Evolution? Who Needs It Now That We Have CRISPR/Cas9s?

by Marcia Stone

By replacing unwanted genes with those more to our liking in germline cells, CRISPR/Cas9 enables us to take charge of our own destiny.

Figure 1. Prostetnic Vogon Jeltz. Source

As reported on Small Things Considered in September 12, 2015, the Vogons in Douglas Adams' Hitchhikers Guide to the Galaxy consider evolution a waste of time and fix everything they think needs fixing with surgery. Now humans can do almost the same thing, albeit on a much smaller scale, with tiny molecular scissors called "Cas9s" carried to their targets by DNA-seeking RNAs known as "CRISPRs."

CRISPR/Cas9s, short for "clustered regularly interspaced palindromic repeats," and the "CRISPR-associated protein, known as "Cas," are used by Archaea and Bacteria to thwart phage- and plasmid-infections by cutting apart their DNAs and saving the bits to help them quickly recognize any future invasion attempts by the same predators. This ancient example of adaptive immunity has recently been borrowed from Streptococcus pyogenes SF370 and engineered by humans to "help cure diseases in which genes are causal or complicit," says Daniel Kevles currently at NYU Law School and Columbia University in New York City.

CRISPR/Cas9 achieves its effects by targeting alien DNA and cleaving it in both somatic cells, which are not passed down from generation to generation, and in germline cells, that are. The system can also replace aberrant genes with more normal ones, if that does not happen naturally.

The race is on to bring CRISPR/Cas9s into clinical use; for example, Jennifer Doudna at the University of California, Berkeley and colleagues are engineering versions that treat Huntington disease and Down syndrome. At the Broad Institute of MIT and Harvard, Feng Zhang and colleagues are designing CRISPR/Cas9s that target genes involved in mental health disorders and cancers. They also share their reagents widely to help others develop useful therapies.

In a first-of-its-kind investigation reported last year, the deletion of a mutated exon 23 by CRISPR/Cas9 in the mdx mouse model of Duchenne muscular dystrophy (DMD) enabled expression of a modified dystrophin gene that resulted in the partial recovery of dystrophin protein in heart and skeletal muscle with a consequent improvement in muscle force. "In contrast to exon-skipping strategies, genome editing can correct disease-causing genetic mutations," says Charles Gersbach from Duke University, Durham N.C. who led a team of scientists from around the world, including the Zhang group, on this project. The work establishes CRISPR/Cas9 as a potential therapy for DMD which currently sentences approximately one of every 5,000 newborn males to a progressively disabling premature death. Importantly, the editing done here only affects muscle cells and is not inherited by future generations.

            In contrast, using CRISPR technology to meddle with germline cells raises a number of ethical considerations. For example, it enables the creation of "designer babies" and even entirely new kinds of people. Thus a report of CRISPR editing in human embryos last April in the journal Protein & Cell caused much controversy and prompted a Summit on Human Gene Editing convened by the national scientific academies of the U.S., the U.K. and China that took place in Washington, D.C. this past December. This research which confirmed suspicions that such experiments were going on was led by Junjiu Huang at Sun Yat-sen University in Guangzhou.

Figure 2. The principle of CRISPR/Cas9-mediated gene disruption. A single guide RNA (sgRNA), consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein (1), binds to a recombinant form of Cas9 protein that has DNA endonuclease activity (2). The resulting complex will cause target-specific double-stranded DNA cleavage (3). The cleavage site will be repaired by the non-homologous end joining (NHEJ) DNA repair pathway, an error-prone process that may result in insertions/deletions (INDELs) that may disrupt gene function. Source

Notably, the Huang group used only non-viable polyspermic embryos, which have an extra set of chromosomes and die naturally after going through the first stages of development. They tested the ability of CRISPR/Cas9 to cleave the endogenous b-globin gene (HBB) in these fatally defective embryos using the tools developed by Zhang and colleagues; the mutated form of HBB causes b-thalassemia.

CRISPR/Cas9 successfully cleaved the targeted b-globin gene indicating it could eventually be a useful therapy for b-thalassaemia. It also confirmed that polyspermic embryos, which are usually discarded by fertility clinics, are a good alternative to viable human embryos. That’s the good news. Unfortunately the efficiency of HBB repair was low, off-target cleavage high as were unexpected mutations. Huang and colleagues therefore call for further improvement in the fidelity and specificity of the CRISPR/Cas9 system as a prerequisite to clinical application.

The expert consensus of the almost 500 participants from a variety of different fields attending the December Summit was that even though CRISPRs offer the ability to eradicate most, or even all, genetic diseases, no one should be rushing into it.

Figure 3. Eight-cell human embryo, at 3 days. Source

This is a course challenged by Prostetnic Vogon Jeltz who points to the extensive experience his people have had making their own changes as needed and how much time they’ve saved by "not waiting for nature to get around to making upgrades."

However, in response J. Jeffrey Morris says "Human attempts to 'intelligently design' ecosystems doesn't leave us with a lot of confidence that we understand nature’s complex web of interactions well enough to manipulate them on a large scale without causing disaster: think kudzu in the [our] South and rabbits in Australia."

"The human genome is a product of evolution simultaneously optimizing hundreds of phenotypes ¾ if we try to alter one or two of those phenotypes we think are important, it’s hard to predict what effect that will have on all those other phenotypes. My intuition tells me that evolution is way smarter than we are and we tinker with it at our peril," cautions Morris, an evolutionist at the University at the University of Alabama in Birmingham.

 

Suggested reading:

Adams, Douglas (1983). Hitchhikers Guide to the Galaxy. Harmony Books, New York. ISBN 0-330-25864-8

Doudna, J.A. and Carpentier, E. (2014) The new frontier of genome engineering with CRISPR-Cas9. Science;346: 1077- DOI: 10.1126/science.1258096

Kevles, D.J. (2015) If you could design your baby’s genes, would you? Politico; pp.1-8.

Laing, P. et al. (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & Cell (5):363–372. DOI 10.1007/s13238-015-0153-5

Morris JJ, Papoulis SE, and Lenski RE.(2014) "Coexistence of Evolving Bacteria Stabilized by a Shared Black Queen Function," Evolution 68; 2960-71.

Morris JJ, Lenski RE, and Zinser ER, (2012):  "The Black Queen Hypothesis: Evolution of Dependencies through Adaptive Gene Loss," mBio 3 e00036-00012.

Nelson, C.E. et al. Science 19.1126/science.aad5143 (205).

 

 

 Marcia Stone is a science writer based in New York City and frequent contributor to the ASM’s Microbe magazine.