54 THE SCIENTIST | the-scientist.com
LAB TOOLSMODIFIED FROM ©ISTOCK.COM, TERA VECTORKnock It Into the Park
Techniques for upping the effi ciency of knocking in genesBY ANNA NOWOGRODZKIA
lmost always, building something
is harder than tearing it down.
Similarly, knocking in genes poses
a greater challenge than knocking them
out. It’s a reality that researchers will have
to overcome in order to get the most out of
gene editing. Knocking in genes allows sci-
entists to study the effects of specific gene
variants, to use reporter genes like green
fluorescent protein to track gene products
in time and space, to probe genome reg-
ulation, and ultimately, to repair disease-
causing genes. “It’s a really effective way to
interrogate every base of a gene,” says Greg
Findlay, an MD/PhD candidate at the Uni-
versity of Washington.
CRISPR-Cas9, a gene editing technology
known for its user-friendliness, can knockgenes in or out. Knocking out a gene involves
inserting CRISPR-Cas9 into a cell using a
guide RNA that targets the tool to the gene of
interest. There, Cas9 cuts the gene, snipping
through both strands of DNA, and the cell’s
regular DNA repair mechanism fixes the
cut using a process called non-homologous
end joining (NHEJ). NHEJ is highly effi-
cient but inaccurate. The process tends to
introduce errors in the form of small inser-
tions or deletions that are usually enough to
knock out the gene.
To knock a gene in, however, the cuts
must be repaired very precisely, with no
extra insertions or deletions. This requires
harnessing a second DNA repair mech-
anism called homology-directed repair
(HDR), which—in mammalian cells, atleast—occurs less efficiently, so its fre-
quency is dwarfed by that of NHEJ. Com-
plicating the process further is the fact that
some gene loci and cell types are inherently
less hospitable to CRISPR-Cas9 editing.
In the past few years, researchers have
developed many new strategies to boost
the efficiency of knocking in genes both
large and small using CRISPR-Cas9, and
along the way they’ve proposed and tested
new applications for this type of gene edit-
ing. Here, The Scientist explores a few of
the most promising approaches.SELECT ITRESEARCHER:Jon Chesnut, senior direc-
tor of synthetic biology R&D, Thermo
Fisher ScientificPROJECT:In developing a gene tagging
kit called Truetag that Thermo Fisher will
put on the market later this year, Ches-
nut used selectable markers to improve
efficiency. A selectable marker—in this case,
an antibiotic resistance gene—is stuck to a
fluorescent protein tag and knocked into
mammalian cells. Those cells are then
grown in culture with the associated anti-
biotic. The resistance gene confers a selec-
tive advantage to the cells that carry it; they
alone are able to grow, and thus those that
grow contain the gene tag of interest. Even
if the efficiency of gene insertion is low,
researchers can use antibiotic selection for
a week or more to end up with a high per-
centage of cells with successful insertions.
Using the antibiotic puromycin or blas-
ticidin with the kit, Chesnut’s team man-
aged to boost the gene insertion rate from
10–30 percent to 90 percent or more in
some cell populations. A few especially dif-
ficult genes went from an insertion rate of
less than 1 percent to greater than 90 per-
cent. It’s important to test multiple doses
of antibiotics on the cell line you plan to