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4.7 Conclusion and Perspectives
Animal models remain the most powerful and widely used genetic tool for studies
of in vivo gene functions and human diseases. The conventional ES cell approaches
are laborious and time-consuming, and the types of the genetic modifications that
can be engineered are limited. The CRISPR/Cas9 system, combined with conven-
tional zygote injection methods, has opened a new era of possibilities for faster and
cheaper animal model production. In our practice, it takes less than a week to pre-
pare the editing reagents for making a simple knockout or small knock-in mouse
model. After zygote collection from donor females, microinjection, and transfer to
recipients, which is a 1-day process, the animals with desired mutations are born in
few weeks. We have experienced firsthand the dramatic change in the practice and
witnessed the power of the genome-engineering technology to shorten the length
required for animal model production and advance the progress of science.
Additionally, the flexibility of the CRISPR/Cas9 system allows for the creationof genetic tools that were previously unobtainable. We are no longer limited to a
simple genetic manipulation (knockout or knock-in of a short distance of DNA) for
a single gene at a time. We routinely knock out as many as four genes with a single
microinjection. We have also deleted or inverted a DNA fragment as large as 133 kb
and 1.7 Mb, respectively, in mouse zygotes directly by a dual sgRNA strategy.
Nonetheless, the size of the DNA segments to be deleted or inverted can be much
larger than our record [ 43 , 60 ]. In addition to strategically adding or removing base
pairs, the ability of the CRISPR/Cas9 system to target specific sequences can be
harnessed to alter the genome in novel ways. When sgRNA is applied with a cata-
lytically inactive Cas9 (dCas9), the complex can target the specific DNA sequence
without cleaving it [ 61 ]. Therefore, one can fuse a functional protein domain or an
effector to dCas9, and then the dCas9/sgRNA complex can bring the effector to the
specific DNA location to exert its function. For instance, dCas9 can be fused to a
cytidine deaminase that converts cytidine to uridine, and this fusion protein can then
be delivered into mouse zygotes along with specific sgRNAs. Kim et al. [ 62 ] showed
that single-nucleotide substitutions (targeted point mutations) are able to be induced
at a specific locus in mice at a high frequency without using a donor oligo. Liu et al.
[ 63 ] showed that when dCas9 is fused to Tet1, which induces DNA demethylation,
dCas9-Tet1 can change the methylation status of a promoter region in postnatal
mice following a lentiviral-mediated delivery. Therefore, the CRISPR/Cas9 system
has greatly expanded the genetic toolbox for biomedical research. Future animal
models will go beyond DNA modification (knockout or knock-in) and give research-
ers the tools to study areas that were not possible to manipulate in the past, such as
epigenetic modifications, RNA editing, chromosome architecture, and genome
organization. However, the CRISPR/Cas9 system is not perfect yet. Refinements in
targeting specificity are still needed. Although off-target mutations are infrequently
detected in CRISPR-targeted rodents [ 9 , 10 , 64 , 65 ], it remains a concern in the
field. Progress has been made to increase the specificity of Cas9 without losing its
on-target activity based on protein structure engineering [ 66 , 67 ]. Other major
C.L. Yuan and Y.-C. Hu