in our 2014 paper.

Mutant recovery

In this thread, we invite comments and questions about the process of mutant recovery.

Mutant detection

In this thread, we invite comments and questions about mutant detection. What's your favorite method?

HRMA

In this thread, we invite comments about high resolution melt analysis (HRMA). How is it working for you? Do you have any questions about this technique? If so, please post them here!