Single Copy Transgene Integration in ROSA26 Safe Harbor

Site specific single copy integration of transgenes is ideal because the precise location of the DNA in the genome Is experimentally predetermined, This control means that transgene insertion will not disrupt endogenous genes important for development (Costantini et al. 1989, Meisler et al. 1992). Use of Cas9 to target the integration site and complete sequencing of the inserted transgene and environs will validate transgene location and structure. In contrast to targeted integration, the examination of random transgene integration events shows complex rearrangements of chromosomal DNA and transgene DNA at integration sites that resemble chromothripsis (Chiang et al., 2012, Dubose et al., 2013). Such unpredictable genetic structures are difficult to define and complicate analysis of Cas9 action on reporter transgenes. To facilitate rapid analysis of on-and off target sites at the DNA level a defined transgene integration site is necessary. The selected site of integration should be a safe harbor site that is permissive for transgene expression directed by heterologous promoters. The surrounding genomic DNA should not contain strong promoter elements that can affect the desired pattern of transgene expression. The ROSA26 locus satisfies these requirements.

Since its initial identification (Zambrowicz et al., 1997) the mouse ROSA26 locus has been recognized as a genomic location that is permissive of gene expression (Chen et al., 2011, Soriano, 1999). For example, there are over 1000 mouse models based on the ROSA26 gene targeting because it is an ideal safe harbor (Blake et al., 2017). Cas9 has been used to target transgenes of various sizes to the mouse ROSA26 locus (Bressan et al., 2017, Chu et al., 2016, Quadros et al., 2015, Yoshimi et al., 2016, Wu et al., 2018). Targeting mouse ROSA26 with reporter transgenes designed to detect Cas9 activity is very straightforward. Targeting the rat ROSA26 locus with Cas9 has also been performed and is also a straightforward process (Charpentier et al., 2018, Ma et al., 2017, Menoret et al, 2015, Yoshimi et al., 2016). The integration of transgenes with Cas9 in mouse and rat ROSA26 is very efficient.

Safe harbor sites for transgene integration defined by retroviral insertions in mouse genomic DNA include ROSA26 (Zambrowicz et al. 1997, Soriano, 1999), Igs7 (TIGRE) (Zeng et al. 2008, Madisen et al., 2015), and Igs2 (Hipp11) (Hippenmeyer et al., 2010, Tasic et al., 2011). Compared to the more than 1000 ROSA26 mouse models, relatively few models exist at Igs2 (17) or Igs7 (26) (Blake et al., 2017). Analysis of the genomic sequences of the ROSA26 integration site shows it is free of repetitive sequences that can make long range PCR inefficient. On the other hand, the Igs2 and Igs7 sites are contaminated with repetitive sequences that that can reduce the efficiency of gene targeting by homologous recombination in ES cells or by HDR in zygotes. Of these three genomic docking sites ROSA26 is the only one that has been targeted by Cas9 or zinc finger nucleases. Thus ROSA26 is preferred to other insertion sites defined by viral mutagenesis.

Other safe harbor sites include the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus and the 3’ untranslated region (UTR) of collagen type 1 alpha chain 1 (Col1a1). These sites have been targeted in mouse ES cell to insert single copy transgenes in 497 Hprt1 targeted mouse models and 169 Col1a1 mouse models (Blake et al., 2017). These sites are permissive for transgene expression (Hooper et al., 1987, Bronson et al., 1996, Yang et al., 2009, Beard et al., 2006, Dow et al., 2012). Hprt1 gene targeting in ES cells is dependent on the repair of a pre-existing 36 kb deletion that disrupts Hprt1 expression in E14Tg2a ES cells and its derivatives. Hprt1 targeting in ES cells is dependent on HPRT repair so that drug selection can be used to enrich for correctly targeted ES cell clones. To precisely reproduce this genomic feature in mice or rats to create a safe harbor site is not consistent with the timeline for deliverables in this proposal. In principle, Cas9 reagents to target the Col1a1 gene targeting site used in ES is possible, however there are no published reports are available to demonstrate feasibility. Repair of Cas9-induced chromosome breaks can result in the deletion of hundreds of base pairs (Shin et al., 2017). If such an event disrupts COL1A1 expression the result can be prenatal lethality in homozygous mutants (Jaenisch et al., 1983) or osteogenesis imperfecta in heterozygotes (Bonadio et al., 1990). In principle the inactivation of a protein coding gene that causes a phenotype is not desirable in the design of a reporter animal model. Analysis of genomic sequences at the Col1a1 integration site shows the presence of repetitive elements that will complicate correct gene targeting. Thus the targeting of either Hprt1 or Col1a1 offer no advantages over targeting the ROSA26 site.

BIBLIOGRAPHY
Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R. 2006. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis. 44:23-8.

Blake JA, Eppig JT, Kadin JA, Richardson JE, Smith CL, Bult CJ, and the Mouse Genome Database Group. 2017. Mouse Genome Database (MGD)-2017: community knowledge resource for the laboratory mouse. Nucl. Acids Res. 45: D723-D729.

Bonadio J, Saunders TL, Tsai E, Goldstein SA, Morris-Wiman J, Brinkley L, Dolan DF, Altschuler RA, Hawkins JE Jr, Bateman JF, Mascara T, Jaenisch R. 1990.  Transgenic mouse model of the mild dominant form of osteogenesis imperfecta. Proc Natl Acad Sci U S A. 87:7145-9.

Bressan RB, Dewari PS, Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, Gogolok S, Skarnes WC, Pollard SM. 2017. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells. Development. 144:635-648.

Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD. 1985. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A. 82:4438-4442.

Bronson SK, Plaehn EG, Kluckman KD, Hagaman JR, Maeda N, Smithies O. 1996. Single-copy transgenic mice with chosen-site integration. Proc Natl Acad Sci U S A. 93:9067-72.

Chen CM, Krohn J, Bhattacharya S, Davies B. 2011. A comparison of exogenous promoter activity at the ROSA26 locus using a ΦiC31 integrase mediated cassette exchange approach in mouse ES cells. PLoS One. 2011;6(8):e23376.

Chiang C, Jacobsen JC, Ernst C, Hanscom C, Heilbut A, Blumenthal I, Mills RE, Kirby A, Lindgren AM, Rudiger SR, McLaughlan CJ, Bawden CS, Reid SJ, Faull RL, Snell RG, Hall IM, Shen Y, Ohsumi TK, Borowsky ML, Daly MJ, Lee C, Morton CC, MacDonald ME, Gusella JF, Talkowski ME. 2012. Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. Nat Genet. 44:390-7, S1.

Chu VT, Weber T, Graf R, Sommermann T, Petsch K, Sack U, Volchkov P, Rajewsky K, Kühn R. 2016. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 16:4

Costantini F, Radice G, Lee JL, Chada KK, Perry W, Son HJ. 1989. Insertional mutations in transgenic mice. Prog Nucleic Acid Res Mol Biol. 36:159-69.

Dow LE, Premsrirut PK, Zuber J, Fellmann C, McJunkin K, Miething C, Park Y, Dickins RA, Hannon GJ, Lowe SW. 2012. A pipeline for the generation of shRNA transgenic mice. Nat Protoc. 7:374-93.

Dubose AJ, Lichtenstein ST, Narisu N, Bonnycastle LL, Swift AJ, Chines PS, Collins FS. 2013. Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene. Nucleic Acids Res. 41:e70.

Hippenmeyer S, Youn YH, Moon HM, Miyamichi K, Zong H, Wynshaw-Boris A, Luo L. 2010. Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron. 68:695-709.

Hooper M, Hardy K, Handyside A, Hunter S, Monk M. 1987. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature. 326:292-5.

Jaenisch R, Harbers K, Schnieke A, Löhler J, Chumakov I, Jähner D, Grotkopp D, Hoffmann E. 1983. Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death. Cell. 32:209-16.

Ma Y, Yu L, Pan S, Gao S, Chen W, Zhang X, Dong W, Li J, Zhou R, Huang L, Han Y, Bai L, Zhang L, Zhang L. 2017. CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre-loxP-mediated lineage tracing. FEBS J. 284:3262-3277.

Meisler MH. 1992. Insertional mutation of 'classical' and novel genes in transgenic mice. Trends Genet. 8:341-4.

Ménoret S, De Cian A, Tesson L, Remy S, Usal C, Boulé JB, Boix C, Fontanière S, Crénéguy A, Nguyen TH, Brusselle L, Thinard R, Gauguier D5,6, Concordet JP, Cherifi Y, Fraichard A, Giovannangeli C, Anegon I. 2015. Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins. Sci Rep. 5:14410.

Quadros RM, Harms DW, Ohtsuka M, Gurumurthy CB. 2015. Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system. FEBS Open Bio. 5:191-7.

Shin HY, Wang C, Lee HK, Yoo KH, Zeng X, Kuhns T, Yang CM, Mohr T, Liu C, Hennighausen L. 2017. CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. Nat Commun. 8:15464.

Soriano P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 21:70-1.

Srivastava A, Philip VM, Greenstein I, Rowe LB, Barter M, Lutz C, Reinholdt LG. 2014. Discovery of transgene insertion sites by high throughput sequencing of mate pair libraries. BMC Genomics. 15:367.

Tasic B, Hippenmeyer S, Wang C, Gamboa M, Zong H, Chen-Tsai Y, Luo L. 2011. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci U S A. 108:7902-7.

Yang GS, Banks KG, Bonaguro RJ, Wilson G, Dreolini L, de Leeuw CN, Liu L, Swanson DJ, Goldowitz D, Holt RA, Simpson EM. Genomics. 2009. Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus. 93:196-204.

Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T.  2016. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun. 7:10431.

Wu Y, Luna MJ, Bonilla LS, Ryba NJP, Pickel JM. 2018. Characterization of knockin mice at the Rosa26, Tac1 and Plekhg1 loci generated by homologous recombination in oocytes. PLoS One. 13:e0193129.

Zeng H, Horie K, Madisen L, Pavlova MN, Gragerova G, Rohde AD, Schimpf BA, Liang Y, Ojala E, Kramer F, Roth P, Slobodskaya O, Dolka I, Southon EA, Tessarollo L, Bornfeldt KE, Gragerov A, Pavlakis GN, Gaitanaris GA. 2008. An inducible and reversible mouse genetic rescue system. PLoS Genet. 4:e1000069.