Generation of Pax6-IRES-EGFP knock-in mouse via the cloning-free CRISPR/Cas9 system to reliably visualize neurodevelopmental dynamics
Introduction
CRISPR/Cas9 mediated genome editing technology is now an essential tool for genetically modified mouse generations: Cas9 nuclease and the guide RNAs directly injected into zygotes cause DNA double-strand break (DSB) at a desired locus, which can then be repaired by error-prone non-homologous end joining (NHEJ) to carry deletions or insertions at the cut site, resulting in knock-out mouse generation (Wang et al., 2013). If a donor DNA with homology sequences to the both ends of the DSB is co-injected, homology directed repair (HDR) can occur to produce mice with targeted insertions (Yang et al., 2013). While knock-out mouse production by CRISPR/Cas9 has proven to be highly efficient from the beginning of 2013 CRISPR explosion (Wang et al., 2013, Mashiko et al., 2013), generation of gene-cassette knock-in mouse via zygote injection has been considered insufficient for practical use due to its low efficiency (Yang et al., 2013, Yang et al., 2014).
Recently, Aida T et al. have reported an improved CRISPR/Cas9 system (Aida et al., 2015) that enables much more efficient production of relatively large functional cassette knock-in mice. In this system, the native form of guide RNAs in bacterium, CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), are chemically synthesized to eventually form a complex with Cas9 protein. Then the ribonucleoprotein (RNP) complex is injected into mouse fertilized eggs along with the targeting vector. The shorter length of crRNA and tracrRNA compared with the originally used chimeric single guide RNA (sgRNA) is appropriate for their chemical synthesis, and now we can easily order them online. Combining with the commercially available recombinant Cas9 protein, absolutely “cloning-free” CRISPR/Cas9 system has become possible (Aida et al., 2015).
Pax6 is a highly conserved transcription factor among vertebrates and is important in various developmental processes such as eye development, early brain patterning, neocortical arealization, and so forth (Shaham et al., 2012, Osumi et al., 2008, O’Leary and Nakagawa, 2002). Visualization of Pax6 expression dynamics in these events could thus offer a useful tool for neurodevelopmental studies.
Here we employ the cloning-free system to generate Pax6-IRES-EGFP knock-in mouse lines expressing EGFP from the endogenous Pax6 locus in IRES-mediated translations. By optimizing the concentration of crRNA/tracrRNA, Cas9 protein, and the targeting vector, efficient targeted insertions of the 1.8 kb reporter gene cassette is achieved to successfully visualize Pax6 endogenous expressions at various developmental stages.
Section snippets
Chemical synthesis of crRNA and tracrRNA
Pax6-crRNA (5′-acuauguggacacagcaguuGUUUUAGAGCUAUGCUGUUUUG-3′) and tracrRNA (5′-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′) were chemically synthesized and purified by HPLC (FASMAC, Kanagawa, Japan).
Cas9 proteins
Recombinant Cas9 proteins (Guide-it Recombinant Cas9 Nuclease, 631442) were purchased from Takara Clontech.
In vitro digestion assay
The genomic region (603 bp) containing the Pax6-crRNA targeted sequences was PCR-amplified (ExTaq, Takara) by the primers listed in Table S1. Cas9 proteins
Generation of Pax6-IRES-EGFP knock-in mouse via zygote injection utilizing the cloning-free CRISPR/Cas9 tools
Until the cloning-free CRISPR/Cas9 system has become available, Cas9 mRNA and sgRNAs were commonly used for zygote injection. Initially we tried the traditional method and experienced the difficulties in obtaining newborns since it was hard to optimize the RNA concentrations at the non-toxic levels for normal embryogenesis. We thus switched to the cloning-free method in which RNP complexes consisting of crRNA/tracrRNA/Cas9 protein are injected into mouse one-cell embryos (Aida et al., 2015).
Discussion
In this study, we generated Pax6-IRES-EGFP knock-in mouse by employing a highly efficient and convenient method referred to as “cloning-free” CRISPR/Cas9 system (Aida et al., 2015). With this new strategy, we didn’t need laborious cloning steps and in vitro transcriptions for Cas9 mRNA and sgRNA anymore. As a noticeable merit, optimization of Cas9 protein/crRNA/tracrRNA concentrations for the best implantation/pup birth rate was much easier than that of Cas9 mRNA/sgRNA concentrations.
Acknowledgements
The authors thank all the lab members in Department of Biochemistry and Cellular Biology @NCNP for their supports. This work was supported by JSPS KAKENHI Grant Numbers 24510280, 16K10004, 17H05967 to Y.U.I, 16H06528 to M.H and T.I, Intramural Research Grants for Neurological and Psychiatric Disorders of NCNP (24-12 and 27-7) to T.I., and the SRPBS from AMED (16dm0107085h0001).
References (14)
- et al.
Pax6: more than meets the eye
Trends Genet.
(1995) - et al.
Fate mapping of the mouse prosencephalic neural plate
Dev. Biol.
(2000) - et al.
Patterning centers, regulatory genes and extrinsic mechanisms controlling arealization of the neocortex
Curr. Opin. Neurobiol.
(2002) - et al.
Area patterning of the mammalian cortex
Neuron
(2007) - et al.
Pax6: a multi-level regulator of ocular development
Prog. Retin. Eye Res.
(2012) - et al.
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering
Cell
(2013) - et al.
One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering
Cell
(2013)
Cited by (7)
Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells
2020, Journal of BiotechnologyCitation Excerpt :For instance, the GFP sequence has recently been fused to genes that code for structural proteins in human induced pluripotent stem cells (iPSCs)—and to the PAX6 gene in mouse embryos. Subsequently, the spatial and temporal expression of the fusion proteins was tracked via fluorescent microscopy (Inoue et al., 2018; Roberts et al., 2017). Transgene KIs can also be utilized for generating stable cell lines for producing recombinant proteins at physiological levels.
Signaling within the pineal gland: A parallelism with the central nervous system
2019, Seminars in Cell and Developmental BiologyCitation Excerpt :We can speculate that further comparative analyses of the intricate mechanisms which dictate specification, differentiation, maturation and migration during neurogenesis and gliogenesis in the CNS will contribute to our understanding of pineal evolution and ontogeny, and will also clarify the role of cell type minorities in pineal physiology. Models such as the Pax6-IRES-EGFP knock-in (KI) mouse, which was generated by Inoue et al. [61] using the cloning-free CRISPR/Cas9 system, are promising tools for exploring the developmental dynamics of Pax6+ cells and the Pax6-controlled genes in both the CNS and the PG. The neurogenic differentiation factor 1 (NeuroD1), also known as β-cell E-box transactivator 2 (BETA2), has emerged as a potential regulator among the TFs involved in the definition and maintenance of the pineal phenotype [12,62–65].
CRISPR/Cas9 technology: applications in oocytes and early embryos
2023, Journal of Translational MedicineDaughter cells inherit YAP localization from mother cells in early preimplantation embryos
2023, Development Growth and DifferentiationDetection of REST expression in the testis using epitope-tag knock-in mice generated by genome editing
2022, Developmental DynamicsReengineering of the CRISPR/Cas System
2022, The CRISPR/Cas Tool Kit for Genome Editing