In Vivo Organ/Tissue Genome Editing in Newborn Pups: Comparison
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Germline manipulation is based on gene delivery to early embryos, such as fertilized eggs (zygotes), through pronuclear microinjection of nucleic acids, electroporation (EP) in the presence of nucleic acids, or transduction in the presence of viral vectors.

  • CRISPR/Cas9
  • genetically modified animal
  • genome editing
  • in vivo genome engineering

1. Introduction

Genetic manipulation in vivo using viral or non-viral vectors has been recognized as a useful and powerful tool for elucidating the role of a gene of interest (GOI), creating genetically modified animals, and rescuing genetic defects caused by mutations in genes that are important for individual life.
Germline manipulation is based on gene delivery to early embryos, such as fertilized eggs (zygotes), through pronuclear microinjection of nucleic acids, electroporation (EP) in the presence of nucleic acids, or transduction in the presence of viral vectors [1,2][1][2]. In most cases, these are performed using zygotes in vitro, which have been isolated from the ampulla region of an oviduct in a pregnant female. To allow these manipulated embryos to develop further, egg transfer (ET) to the recipient female reproductive tract (in a pseudopregnant state), such as the oviduct or uterus, is a prerequisite. However, the preparation of the recipient female is laborious, and ET itself requires special skills. To avoid such ex vivo handling of zygotes, novel techniques, such as Genome Editing via Oviductal Nucleic Acids Delivery (GONAD) or improved GONAD (i-GONAD) [3[3][4],4], which enable in situ genome editing of zygotes without the need to isolate zygotes, have been developed. These do not require the ex vivo handling of embryos and are performed solely in vivo (within the oviductal lumen of a pregnant female). Breath-controlled micropipette-based instillation of genome editing components into the oviductal lumen of a pregnant female on day 0 or 1 of pregnancy (day 0 of pregnancy is defined as the day when the vaginal plug is recognized in the morning) takes place under a dissecting microscope, followed by EP towards the entire oviduct. Notably, in this case, it does not require any recipient females, which are usually needed for the ET of ex vivo manipulated embryos. In summary, the creation of knockout (KO) or transgenic (Tg) animals is still recognized as a standard approach for studying the function of a GOI in vivo. However, systematic functional screening of candidate genes and maintenance of established lines are time-consuming and costly. Moreover, various compensatory mechanisms often make it difficult to interpret the results.
In contrast to the germline manipulation, a new strategy called “in vivo somatic cell genome editing” [5] or “somatic-Tg mice” [6] is emerging as an alternative to the abovementioned germline manipulation, which generates somatic gene modification in the fetal stage through in utero injection of nucleic acids, or in the adult stage through tail vein injection or local administration of nucleic acids. This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period. However, in the case of in utero genetic manipulation, surgery on a pregnant female is always required, which often causes fetal death after surgery and requires special skills to introduce nucleic acids to the target site of a fetus under a dissecting microscope [7]. For in vivo gene manipulation in the adult stage, a tail-vein-based gene introduction approach requires a large amount of nucleic acids to achieve somatic cell genome editing throughout the entire body. Furthermore, it often causes immunological rejection of certain types of nucleic acids (as exemplified by viral vectors) after repeated administration.

2. Concept for In Vivo Organ/Tissue Genome Editing

Genome editing, as exemplified by the CRISPR/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has been well established as a useful genome engineering tool that enables researchers to examine gene function, create animal models of human diseases, modulate immunological systems and carcinogenesis, suppress viral infections, and improve genetically transformed organisms [8]. The CRISPR/Cas9 system employs a bacterial-derived Cas9 endonuclease and short guide RNA (gRNA), which is complementary to the target site in the genome [9]. When these two components are introduced into a cell, they form a complex called ribonucleoprotein (RNP), which binds to a specific chromosomal locus. After RNP binding, double-stranded (ds) DNA breaks (DSBs) occur only 3–4 bp in front of the protospacer adjacent motif (PAM) [9]. The two edges generated after the DSB are repaired through the DNA repair machinery of the cell, namely, non-homologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ), and homology-directed repair (HDR) pathways. In the absence of a DNA donor, the two edges are rejoined through NHEJ, which often causes the alteration of nucleotides near the DSB site, leading to random insertions or deletions of nucleotides, called “indels”. In the case of MMEJ, it employs naturally occurring microhomology of 5–25 bp present on either side of the DSB to mediate end-joining [10]. The outcome of MMEJ is a reproducible deletion of intervening sequences while retaining one copy of the microhomology. For this reason, MMEJ is normally considered to be mutagenic, because of an overall loss of genetic information through precise deletion [11]. In the case of HDR, it requires a DNA donor (in which homologous regions exist on both sides), and the broken ends are repaired by homologous recombination using a DNA template. However, HDR’s efficiency is low, and it is only active in dividing cell types, limiting the range of proper HDR applications [9]. Notably, two classes of CRISPR/Cas9-mediated DNA base editors (BEs)—cytosine base editors (CBEs) and adenine base editors (ABEs)—have recently been developed to overcome these limitations [12,13,14][12][13][14]. BEs can directly install point mutations in cellular DNA without inducing DSBs. According to Kantor et al. [15], CBEs are targeted to a specific locus by a gRNA using Cas9 nickase (nCas9) or catalytically inactive “dead” Cas9 (dCas9) fused to a cytidine deaminase. CBEs can convert cytidine to uridine within a minute editing window near the PAM site. Uridine is converted to thymidine via base excision repair, creating a C-to-T or G-to-A change on the opposite DNA strand. ABEs have been engineered to convert adenosine to inosine, which is treated by the cell in a manner similar to guanosine, creating an A-to-G (or T-to-C) change. Adenine deaminases do not exist in nature but have been created by the directed evolution of Escherichia coli TadA, a tRNA adenine deaminase. Similar to the CBEs, the evolved TadA domain was fused to a Cas9 protein to create the ABE. Prime editors (PEs) are the latest additions to the CRISPR genome engineering toolkit. They use an engineered reverse transcriptase (RT) fused to Cas9 nickase and a prime editing guide RNA (pegRNA), which significantly differs from regular sgRNAs [16]. The pegRNA consists of two components: (a) a sequence complementary to the target sites that direct nCas9 to its target sequence, and (b) an additional sequence spelling the desired sequence changes [16]. The 5′ of the pegRNA binds to the primer binding site region on the DNA, resulting in the exposure of the non-complimentary strand. The unbound DNA of the PAM-containing strand is nicked by Cas9, creating an RT primer that is linked to nCas9. The nicked PAM strand is then extended by RT using the interior of the pegRNA as a template, consequently modifying the target region in a programmable manner [16]. BEs and PEs are effective tools for enabling precise nucleotide substitutions in a programmable manner, without requiring DSBs or a donor template. Notably, both BEs and PEs have remarkable potential as therapeutic tools for correcting disease-causing mutations in the human genome [15]. For example, Song et al. [17] demonstrated the successful delivery of RNA-encoded ABEs into the livers of tyrosinemia type 1 model mice, thereby correcting splice-variant mutations and rescuing the phenotype. Genome editing of germline cells (i.e., primordial germ cells, gamete progenitors, and gametes) and preimplantation-stage embryos, including fertilized eggs (zygotes), has been extensively used to obtain genetically engineered animals and is referred to as “germline editing” [5]. As mentioned previously, genome editing of zygotes has now been extensively performed through pronuclear microinjection of genome editing reagents, EP in the presence of genome editing, and direct in situ genome editing of zygotes present in the oviductal lumen of a pregnant female, which is called “GONAD or i-GONAD” [3,4][3][4]. On the other hand, genome editing of adult organs/tissues has been achieved for genome modifications in various organs/tissues, using physical interventions (e.g., in vivo EP, tail vein injection (also called “intra-venous (i.v.) injection via tail vein”), and nanoparticle-based non-viral gene delivery or viral vector delivery [18,19,20][18][19][20]. Recently, tremendous advances have been made in genome editing at the adult stage for genome modification in various organs and tissues. For example, post-mitotic neurons have been genome-edited through intracerebral delivery of a recombinant adeno-associated virus (rAAV) carrying CRISPR components [21,22][21][22] or nanoparticle delivery of CRISPR [23,24][23][24]. Hepatocytes have been genome-edited in vivo through i.v. injection of AD vectors, naked plasmid DNA carrying CRISPR components [25], or i.v. injection of rAAVs carrying genome editing components [26]. Immune cells have been genome-edited in vivo through the i.v. injection of nanoparticles carrying CRISPR reagents targeting neutrophils [27], macrophages [28], or T cells [29]. Cancer cells are also targets of CRISPR-based genome editing [30,31,32][30][31][32]. However, therapeutic approaches using the abovementioned genome editing tools in the adult stage often require large amounts of reagents to be administered in vivo, with possible immunological responses against the therapeutic reagents upon repeated administration, and limited ability to deliver them to juvenile cells such as stem cells or actively proliferative cells.

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