Method of the Year 2014 – CRISPR/Cas9 genome editing

by Alexey Bersenev on December 31, 2014 · 0 comments

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Happy New Year, dear readers! Every year we are picking the “Method of the Year“. The methods in this contest should be relatively new (at least for human applications) and have huge potential impact in stem cell research/ cell therapy/ regenerative medicine field.

The winner of 2014 – CRISPR/Cas9-based techniques for genome editing of stem cells!
About 2 weeks ago, I asked you to help me to pick the best method of 2014, and here is the results of the poll. CRISPR was unbeatable, solid as rock! No contest!

Now, I’d like to introduce our guest contributor Erik Westin. He nicely summarized highlights of CRISPR-based genome editing field in 2014.

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Last year CRISPR had taken off as one of the most important scientific breakthroughs of 2013 with widespread applications (a Science runner-up to CAR T-Cell research; a runner-up on this website to SCNT). Now that the CRISPR tires have been kicked by the biological research community a number of things are becoming apparent. The first is that the facile nature of CRISPR, related to other genome editing methodologies like zinc finger nucleases, lends itself well to labs that are interested in genome editing while also requiring a low-barrier-to-entry type of method. This is apparent in the vast number of publications related to CRISPR (277 in 2013, growing to 641 in 2014 on Pubmed this year and 21 in 2015 [as of 12/30/14]; 110/117/7 for ZFNs in 2013/2014/2015 for comparison). A Google query for CRISPR frequency (vs ZFNs and a related respective companies Editas and Sangamo) reveals a strong trend favoring CRISPR, likely fueled by a number of successful applications of CRISPR in the scientific literature. A second point that can be made is that diminution of off-target effects appears to be real and achievable by trading in the more efficient double-strand DNA Cas9 endonuclease for the modified endonucleases that create single-strand nicks. A number of papers have reported some very promising results in this regard. A third point that was made this year is that CRISPR is a technique pushing the envelope regarding therapeutics. A couple highlights include the targeting of the B2M and CCR5 loci in hematopoietic cells by the Cowan and Rossi labs as well as the generation of a CRISPR-modified primate from the editing of a single cell. I missed out on the ZFN craze of the mid 2000’s but imagine that 2014 was similar to the aftermath of first ZFN publications for CRISPR, but on steroids.

Some drawbacks do still exist for CRISPR though. CRISPR had a bill of sale early on that appeared to include high efficiency and thereby high recovery rates of modified cells but this may not be the case for a number of reasons. A couple of papers have reported increased success in regards to targeting but they involve slightly modified protocols such as the use of protein or cell cycle synchronization. Papers like these appear to me to be an implicit indication that targeting is likely an issue for that vast majority of labs using primary cells. My personal preference at this time is to recover genome edits without the use of large antibiotic selection constructs however the recovery rate for this methodology is not yet optimal in my hands. One particular methods paper that I would like to highlight is the coupling of Taqman probes and sib-selection protocols published by the Conklin lab. By screening millions of cells, this protocol assists in the isolation of modified iPSC clones over the course of a small number of passages. This protocol may be of interest to those wishing to minimize off-target effects via double-nickase methods that are inherently less efficient.

Finally, given the growth and interest in CRISPR as of late, a particular interesting off-the-field fight over the CRISPR patent rights is taking place. A recent article highlights the schism that taking place among labs towards deciphering the laboratory origins of CRISPR for future intellectual property rights. Feng Zhang and the Broad Institute were awarded patent rights in the United States in 2014 and subsequently licensed it to Editas (and a number of universities). Jennifer Doudna had been a co-founding member of Editas however left after the Broad Institute was awarded this patent and issued her own pending patent to another CRISPR startup, Intellia. A third patent already exists in Europe originating from Doudna’s collaborator, Emmanuelle Charpentier, that was sold to CRISPR Therapeutics. According to the article, intellectual property rights may ultimately boil down to the lab whose notebooks prove that they conceived of using this bacterial immune system towards genome editing and therapeutic use first.

2014 appeared to be the year that ZFNs achieved after their initial success however without the impressive scale that CRISPR achieved. It will be interesting to see what 2015 holds for CRISPR as this genome editor exits the shadow left by previous editing methodologies and comes into its own.

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