YALE JOURNAL OF BIOLOGY AND MEDICINE 90 (2017), pp.653-659.

Perspectives

Genome Editing: Past, Present, and Future
Dana Carroll*
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UTThe CRISPR-Cas genome editing tools have been adopted rapidly in the research community, and they
are quickly finding applications in the commercial sector as well. Lest we lose track of the broader
context, this Perspective presents a brief review of the history of the genome editing platforms andconsiders a few current technological issues. It then takes a very limited view into the future of thistechnology and highlights some of the societal issues that require examination and discussion.
INTRODUCTION
This is a marvelous time for genetics, due largely toadvances in genetic analysis and genetic manipulation.
The impact of innovations in high-throughput DNA sequencing and in genome editing have been felt broadly,from work on model organisms, to evolutionary studies,to improvement of food organisms, to medical applications.
Classically, genetic studies relied on the discovery and analysis of spontaneous mutations. This dependence was true of Mendel, Morgan, Avery, et al. In
the mid-twentieth century, Muller [1] and Auerbach [2]demonstrated that the rate of mutagenesis could be enhanced with radiation or chemical treatment. Later methods relied on transposon insertions that could be induced
in some organisms; but these procedures, like radiationand chemical mutagenesis, produced changes at randomsites in the genome. The first targeted genomic changes were produced in yeast and in mice in the 1970s and1980s [3-6]. This gene targeting depended on the processof homologous recombination, which was remarkably

precise but very inefficient, particularly in mouse cells.
Recovery of the desired products required powerful selection [7] and thorough characterization. Because of the
low frequency and the absence of culturable embryonicstem cells in mammals other than mice, gene targeting
was not readily adaptable to other species.
The current genome editing technologies resolved
this issue, making directed genetic manipulations possible in essentially all types of cells and organisms [8,9].
In addition, these methods confirmed Nobel laureate
Sydney Brenner’s notion that, “Progress in science depends on new techniques, new discoveries and new ideas,
probably in that order.” (http://www.azquotes.com/author/24376-Sydney_Brenner) In this short article, I want
to review where the genome editing platforms came fromand speculate about where we are headed through theiruse. I will leave description of the technologies themselves to other contributors.
GENOME EDITING PLATFORMS*To whom all correspondence should be addressed: Dana Carroll, Department of Biochemistry, 15 N. Medical Dr. East, room 4100,
Salt Lake City, UT 84112-5650 USA. Tel: 801-581-5977; Fax: 801-581-7959; Email: dana@biochem.utah.edu.
†Abbreviations: DSB, DNA double-strand break; ZFN, zinc-finger nuclease; TALEN, transcription activator-like effector nuclease;
CAR, chimeric antigen receptor; HDR, homology-dependent repair; NHEJ, nonhomologous end joining; CRISPR, clustered regularlyinterspaced short palindromic repeats.
Keywords: Genome editing, DNA repair, CRISPR-Cas, zinc-finger nucleases, TALENs
Author Contributions: DC wrote the article.
Copyright © 2017

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The secret to high-efficiency genome editing is theability to make a targeted DNA double-strand break(DSB†) in the chromosomal sequence of interest. Realization that such a break would stimulate gene targetingand local mutagenesis did not arise de novo, but camefrom research on DNA damage and repair. Recombination between homologous sequences is stimulated inmeiosis by intentional DSBs [10], and DSBs generated
by ionizing radiation lead to sister chromatid crossovers[11]. Model experiments with highly specific nucleases
showed stimulation of homologous repair in yeast andmammalian cells and pointed the way for programmablegenome editing [12-15]. Broken ends are also rejoinedby a process called nonhomologous end joining (NHEJ)[16]. The ends are often joined precisely, restoring theoriginal sequence; but occasionally errors are made, leading to local small insertions and deletions (indels). Whenthese mutations occur in a gene, they will frequently inactivate it.
We are currently endowed with three powerful classes of nucleases that can be programmed to make DSBs
at essentially any desired target: zinc-finger nucleases(ZFNs), transcription activator-like effector nucleases(TALENs) and CRISPR-Cas [8]. Although the latter platform now dominates in research laboratories around theworld, the other two are still in use for research and invarious agricultural and medical arenas. All of these platforms arose from investigations into natural biologicalprocesses and not from intentions to find genome editing
reagents.
ZFNs are hybrids between a DNA cleavage domainfrom a bacterial protein and sets of zinc fingers that wereoriginally identified in sequence-specific eukaryotic transcription factors. TALENs employ the same bacterial
cleavage domain, but link it to DNA recognition modules
from transcription factors produced by plant pathogenic bacteria. CRISPR-Cas is a prokaryotic system of acquired immunity to invading DNA or RNA.
Let us take a closer look at components of each of theplatforms. ZFNs: The first eukaryotic sequence-specifictranscription factor to be characterized was found to havezinc-binding repeats in its DNA-binding domain [17].
Related sequences from other transcription factors wereshown to be peptide modules that made stereotyped contacts with base pair triplets [18]. Changing a few residues
in a single zinc finger altered its DNA-recognition specificity, and fingers could be devised to recognize many different DNA triplets [19]. TALENs: Some plant pathogenic bacteria secrete into host cells proteins that bind to andregulate the activity of host genes to promote the infection. There is a simple and robust one-to-one code of recognition between modules in the protein and base pairs in
the DNA target [20,21]. ZFNs and TALENs: Some bacterial restriction enzymes cut DNA a few base pairs awayfrom their recognition sites. This is because they havephysically separable binding and cleavage domains [22].
The cleavage domain has no inherent sequence specificity, and it can be linked to novel DNA-binding domains,which alters where it cuts [23,24]. One such domain was
linked to zinc finger arrays and TALE arrays to generete
ZFNs and TALENs.
CRISPR-Cas: This story begins with the discovery
of a cluster of odd, short repeats in a bacterial genome[25]. Between those clustered regularly interspaced shortpalindromic repeats (CRISPR) are short sequences thatwere eventually shown to match viral genomes [26-28].
Some CRISPR-associated (Cas) proteins encoded adjacent to the repeat clusters mediate capture of these viralsequences, while others mediate cleavage and inactivation of invading viral genomes, guided by short RNAs
(crRNAs) transcribed from the CRISPR arrays [29,30].
The final piece of the puzzle was the identification of thesmall trans-acting RNA (tracrRNA) that participates inboth processing of the crRNAs and cleavage of the invading DNA in Streptococcus pyogenes [31]. Putting together the crRNA with tracrRNA and the one protein needed
for cleavage in this system (Cas9) led to the editing reagent that is now most widely used [32].
In summary, powerful tools come from unexpectedsources.
GENOME EDITING ISSUES
Remarkably, all that the genome editing nucleases
do is to make a break in chromosomal DNA. The key, ofcourse, is that the break is targeted and thus very specific. Everything that happens after the break, however, depends on cellular DNA repair machinery. The two broad
pathways of DSB repair are homology-dependent repair(HDR) in which a donor sequence matching the target iscopied, and NHEJ in which putting ends back together
can lead to mutations at the break site [8,9]. Most somatic
cells in higher eukaryotes generate mutations via NHEJ
more frequently than they copy sequences from a user-supplied donor. This bias is acceptable if all you want
to do is to knock out a protein coding sequence, but not
so good if you want to introduce sequences of your own
choice. Limited success has been achieved in modulating the ratio between homologous and non-homologous
products [33,34], but no general solution is yet at hand,
and the ratio in some cell types is very biased toward
NHEJ. Several recent reports suggest that small-molecule inhibitors of key NHEJ activities may be effective[35-37], but more research is needed to produce simple
and reliable reagents. Another way to influence the efficiency of homology-dependent events is through designof the donor DNA [38], linkage of the donor sequence tothe guide RNA [39], and consideration of specific mecha-

Carroll: Genome editing: past, present, and future

nisms to mediate sequence insertions [40,41].
All of the nuclease platforms can be very effective,
but none of them has perfect specificity. Recent modifications of both Cas9 protein and guide RNA have enhanced their discrimination against secondary targets[42]. How much one cares about off-target cleavage andmutagenesis depends on the application. In many model organisms, there are ways to validate the effects of an
introduced sequence change, including making independent mutations in the same gene, crossing into a clean
background, and complementing with a wild type gene.
In cases of organisms that can be rapidly expanded, likecrop plants, founder genomes can be fully sequenced, andfounder phenotypes can be analyzed thoroughly. Even insome medical applications, off-target mutations may betolerable, as long as they do not lead to a novel clinicalcondition.
LOOKING AHEAD
Research advances. It is safe to say that genome editing will continue to be a widely-used tool in research andin commercial and medical applications. One questionthat arises is whether CRISPR-Cas is the last word in programmable nucleases, or perhaps there is something evenbetter on the horizon. With limited vision into the future,
it is difficult to imagine a protein-based system that is fundamentally simpler than recognition by base pairing andcleavage by a single protein. Perhaps the protein could besmaller and be endowed with additional beneficial properties, but that constitutes variations on the same theme
rather than something completely novel. Maybe a fully
chemically-based reagent could be developed, based on
small synthetic compounds that combine DNA recognition with DNA cleavage. Research toward this end has
been going on for decades – from triplex-forming oligonucleotides [43], to peptide nucleic acids [44], to polyimines [45] – without producing a platform with adequatecleavage efficiency and recognition range. It seems likelythat if novel methods emerge, they will come, like thecurrent ones, from research into natural processes, notfrom an intent to improve on CRISPR.
A variation on the theme of DSB-induced genome
editing is the introduction recently of CRISPR-mediatedbase editing [46-49]. This platform makes use of Cas9
nickase, that cuts only one strand of the target DNA,linked to a base-modifying activity. Conversion of C to
U within a few base pairs of the RNA-guided binding site
leads to specific coding changes in that very narrow area.
Future uses of this approach include fusions to alternative
activities and modeling and correction of human disease
alleles.
Medical applications. A few somatic therapies thatinvolve genome editing have been approved for Phase655

I clinical trials. The earliest trials used ZFNs to knockout the CCR5 co-receptor gene in T cells of HIV-positive patients [50], thereby making the T cells resistant tothe virus. The results were encouraging, and an extension
to earlier hematopoietic precursors is planned. TALENs
have been used to enhance the efficacy of therapeutic
CAR T cells [51], and at least two trials using CRISPR-Cas9 for this purpose have been approved [52,53].
These examples rely on editing of cells in the laboratory – in some cases cells derived from the person being
treated – and transfer to the patient. Such ex vivo treatments allow facile delivery of the editing reagents andpreliminary characterization of the edited cells. As stem
cell therapies are developed, genome editing is a natural adjunct. Particularly when stem cells are derived inculture from somatic cells of an affected individual, correction of an offending mutation would fall to one of theediting platforms.
In many cases, cell-based therapy is not possible.
Clinical trials for treatment of hemophilia and two lysosomal storage diseases, based on in vivo delivery of ZFNs
with viral vectors, are under way (see clinicaltrials.gov,
and search “Sangamo”). These rely on gene editing in theliver, a comparatively accessible organ. Delivery to otherin vivo sites will require novel vector and non-vector approaches, and possibly the development of well-behaved
stem cells for particular tissues. Very active research is
directed toward treatments for other genetic diseases, including sickle cell disease and muscular dystrophy. In allcases, whether based on ex vivo or in vivo treatment, both
safety and efficacy must be demonstrated.
Germline editing. Stimulated by recognition of theease of CRISPR-based editing and the possibility of misuse of the technology, there is considerable current interest in prospects for human germline genome editing.
Such applications would involve delivery of the editing
reagents to embryos created by in vitro fertilization. In
the future, it may be feasible to engineer gametogenic
precursor cells in prospective parents instead. The advantage to germline correction of disease alleles is that they
will forever be gone from the lineage of the treated individual. The risk at present is that the attempt to correct
may do more harm than good. Current genome editing
technology does not have sufficient efficiency and specificity to be reliably safe. Mutations generated at non-target sites in the genome will also affect the treated person
and be transmitted through subsequent generations, andtheir effects will not always be benign or predictable, norwill they be readily reversible.
Continuing research will make germline editing safer and more effective, and it seems inevitable that it will
eventually be used. In the meantime, broad discussion of
the ethical issues raised by the prospect should be continued [54]. A thoughtful summary of the practical aspects

656

Carroll: Genome editing: past, present, and futureof both somatic and germline therapies is provided by
Kohn et al. [55].
Gene drives. An application of genome editingthat has begun to attract attention is the use in a genetic process called gene drive. In brief, a genetic element
can spread itself rapidly through a breeding populationby copying itself into genomes that previously lacked it.
Even if this element causes a moderately deleterious phenotype, it can expand in frequency. Natural gene drives
have been identified, but current interest is focused on
ones that are mediated by CRISPR-Cas9 [56]. Synthetic gene drives have been developed in mosquitoes thatserve as vectors for tropical diseases, including a systemthat produces sterility in females [57] and one that inactivates genes required for parasite growth [58]. In principle, these approaches could dramatically reduce diseasetransmission in areas where disease treatment is challenging. The enormous burden of mosquito-borne diseases onhuman lives and health, particularly in the developingworld, provides strong motivation for containing or eliminating the vectors.
The prospect of intentionally, or even unintentionally, releasing organisms carrying gene drives has evoked
appropriate concern [59,60]. It is very difficult to predictthe consequences for a broad ecosystem of depleting or
removing one of its residents. If a particular mosquito
population disappears, what will be the impact on organisms that rely on it, perhaps fish, birds, or plants? Other
species will soon fill a vacant niche, but will they havethe same influence on their surroundings? Will the drive
itself become ineffective by mutation or by adaptation of
the target organisms? Reversible gene drives are being
developed [56], but their efficacy has not been tested.
Unfortunately, small-scale laboratory tests will be poor
predictors of effects in a natural environment, and we will
not know the full impact of gene drives intended for benefit until they have actually been released.
Agriculture. Turning to agriculture, both livestockand crop plants are current targets for genome editing.
The organisms produced are literally genetically modified, but they differ from earlier GMOs in important ways
[61]. In most cases, no genetic material from another species is introduced, and when it is, it is inserted in a precise
genomic location. The changes that are introduced arevery often ones that could have occurred naturally, andwhole genome sequencing can be done on edited organisms to look for off-target mutations. Because both seeds
and semen can be dispersed rapidly into succeeding generations, validated genomes will quickly generate large
populations of modified plants or animals.
Among current examples of edited crops are disease
resistance in wheat [62], potatoes that don’t sweeten on
1

storage [63], and soy plants that produce healthier oil[64]. The prospects for developing other healthier crops
are bright. To address economic and animal welfare issues, dairy cows have been generated that lack horns, due
to a genetic modification [65]. Cows [66], sheep [66,67],
pigs [68], and other food animals that carry more muscle mass (i.e., meat) have also been produced by disruption of a single gene. Genome editing has the advantage
over breeding selection that a trait can be introduced in asingle generation without disrupting a favorable genetic
background. The same beneficial modification can be introduced into different breeds or cultivars that are adapted
to different environments, without leading to monoculture. A key question is whether the precise and largely
natural genome modifications made by editing will findgreater public acceptance than earlier GMOs. As the current resistance is based more on distaste for commercial
greed and dominance than on evidence of adverse effects,
there is a substantial hurdle to cross.
Beyond food modifications, large animal models ofhuman disease are being produced to facilitate physiological analysis, drug testing and other therapies. It seems
likely that genome editing will be applied to companion
species, generating new breeds of dogs and cats and correcting genetic susceptibilities in current breeds. Additional work will be needed to uncover the genetic causes
for desirable traits, but genetic research in dogs, at least,
is making good headway.
Societal issues. Finally, I want to address societal issues that apply to medical and agricultural applicationsof genome editing. Who will decide what products ortreatments are developed, and who will decide who getsthem? I call these issues Attribution and Distribution.
In the medical realm, what therapies will be developed based on whom we decide needs to be “fixed”? Devastating diseases, like Huntington’s disease and muscular
dystrophy, are obvious candidates. What about hereditarydeafness or short stature? People with these conditions
are often high-functioning, have strong communities, anddo not feel themselves to be in need of “correction” [69].
To take an absurd example, is skin color a condition thatneeds altering? This brings us to purely cosmetic changes that some may find desirable – hair color, eye color,height, athletic ability (assuming we know how to engineer these traits genetically). Should these applications
be pursued?1
Once methods are developed, who will benefit? Human therapies based on genome editing are currently
complex and expensive. Will only the wealthy be able
to afford them? Could we distribute a genetic therapy forsickle cell disease to the large populations in Africa andAsia that are most affected?

Two very recent papers highlight the interest in human germline editing and the value of research on human embryos [70,71].

Carroll: Genome editing: past, present, and future

These considerations apply to food organisms as
well. Will nutritional improvements be made in specialty
crops for the developed world, or in staples that predominate in the developing world? In both plants and animals,
will we engineer resistance to diseases that are endemic
in wealthy, temperate regions or to ones that limit production in developing regions? Ultimately, who will pay
for development and distribution of improved crops andlivestock – only the marketplace? Or will generous benefactors emerge?
Things are moving fast in genome editing. Manydifferent applications are being pursued, and the only
limit seems to be our imagination. In the midst of thisexcitement, we need to consider what are the best uses of
the technology, what adjustments are needed to make thetechnology safe and effective, and how its advances will
be provided to those who would benefit most. Currently,
these decisions are driven by market forces, not humanitarian considerations. Are we comfortable with this, or do
we need governmental participation at the national andinternational levels to change the situation? Count me as
an advocate for the latter.
Acknowledgments: Many talented people have contributed to the development of genome editing – too many
to be cited individually. I want to acknowledge that myunderstanding of the ethical and societal issues has
been enhanced by interactions with Prof. Alta Charo andby presentations by Profs. Ruha Benjamin and Catherine
Bliss at the International Summit on Human Gene Editingin Washington, DC, in December 2015. I thank Prof. Paul
Sigala and an anonymous reviewer for their comments
on earlier versions of the manuscript. Work in my own
laboratory is supported by grant GM078571 from the US
National Institutes of Health.
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