High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

Abstract

Summary An efficient Agrobacterium ‐mediated site‐specific integration ( SSI ) technology using the flipase/flipase recognition target ( FLP / FRT ) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line ( RTL ) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom ( Bbm ) and Wuschel2 ( Wus2 ) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL 1 resulted in higher transformation frequency than strain LBA 4404 THY ‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL 1; 0.65% in LBA 4404 THY ‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.

Site‐specific recombinase genome engineering toolkit in maize

“…Additionally, it is commonly accepted that recombinase coding sequences are degraded and eventually lost; however, Srivastava and Ow (2001) identified 40% of excision modifications contained a stably integrated Cre recombinase coding sequence after bombardment (Wang et al, 2011). Similarly, Anand et al (2019) reported the presence of FLP and/or Cre in T 0 plants when using Agrobacterium as the mode of delivery to induce a recombinase‐mediated cassette exchange reaction. Taken together, transient expression of recombinases using Agrobacterium or bombardment adds the benefit of timely modifications; however, it requires tissue culture and subsequent screening to obtain a desired plant background.…”

“…The first application using Cre to excise a stably integrated selectable marker gene from tobacco occurred in the early 90s (Dale & Ow, 1990). Since then, recombinases have been used in a number of plant systems including rice (Hoa, Bong, Huq, & Hodges, 2002; Hu et al, 2007; Radhakrishnan and Srivastava, 2005), tobacco (Albert, Dale, Lee, & Ow, 1995; Dale & Ow, 1991), wheat (Srivastava, Anderson, & Ow, 1999), tomato (Stuurman, Vroomen, Nijkamp, & Haaren, 1996), barley (Kapusi, Kempe, Rubtsova, Kumlehn, & Gils, 2012), soybean (Li et al, 2009), Arabidopsis (Hong, Lyznik, Gidoni, & Hodges, 2000; Thomson, Chan, Thilmony, Yau, & Ow, 2010; Vergunst & Hooykaas, 1998), and maize (Anand et al, 2019; Kerbach, Lörz, & Becker, 2005; Lyznik, Rao, & Hodges, 1996; Srivastava & Ow, 2001; Zhang et al, 2003). In these studies, recombinase enzymes have been used for the purpose of selectable marker removal, transgene targeting to predetermined locations, or resolution of multiple transgene concatemers.…”

“…While most studies use bidirectional recombinases, the use of mutant binding sites or transient expression forces unidirectional activity and fixes desired modifications, such as targeted integrations (Albert et al, 1995); however, integration stability using double‐mutant sites with FLP/ FRT has not been successful, and R/ RS has no known mutant sites (Wang, Yau, Perkins‐Balding, & Thomson, 2011). Alternatively, a strategy referred to as recombinase‐mediated cassette exchange (RMCE) has been used successfully to direct transgenes to predetermined locations using FLP/ FRT and R/ RS systems (Anand et al, 2019; Ebinuma, Nakahama, & Nanto, 2015).…”

Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus

“…Each method has its own advantages and disadvantages. While recombinase-mediated methods are highly efficient in targeted integrations, e.g., with Cre-lox, FLP-FRT and other site-specific recombination systems, they require placement of recombination target sites that can subsequently be targeted for gene stacking (Anand et al, 2019;Li et al, 2009;Ow, 2003;Srivastava and Gidoni, 2010). The CRISPR/Cas9 method, on the other hand, can target virtually any site in the genome, but the non-homologous mode of DSB repair in plant cells overrides the integration of exogenous DNA into the DSB site (Puchta and Fauser, 2014;Voytas, 2013).…”

“…Other site-specific recombination systems that show high efficiency in plant cells include FLP-FRT, phiC31-att and Bxb1-att systems (Anand et al, 2019;Hou et al, 2014;Li et al, 2009;Nandy et al, 2011;Ow, 2011;Thomson and Ow, 2006). Recombinase-mediated gene stacking has been demonstrated in soybean by inserting gene silencing and overexpression constructs through two rounds of transformation (Li et al, 2010).…”