Abstract
Despite its convenience and precision, CRISPR-based gene editing approaches still suffer from off-target effects and low efficiencies, which are partially rooted in Cas9, the nuclease component of the CRISPR/Cas9 system. In this study, we showed how mouse genome editing efficiency can be improved by constitutive and inheritable expression of Cas9 nuclease. For this goal, a transgenic mouse line expressing the Cas9 protein (Cas9-mouse) was generated. For in vitro assessment of gene editing efficiency, the Cas9-mice were crossed with the EGFP-mice to obtain mouse embryonic fibroblasts (MEF) expressing both EGFP and Cas9 (MEFCas9-EGFP). Transfection of these cells with in vitro transcribed (IVT) EGFP sgRNA or phU6-EGFPsgRNA plasmid led to robust decrease of Mean Fluorescent Intensity (MFI) to 8500 ± 1025 a.u. and 13,200 ± 1006 a.u. respectively. However, in the control group, in which the MEFEGFP cells were transfected with a pX330-EGFPsgRNA plasmid, the measured MFI was 16,800 ± 2254 a.u. For in vivo assessment, the Cas9-zygotes at two pronuclei stage (2PN) were microinjected with a phU6-HhexsgRNA vector and the gene mutation efficiency was compared with the wild-type (WT) zygotes microinjected with a pX330-HhexsgRNA plasmid. The analysis of born mice showed that while the injection of Cas9-zygotes resulted in 43.75% Hhex gene mutated mice, it was just 15.79% for the WT zygotes. In conclusion, the inheritable and constitutive expression of Cas9 in mice provides an efficient platform for gene editing, which can facilitate the production of genetically-modified cells and animals.
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References
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Farzaneh F, Mirzapoor Z, Jahangirian E, Heidari F, Hashemi E, Rahim-Tayefeh A, Fatemi N, Jamshidizad A, Dashtizad M, Shamsara M (2019) The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice. 3 Biotech 9:255
Ghassemi B, Shamsara M, Soleimani M, Kiani J, Rassoulzadegan M (2018) Pipeline for the generation of gene knockout mice using dual sgRNA CRISPR/Cas9-mediated gene editing. Anal Biochem 568:31–40
Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I (2014) Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep 4:4513
Karikó K, Muramatsu H, Ludwig J, Weissman D (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 39:e142
Kim MJ, Ahituv N (2013) The hydrodynamic tail vein assay as a tool for the study of liver promoters and enhancers. Methods Mol Biol 1015:279–289
Kubo A, Kim YH, Irion S, Kasuda S, Takeuchi M, Ohashi K, Iwano M, Dohi Y, Saito Y, Snodgrass R, Keller G (2010) The homeobox gene Hex regulates hepatocyte differentiation from embryonic stem cell-derived endoderm. Hepatology 51:633–641
Lino CA, Harper JC, Carney JP, Timlin JA (2018) Delivering CRISPR: a review of the challenges and approaches. Drug Deliv 25:1234–1257
Mehravar M, Shirazi A, Nazari M, Banan M (2019) Mosaicism in CRISPR/Cas9-mediated genome editing. Dev Biol 445:156–162
Nagy A, Gertsenstein M, Vintersten K, Behringer R (2003) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Pérez-González A, Caro E (2019) Benefits of using genomic insulators flanking transgenes to increase expression and avoid positional effects. Sci Rep 9:8474
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308
Rincón-Arano H, Furlan-Magaril M, Recillas-Targa F (2007) Protection against telomeric position effects by the chicken cHS4 beta-globin insulator. Proc Natl Acad Sci USA 104:14044–14049
Schirmbeck R, Reimann J, Kochanek S, Kreppel F (2008) The immunogenicity of adenovirus vectors limits the multispecificity of CD8 T-cell responses to vector-encoded transgenic antigens. Mol Ther 16:1609–1616
Sosa MAG, Gasperi RD, Elder GA (2010) Animal transgenesis: an overview. Brain Struct Funct 214:91–109
Staahl BT, Benekareddy M, Coulon-Bainier C, Banfal AA, Floor SN, Sabo JK, Urnes C, Munares GA, Ghosh A, Doudna JA (2017) Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol 35:431–434
Wu Z, Yang H, Colosi P (2010) Effect of genome size on AAV vector packaging. Mol Ther 18:80–86
Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, Cai W, Yang G, Bronson R, Crowley DG, Zhang F, Anderson DG, Sharp PA, Jacks T (2014) CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514:380–384
Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, Koteliansky V, Sharp PA, Jacks T, Anderson DG (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 32:551–553
Zhang L, Zhou J, Han J, Hu B, Hou N, Shi Y, Huang X, Lou X (2016) Generation of an oocyte-specific Cas9 transgenic mouse for genome editing. PLoS ONE 11:e0154364
Funding
This project was supported financially by research grant number 97-4-20-13610 from Cellular and Molecular Research Centre, Iran University of Medical Sciences and the National Institute for Medical Research Development (NIMAD) grant number 962825. The funders provided only financial support for the conduct of the research.
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JK and MSH designed the study and wrote and edited the draft of the manuscript. BG and MJ conducted the experiments and wrote the manuscript. MS helped JK and MSH supervise the project. GS and MK helped carry out the experiments. JK is the corresponding author of this manuscript.
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The study protocol was approved by the Research Ethics Committee of Iran University of Medical Sciences with ethical code number IR.IUMS.REC.1398.770. All of the experiments conducted on mice were in compliance with the ARRIVE guidelines and in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
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Ghassemi, B., Jamalkhah, M., Shokri, G. et al. Improved efficiency of genome editing by constitutive expression of Cas9 endonuclease in genetically-modified mice. 3 Biotech 11, 56 (2021). https://doi.org/10.1007/s13205-020-02580-z
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DOI: https://doi.org/10.1007/s13205-020-02580-z