Methods for genotyping #CRISPR -generated mutations/edits: interesting alternatives.

Here are two papers with nice methods that are alternatives to the commonly used heteroduplex nuclease assays, e.g. Surveyor/Cel/T7E1.    First, a very new paper showing that simple PAGE gels can be pretty good for this.   Second, a paper from January that describes an in vitro method to use Cas9 itself + guide RNA to test PCR products for changes in "cuttability" following mutagenesis/editing.    

An Efficient Genotyping Method for Genome-modified Animals and Human Cells Generated with CRISPR/Cas9 System.

Zhu X, Xu Y, Yu S, Lu L, Ding M, Cheng J, Song G, Gao X, Yao L, Fan D, Meng S, Zhang X, Hu S, Tian Y.

Sci Rep. 2014 Sep 19;4:6420. doi: 10.1038/srep06420.

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.

Kim JM, Kim D, Kim S, Kim JS.

Nat Commun. 2014;5:3157. doi: 10.1038/ncomms4157.

The second paper gets around the problem of what if there are homozygous novel variants in some samples, as these are insensitive to simple heteroduplex assays unless you mix them with known wild type products first - which can be tedious and seems sort of wasteful to me personally.

Another gene-therapy-ish #CRISPR paper: germline correction of Duchenne muscular dystrophy mutation in mice.

 In this paper editing was performed in Mdx mutant mouse zygotes. Mdx is a classic mouse model for DMD.    Since Mdx is a point mutation that causes a premature stop codon in the dystrophin gene it was a natural target for CRISPR-mediated genomic editing.   

Science. 2014 Sep 5;345(6201):1184-8. doi: 10.1126/science.1254445. Epub 2014 Aug 14.Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN.

Muscular dystrophies fall in the groups of disorders that may be particularly amenable to gene therapy, as restoring gene products to even a fraction of deficient myofibers within a muscle may provide some improvement in overall muscle function.

Somewhat wistfully, this reminded me of the first transgenic "rescue" of the Mdx mouse by Greg Cox et al. in Jeff Chamberlain's lab at Michigan, back in 1993…

Nature. 1993 Aug 19;364(6439):725-9.Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity.Cox GA, Cole NM, Matsumura K, Phelps SF, Hauschka SD, Campbell KP, Faulkner JA, Chamberlain JS.

#CRISPR paper: Disruption of PCSK9 in liver lowers cholesterol in mice.

PCSK9 is an attractive target as inhibitors reduce serum cholesterol and inactivating mutations are associated with reduced cholesterol as well; conversely gain-of-function mutations are associated with familial hypercholesterolemia.  Knockdown approaches in adult mouse liver had been done previously for PCSK9 but not yet with CRISPR.

Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing

Qiurong Ding, 

Alanna Strong, 

Kevin M. Patel, 

Sze-Ling Ng,

Bridget S. Gosis, 

Stephanie N. Regan, 

Chad A. Cowan, 

Daniel J. Rader, 

Kiran Musunuru

.   Circ Res. 2014 Aug 15;115(5):488-92. doi: 10.1161/CIRCRESAHA.115.304351. Epub 2014 Jun 10.

Paper demonstrating #CRIPSR to correct human beta-thalassemia mutations in vitro.

  This made use of the piggyBAC transposon system to select for inserted clones; of the Puro+ clones, ~23% had undergone homologous recombination at HBB; of these, about 75% had recombined in such a way as to replace a mutation with wild type sequence.   

The piggyBAC transposon can be excised cleanly after the fact, allowing in vitro selection with a "clean getaway".   So this is very nice for certain in vitro applications. 
  
Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Xie F, Ye L, Chang JC, Beyer AI, Wang J, Muench MO, Kan YW.  Genome Res. 2014 Sep;24(9):1526-33. doi: 10.1101/gr.173427.114. Epub 2014 Aug 5.

Paper: More details about what sequence features make a CRISPR target highly "cuttable".

This was interesting for several nice reasons.  First, my interpretation is that this suggests that while most CRISPR targets have some level of acceptable targeting by Cas9/sgRNA, there is a subset of sites that are *very* susceptible.  Therefore, if you want to make a null allele in a gene it's a good idea to score all the possible sites first with this sort of algorithm.

Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.  Doench et al, Nat Biotechnol. 2014 Sep 3. doi: 10.1038/nbt.3026. [Epub ahead of print].

Second, they of course have made their scoring algorithm available as a web tool: http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design

I took a crack at it with a small DNA sequence from mouse that I know has a decent CRISPR target.  Interestingly, my pre-validated target had a lousy score (~0.05 on a scale from 0 to 1)  despite my knowledge that it works pretty well in my hands.  I take this to mean that of course, no scoring algorithm is perfect, but also that most targets actually fall into the so-so category of activity.   This jibes with data in this paper somewhat and I won't jump to conclusions based on my N=1!   I'd be interested to hear other people's results from running their targets through this algorithm.

Paper: #CRISPR optimization in mouse ESCs, with 50 vs 200 bp arm comparison for HDR. Longer arms better.

 This was just out in PLoS ONE: 
Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System, by Li et al.   The final figure shows a direct comparison between 50 and 200 bp homology arms for GFP knock-in via CRISPR-mediated HDR;  no big surprise, but clearly 200 bp had greater efficiency to 50 bp, by about an order of magnitude.   This is useful info for anyone who is designing "large" knock-in cassettes and wondering if it's important to put long arms on the cassettes; although ~60 bases is commonly used for ssDNAs in HDR (i.e. for peptide tag or loxP insertion), it's important to lengthen them for longer cassettes.    I suggest ~1 kb.  (my gut feeling as of today).