Gene
editing is a category of new methods that can be used to precisely
edit or change the genetic code. As the name ‘gene editing’
suggests, these technologies enable researchers to add, delete,
or replace letters in the genetic code. In the same way that
spell check identifies and corrects single letter errors in
a word or grammar errors in a sentence, gene editing can be
used to identify and change the letters that make up the genetic
code (i.e. DNA) within an individual.
Gene
editing has many potential applications. For example, it can
be used to correct diseases and disorders that have a genetic
basis. It could also be used to change a less desirable form
of a gene (called an allele) to a more desirable allele without
the need to introgress (repeatedly backcross) or bring in that
allele through outcrossing with an animal that carries the desirable
allele. Therefore gene editing is really more like precision
breeding where breeders can introduce the specific sequences
that they would like to select for using gene editing technologies.
Gene
editing is different from traditional genetic engineering. Continuing
with the analogy of a word processor, genetic engineering enables
a gene sequence of foreign DNA to be cut and pasted from one
species to another; typically the location where the new DNA
sequence inserts into the genome is random. Gene editing can
add, delete, or replace a series of letters in the genetic code
at a very precise location in the genome.
The
basic idea behind gene editing is that molecular scissors called
nucleases are used to cut DNA at a specific location in the
genome based on recognition of the specific, unique target DNA
sequence. The cut site is then repaired using the DNA repair
mechanisms of the cell. These repairs can be directed to introduce,
delete, or replace a series of letters in the genetic code.
This essentially enables the introduction of known, desired
alleles based on what is understood about naturally-occurring
genetic variation in the target species.
Without
the addition of template DNA, the double stranded breaks created
at a precise location in the genome by the nucleases are repaired
by the cell’s natural DNA repair mechanism, and this typically
results in single nucleotide changes, deletions or small (1-2
nucleotide) insertions at the DNA cut site. In this case, although
the location of the cut site is very precise, the exact change
that occurs when the DNA is repaired is random and so a number
of different outcomes representing minor sequence changes are
possible.
Supplied
with a nucleic acid template, however, the double stranded breaks
initiated by the nucleases are repaired via the cell’s
homologous recombination repair pathway whereby the template
dictates the sequence resulting from the repair, allowing the
introduction of the DNA sequence dictated by the template into
the host genome. Such changes might range from nucleotide-specific
changes, to large (whole gene) insertions or substitutions depending
upon the template. The end result of this maybe a targeted SNP
edit (e.g. the nucleotide A at a given location in the genome
is deliberately replaced by T), the replacement of one naturally
occurring allele with another naturally occurring allele at
target genetic gene locus in that species, or the introduction
of a novel DNA sequence as encoded by the template at the target
location in the genome.
There
are many potential uses of this technology ranging from human
medicine to plant and animal breeding.
HOW
MIGHT GENE EDITING BE USED IN ANIMAL BREEDING?
The
currently available set of gene editing technologies (zinc finger
nucleases (ZFNs), transcription activator-like effector nucleases
(TALENs), and clustered regulatory interspersed short palindromic
repeats (CRISPRs) associated system) have been used for a relatively
small number of livestock applications to date.
Gene
editing has been used to produce genetically hornless Holstein
dairy cattle by replacing the Holstein ‘horned’
allele with the naturally-occurring Angus ‘polled’
allele at the gene that is responsible for horn development
and to generate pigs with a single base deletion in a gene that
may confer resilience to African Swine Fever Virus. Recently
a paper was published showing that gene edited pigs were protected
from porcine respiratory and reproductive syndrome (PRRS) virus,
a particularly devastating disease of the global pork industry.
It has also been used to introduce changes in the myostatin
gene in sheep and cattle. As the Latin origin of the word myostatin
(muscle/stop) suggests, turning off this gene results in muscle
growth. Naturally-occurring mutations in this gene have historically
been selected by conventional animal breeders and are the genetic
basis for the ‘double muscled’ phenotype that is
seen in cattle breeds like the Belgian Blue, and the ‘bully’
phenotype in whippet dogs.
In
this way, gene editing really mimics the natural processes that
form the basis of selective breeding programs, and for that
matter, natural selection. Breeders work with the genetic variation
that exists within a species, and that genetic variation ultimately
arises from naturally-occurring mutations. Although the word
mutation’ sounds negative, it simply refers to variations
in DNA sequences. These variations, or mutations, are responsible
for virtually all genetic differences which exist between individuals,
such as having blue eyes instead of brown.
Although
different mammals have many of the same genes, many people do
not appreciate that the genetic code that makes up those genes
differs among animals of different breeds, and even among animals
within the same breed. In fact, with the exception of identical
twins, there are literally millions of DNA sequence variations
between two individuals of any species. For example, an enormous
number of genetic variants have accumulated within cattle since
the advent of domestication and selective breeding due to the
naturally-occurring processes that lead to a small number of
mutations each generation. In one recent analysis of whole-genome
sequence data from 234 taurine cattle representing 3 breeds,
more than 28 million variants were observed, including insertions,
deletions and single nucleotide variants. A small fraction of
these mutations are those that have been selected by breeders;
most of them are silent and have no impact on traits of importance
to breeding programs. Occasionally, such mutations result in
a genetic condition such as red or black coat color or an undesirable
disease condition such as dwarfism.
HOW
MIGHT GENE EDITING INTERSECT WITH CONVENTIONIAL BREEDING?
Data
coming out of some of the large-scale genomic and sequencing
projects are revealing situations in which the sequence of one
naturally-occurring allele results in superior performance than
observed when animals inherit an alternative allele of that
gene. It is envisioned that it might be possible to edit an
animal’s genome to the superior allele, and to do that
at several genomic locations, or for several different genes.
The advantage of gene editing over conventional selection to
move these naturally-occurring alleles from one animal to another
is that favourable alleles rarely all occur in one single individual
and editing offers the opportunity to increase the frequency
of desirable alleles in an individual or a breed more rapidly
than could occur through conventional breeding.
One
could potentially envision editing several alleles for different
traits – such as disease resistance, polled and to correct
a known genetic defect – all while using conventional
selection methods to keep making genetic progress towards a
selection objective. One study found that combining gene editing
with genomic selection could improve the response to selection
four fold after 20 generations.
It
should be remembered that complex traits are typically impacted
by many different genes. It is not likely that all of the genes
impacting such traits are known, nor is it typically evident
which might be the desirable molecular edits for these genes
(i.e. what is the sequence of the desirable allele). It is likely
that editing will be focused on large effect loci and known
targets to correct genetic defects or decrease disease susceptibility,
and conventional selection will continue to make progress in
selecting for all of the many small effect loci that impact
the complex traits that contribute to the breeding objective.
Gene
editing offers an approach to translate the thousands of SNP
markers discovered through livestock sequencing projects, the
information obtained from numerous genome wide association studies,
and the discovery of causative SNPs (Quantitative Trait Nucleotides;
QTNs) into useful genetic variation for use in animal breeding
programs.
WILL
GENE EDITING BE REGULATED?
At
the current time it is unclear whether gene editing will be
formally regulated as is the case with animals that have been
produced using genetic engineering. Animal breeding per se is
not regulated by the federal government, although it is illegal
to sell an unsafe food product regardless of the breeding method
that was used to produce it. Gene editing does not necessarily
introduce any foreign genetic DNA or ‘transgenic sequences’
into the genome, and many of the changes produced would not
be distinguishable from naturally-occurring alleles and variation.
As such, many applications will not fit the classical definition
of genetic engineering. For example, many edits are likely to
edit alleles of a given gene using a template nucleic acid dictated
by the sequence of a naturally-occurring allele from the same
species (e.g. the hornless Holstein example described earlier
used template sequence based on the polled allele from Angus).
As such there will be no novel DNA sequence present in the genome
of the edited animal, and likewise no novel phenotype associated
with that sequence. It is not evident what unique risks might
be associated with an animal that is carrying such an allele
given the exact same sequence and resulting phenotype would
be observed in the breed from which the allele sequence was
derived.
It
is possible that nucleases might introduce double stranded breaks
at locations other than the target locus, and thereby induce
alterations elsewhere in the genome. Such off-target events
are analogous to spontaneous mutations and can be minimized
by careful design of the gene editing reagents.
Governments
and regulators globally are currently deliberating about how
or if gene-edited animals should be regulated. It is likely
that gene editing will be considered on a case-by-case basis
depending upon the novelty of the edited DNA sequence and the
resulting attributes or phenotype displayed by the animal. Although
gene editing is a very versatile tool, many applications will
likely result in animals carrying desirable alleles with sequences
that originated in other breeds or individuals from within that
species. As such, this process is directly analogous with conventional
breeding. There is a need to ensure that the extent of regulatory
oversight is proportional to the unique risks, if any, associated
with the novel phenotypes. This question is of course important
from the point of view of technology development, innovation
and international trade.
