Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

494 Chapter 8: Analyzing Cells, Molecules, and Systems
for about 90% of the differences between one person’s genome and another’s. But
when we try to tie these common variants to differences in disease susceptibility
or other heritable traits, such as height, we find that they do not have as much
predictive power as we had anticipated: thus, for example, most confer relatively
small increases—less than twofold—in the risk of developing a common disease.
In contrast to polymorphisms, rare DNA variants—those much less frequent
in humans than SNPs—can have large effects on the risk of developing some common
diseases. For example, a number of different loss-of-function mutations,
each individually rare, have been found to increase greatly the predisposition
to autism and schizophrenia. Many of these are de novo mutations, which arose
spontaneously in the germ-line cells of one or the other parent. The fact that these
mutations arise spontaneously with some frequency could help explain why
these common disorders—each observed in about 1% of the population—remain
with us, even though the affected individuals leave few or no descendants. These
rare mutations may arise in any one of hundreds of different genes, which could
explain much of the clinical variability of autism and schizophrenia. Because they
are kept rare by natural selection, most such variants with a large effect on risk
would be missed by genome-wide association studies.
Now that DNA sequencing has become fast and inexpensive, the most efficient
and cost-effective way to identify these rare, large-effect mutations is by sequencing
the genomes of affected individuals, along with those of their parents and siblings
as controls.
Reverse Genetics Begins with a Known Gene and Determines
Which Cell Processes Require Its Function
As we have seen, classical genetics starts with a mutant phenotype (or, in the case
of humans, a range of characteristics) and identifies the mutations, and consequently
the genes, responsible for it. Recombinant DNA technology has made
possible a different type of genetic approach, one that is used widely in a variety
of genetically tractable species. Instead of beginning with a mutant organism and
using it to identify a gene and its protein, an investigator can start with a particular
gene and proceed to make mutations in it, creating mutant cells or organisms
so as to analyze the gene’s function. Because this approach reverses the traditional
direction of genetic discovery—proceeding from genes to mutations, rather
than vice versa—it is commonly referred to as reverse genetics. And because the
genome of the organism is deliberately altered in a particular way, this approach
is also called genome engineering or genome editing. We shall see in this chapter
that this approach can be scaled upward so that whole collections of organisms
can be created, each of which has a different gene altered.
There are several ways a gene of interest can be altered. In the simplest, the
gene can simply be deleted from the genome, although in a diploid organism,
this requires that both copies—one on each chromosome homolog—be deleted.
Although somewhat counterintuitive, one of the best ways to discover the function
of a gene is by observing the effects of not having it. Such “gene knockouts”
are especially useful if the gene is not essential. Through reverse genetics, the gene
in question (even if it is essential) can also be replaced by one that is expressed in
the wrong tissue or at the wrong time in development; this type of manipulation
often provides important clues to the gene’s normal function. For example, a gene
of interest can be modified to be expressed at will by the experimenter (Figure
8–52). Finally, genes can also be engineered so that they are expressed normally
in most cell types and tissues but deleted in certain cell types or tissues selected
by the experimenter (see Figure 5–66). This approach is especially useful when a
gene has different roles in different tissues.
It is also possible to make subtler changes to a gene. It is sometimes useful to
make slight changes in a protein’s structure so that one can begin to dissect which
portions of a protein are important for its function. The activity of an enzyme, for
example, can be studied by changing a single amino acid in its active site. It is
also possible, through genome engineering, to create new types of proteins in an