35 128
egg extracts. The method allows for the generation of complex and customized libraries from
any source of DNA via routine molecular biology methods.
CRISPR/Cas9 for Generating Animal Models
Genetically engineered animal models are crucial for the study of complex cellular and physio-
logical processes. While mouse models have been widely used, the CRISPR/Cas9 gene-editing
approach has been established in many other animal models, including worm [73],
fl
y [74],
fi
sh
[75,76], rat [77], rabbit [78,79], goat [80], sheep [81], dog [82], pig [83], and monkey [84]. The
expansion of transgenic animal models beyond mouse is advantageous to biomedical research
because it can accelerate the development of new therapeutic strategies.
CRISPR provides an easier approach to establish these transgenic animal models compared
with previous gene-editing tools. Traditional approaches to construct transgenic mice via
insertional mutagenesis or TALEN-mediated gene editing are time consuming, costly, and
inef
fi
cient. The robustness and high ef
fi
ciency of CRISPR
[5_TD$DIFF]
/Cas9 simplify the process for creating
model systems [85,86]. Moreover, nucleic acids encoding the Cas9 protein and target-speci
fi
c
sgRNAs can be conveniently injected into embryos to generate gene-modi
fi
ed mice with
deletions of multiple genes, mutations in de
fi
ned genes, or insertions of
fl
uorescence reporters
or other peptide tags to endogenous genes. For example, co-injection of Cas9 mRNA and
sgRNAs targeting
Tet1
and
Tet2
into zygotes generated mice with biallelic mutations in both
genes with an ef
fi
ciency of 80% [85]. Furthermore, co-injection of Cas9 mRNA and sgRNAs with
mutant oligos generated precise point mutations simultaneously in two target genes, while co-
injecting Cas9 mRNA and sgRNAs into one-cell-stage cynomolgus monkey embryos generated
founder animals harboring two gene modi
fi
cations [84].
The establishment of a Cre-conditional Cas9 knock-in mouse has broadened the applications of
Cas9
in vivo
[87]. The Cas9 knock-in mouse is a great resource to rapidly generate mutations in a
subpopulation of cells
in vivo
, and test how mutations cause disease phenotypes. Different
methods based on adeno-associated virus (AAV), lentivirus, or nanoparticles can be used to
deliver sgRNAs into multiple cell types, such as neurons, immune cells, and endothelial cells, in a
Cas9 knock-in mouse to model the dynamics of signi
fi
cantly mutated genes in lung adenocar-
cinoma [87]. Another work demonstrated that the Cre-conditional Cas9 knock-in mouse
phenocopied Cre-mediated genetic deletion of genes in Cre/LoxP mouse models in studying
pancreatic ductal adenocarcinoma [88]. Via retrograde pancreatic ductal injection of lentiviral
vectors expressing Cre and an sgRNA into Cre-conditional Cas9 knock-in mice, the authors
showed knockout of
Lkb1
together with manipulated expression of oncogenic
Kras
. However,
due to the heterogeneity of delivery and Cas9-mediated gene editing, caution is required when
interpreting results.
In addition to using a Cas9 knock-in mouse model, viral vectors encoding Cas9 and an sgRNA
can be directly delivered into wild-type mice or Cre/loxP mouse models to probe gene function.
One study used AAV vectors encoding Cas9 and sgRNAs to target a single gene or multiple
genes in the normal adult mouse brain
in vivo
[89]. Characterizing the effects of gene mod-
i
fi
cations in postmitotic neurons revealed similar phenotypes as observed in gene knockout
mice. Another work used a lentiviral system that delivers both the CRISPR system and Cre
recombination to examine CRISPR-induced mutation of genes in the context of well-studied
conditional Cre/
loxP
mouse models of lung cancer and other cancer types [90]. In other research
to study cancer genes in the mouse liver, a hydrodynamic injection was used to deliver a plasmid
DNA expressing Cas9 and sgRNAs that directly targeted the tumor suppressor genes (
p53
or
PTEN
) alone and in combination into the liver. The authors demonstrated the feasibility of Cas9-
mediated mutation of tumor suppressor genes in the liver as an avenue for the rapid develop-
ment of liver cancer models [91]. However, similar to the Cas9 knock-in mouse, the virally
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