Production of genetically engineered mice with higher efficiency, lower mosaicism, and multiplexing capability using maternally expressed Cas9

Abstract

The CRISPR/Cas9 system is widely used to generate gene-edited animals. Here, we developed an efficient system for generating genetically modified mice using maternal Cas9 from Cas9 transgenic mice. Using this system, we achieved lower mosaicism and higher rates of knock-in success, geneediting, and birth compared to the similar parameters obtained using exogenously administered Cas9 (mRNA/protein) system. Furthermore, we successfully induced simultaneous mutations at multiple loci (a maximum of nine). Our novel gene-editing system based on maternal Cas9 could potentially facilitate the generation of mice with single and multiple gene modifications. Genetically modified (GM) organisms, with specific genes altered (added or ablated), are widely used for modeling human and animal diseases. They are particularly useful for understanding the molecular mechanisms of diseases and for the development of novel disease treatments 1,2. In the past three decades, GM animals have been created by microinjecting transgenes into zygote nuclei (zygote microinjection) or by injecting the blastocoel with GM embryonic stem cells engineered to exhibit altered expression of a specific gene by gene targeting technology 3,4. It is now easier than ever to create GM animals by using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated-9 (CRISPR/Cas9) genome editing system 5-12 due to its wide applicability, high efficiency, and design simplicity 13-15. The most commonly used approach for generating GM animals with the CRISPR/Cas9 system is by microinjecting the CRISPR/Cas9 components, such as Cas9 DNA, mRNA or protein, guide RNA (gRNA), and, in some cases, a homology direct repair (HDR) template, into the zygote 16-20. gRNA efficiently induces Cas9-mediated double strand breaks at desired target sites, which stimulate DNA repair by at least two distinct mechanisms: non-homologous end joining and HDR 21-23. However, the zygote microinjection-based genome editing system has inherent problems. For example, frequent mosaicism for inducing insertion/deletion (indel) at the target locus is seen in almost all individuals obtained using this approach 24-26. Given that transcription and translation are suppressed in mouse zygotes, the translation of the introduced Cas9 DNA/mRNA into its active enzymatic form is likely delayed until after the first cell division, causing unequal genome editing in individual blastomeres 27. It has been recently reported that direct Cas9 protein expression in early-stage mouse zygotes reduces the occurrence of mosaic mutations 28. In addition, the amount of CRISPR/ Cas9 reagents injected into the zygote is limited as high volumes are often associated with developmental arrest of the embryos 29 , reducing the possibility of simultaneous modifications in multiple genes using the CRISPR/Cas9 system 11. Previously, we tackled these problems by generating systemically Cas9-expressing transgenic (Tg) (sCAT) mice that produce maternal Cas9 (maCas9), which exhibits nuclease activity, during...

Electroporation-Mediated Genome Editing of Livestock Zygotes

“…Others include a 103 ssODN donor targeting the Fgf10 gene (Hashimoto et al, 2016), and a 128 bp oligonucleotide targeting the Aicda gene (Wang et al, 2016). Sakurai et al (2020) utilized oocytes from transgenic mice expressing maternal Cas9 (maCas9) to generate gene-edited embryos and pups. The group compared mutation rates between embryos and pups following zygote transfections either with gRNA alone or with both Cas9 and gRNA.…”

In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

“…This is in contrast to the case of genome-edited mice. For instance, since Kaneko et al [21] first reported the successful use of this technology in 2014, several genome-edited mice have been produced by numerous laboratories worldwide [22][23][24][25][26][27][28][29][30][31][32][33][34]. Tanihara et al [13] pioneered in demonstrating in vitro EP as a promising tool for producing GM piglets with high efficiency.…”

“…In the event of the GM animals developing mutations (or transgene insertion) only in one allele, obtaining homozygous knock out (KO) animals is time-consuming (at least >0.5 years in the case of pigs) and cumbersome [35]. In mice, in vitro EP is beneficial for producing animals with bi-allelic KO phenotypes with relatively high efficiency, as the GECs are incorporated into the embryo with lesser bias [34]. This is in contrast with the findings of our previous work, in which a cytoplasmic injection administered to obtain genome-edited porcine embryos resulted in frequent mosaic mutations in the target locus [36].…”

Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes

“…Maternally contributed Cas9 protein within the fertilized zygote is sufficient for mutagenesis, and only the sgRNA and repair template need be introduced. Indeed, maternally supplied Cas9 was found to result in a more consistent and efficient mutagenesis 14 , 16 . In this study we extend our side-by-side comparison of delivery methods to examine the effect of introducing sgRNA and repair template into zygotes derived from Cas9-expressing females by electroporation.…”

“…We and others have shown previously that transgenic Cas9-expressing embryo donors can be used effectively for mouse model production, obviating the need to provide exogenous Cas9 [14][15][16] . Maternally contributed Cas9 protein within the fertilized zygote is sufficient for mutagenesis, and only the sgRNA and repair template need be introduced.…”