July 3, 2016

CRISPR craze: Applications of genome editing

by David Warmflash

CRISPR-Cas9 genetic editing is in the news a lot, especially in connection with concerns that it will usher in an era of designer babies. The capability for editing the genome of human embryos for non-medical purposes could lead to an ethical conundrum — especially considering the potential for gray areas, such as enhancing an embryo’s immune system to resist HIV or even the amount of skin pigmentation. The latter may sound cosmetic until you consider that pigmentation affects health factors such as vitamin D production and susceptibility to sunburn and skin cancer.

human genome
Graphical representation of the human genome organized into chromosomes.

But there are many other CRISPR applications already in use and in development that stand to revolutionize biology and medicine with far less controversy than the prospect of designing one’s offspring. Before looking at some of those, let’s discuss a little about what we even mean by CRISPR-Cas9 (“CRISPR” for short).

Over the last few decades, genetic engineers have worked with a specific handful of genetic editing tools – proteins called endonucleases – because they are able to make cuts in DNA. One class of these proteins is called “zinc fingers” and another is called “transcription activator-like effector nucleases” (aka, TALENs). Both zinc fingers and TALENs can be designed to find and cut specific sequences of nucleotide bases within a DNA molecule. This enables “editing” of a specific spot in an organism’s genome, but specific TALENs and zinc fingers must be designed for each particular genetic editing project. CRISPR stands for “clustered regularly interspaced short palindromic repeats,” a complex term for DNA sequences that enable microorganisms to protect themselves against viruses that could destroy them; “Cas” refers to an endonuclease protein that is associated with CRISPR – one that finds and cuts DNA sequences similar to TALENs and zinc fingers, except that a CRISPR-Cas system is programmable. Scientists first noticed the system in E. coli, but similar microbial immune systems exist throughout the Bacteria and Archea domains of life. Using one or more Cas proteins as cutting tools and different strands of RNA to locate genes of invading viruses, microorganisms with such an immune system can chop up viral DNA before the virus harms them.

It took a few decades for scientists to figure out what the CRISPR sequences were doing, but a few years ago teams of researchers figured out how to hijack the system from a microorganism (specifically the CRISPR-Cas9 system from Streptococcus pyogenes) and adapt it to other purposes. If microorganisms used Cas proteins to cut viral DNA, and were guided to the viral genes by RNA, the scientists reasoned that the system could be applied to cut any DNA.

Streptococcus pyogenes
Photomicrograph of Streptococcus pyogenes bacteria, 900x magnification. Courtesy of the CDC.

To simplify the system, researchers combined the various RNA strands into one strand, called chimeric RNA. By choosing a specific sequence for that one piece of RNA, you can easily create a CRISPR-Cas system that is customized to make a double-stranded cut in the genome of any organism. This is far more powerful than zinc fingers and TALENs, which require a completely different endonuclease for each sequence that needs to be cut.

Simply knowing the target sequence and inserting an RNA that matches that sequence enables deletion of the sequence and even replacement of the sequence. Essentially, CRISPR-Cas is like the search and replace features on your word processing application, but for letters in DNA. It’s so powerful that sometimes it’s called “molecular scissors.”

Graphical overview of CRISPR Cas9 plasmid construction. ©Nielsrca

What could you do with such a tool? Examples are almost endless, but here are a few:

  1. You can make humanized mice, meaning mice that carry certain human genes causing their tissues to react to experimental drugs and other treatments similar to how human tissues would react. Use of humanized mice is not only advancing medical research, but, because the humanized mice are a better model of human disease compared with standard laboratory mice, researchers are finding that they don’t need to use larger animals like dogs and pigs as much as they needed them in the past.
  2. Using CRISPR-Cas9 to modify pigs to be more like humans biochemically and immunologically, researchers are developing pigs that will be able to grow human organs inside their bodies. This approach could a long way toward mitigating, or even eliminating, the shortage of donated organs. Combined with advances in organ preservation, it could even lead to organ banking, the ability to pull an organ out of cold storage, as we do currently with blood products, with no need for patients to be on a waiting list.
  3. The technology is also being used in multiple farming applications. With pigs, for instance, CRISPR is being applied to tune down the gene for a protein called myostatin. This protein normally inhibits muscle growth, so the altered pigs grow to be extremely muscular, kind of like bodybuilders, an advantage to pork farmers.

These applications are just the tip of the iceberg and, in the near future, we may see genome editing via CRISPR used to take control over entire species. Why might we want to do that? One reason is to get a hold on diseases that are transmitted by insects. This is a major topic in international public health, it has implications for the Olympic Games in Brazil, and will be the topic of the next post.

David Warmflash

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David is an astrobiologist and science writer. He received his M.D. from Tel Aviv University Sackler School of Medicine, and has done post doctoral work at Brandeis University, the University of Pennsylvania, and the Johnson Space Center, where he was part of the NASA's first cohort of astrobiology training fellows. He has been involved in science outreach for more than a decade and since 2002 has collaborated with The Planetary Society on studying the effects of the space environment on small organisms.