custom RMCE landing sites-CRISPR/cas9

Landing sites can be generated by traditional CRISPR/Cas9 insertion techniques.  The inserts are large and include a self excision cassette (SEC) for selection using hygromycin or screening via sqt-1(e1350) Rol phenotype.  Once inserted the SEC must be removed by heat shock to create the final landing site strain.  We have generated landing site plasmids that are designed to create CRISPR/cas9 repair templates using SapI Golden Gate cloning (Dickinson et al. 2018).  Here we  only describe how to make the repair template.  We refer the readers to Dickinson et al. (2015) and Nonet (2021) for protocols for the CRISPR/cas9 injections and work-up.

Protocol Overview

 Creating the targeting vectors using Golden Gate cloning is accomplished by either 1) by amplifying the  homology arms (600 bp to 1 kb) using oligonucleotides with SapI sites on the 5′ end of the oligonucleotides and performing a 4 part Golden Gate Reaction using the vector pSapI, the two arm PCR products and the landing site construct, or 2) first amplifying the arms using oligonucleotides that have BsaI sites on the 5′ ends, then performing a BsaI Golden Gate reaction for each arm to create arm clones in DR274 5′ arm and DR274 3′ arm, then performing a second SapI Golden Gate reaction with pSapI, the two arm clones, and the landing site construct.  Both approaches work and the only reason to clone the arms is if you plan to create many different targeting vectors with the same arms.  The only limitations of this approach if you use only SapI, is that neither arm can have an internal SapI site.  If one is subcloning the arms using BsaI then the arms also cannot have an internal BsaI site.

Figure 1. Construction of a novel landing site using CRISPR/cas9. To create a novel landing site one must first create a targeting vector that contains the landing site [with an added Self Excision Cassette (SEC)] that contains homology arms. We find the simplest way to do this is using a Golden Gate reaction using either cloned arms or PCR products of the arms (not shown). This templates is then co-injected along with an sgRNA and a source of Cas9 to identify insertions at the locus using either a screen for Rol animals or a selection for hygR animals. The SEC is then excised with a heat shock to create the final landing site. Typically, we find we need to inject ~ 20-30 animals on average to identify an insertion. The insertion should be molecularly characterized to insure it is complete. The example shown is to create a myo-2p rapid RMCE landing site
Plasmid Reagents

DR274 CT-NT loxP FRT FRT3 landing [map] [seq] [addgene] (for two component sites)

DR274 CT-NT FLP LoxP FRT FRT3 landing [map] [seq] [addgene] (for one component sites)

DR274 CT-NT loxP myo-2 FRT mex-5 FLP mNG landing [map] [seq] (for rapid RMCE sites)

pSapI [map] [sequence]

DR274 5′ arm-BsaI [map] [sequence]

DR274 3′ arm-BsaI [map] [sequence]

Golden Gate Reactions

 Golden Gate (GG) reactions are performed by mixing 50 fmol of the vector, and 60 fmol of each insert plasmid or PCR fragment and diluting to 10 ul with TE (10 mM Tris, 0.1 mM EDTA pH 8.0).   1 µl of the DNA mix is added to 1 µl of 6X Sap buffer (300 mM Tris-HCl, 25 mM KAc, 60 mM MgCl2, 60 mM dithiothreitol, 6 mM ATP,  pH 7.5 @ 25°C), 3.5 µl H20, 1/2 µl of SapI or BsaI, and 1/4 µl T4 DNA ligase.   The reactions are incubated 15 min at 37°C, 5 min at 16°C, followed by 10 cycles of 2 min at 37°C, and 2 min at 16°C, followed by 5 min at 37°C, and 20 minute at 65°C. 1/2 µl of the reaction is transformed into DH5α.  Typically, hundreds to thousands of transformants are obtained and the majority are correctly assembled clones.

Primers

5’SapIf aaGCTCTTCNTGG(N)x   |    5’SapIr ttGCTCTTCNCGC(N)x

3’SapIf aaGCTCTTCNACG(N)x   |    3’SapIr ttGCTCTTCNTAC(N)x

5’BsaIf aGGTCTCNGTGG(N)x   |   5’BsaIr tGGTCTCNTCGC(N)x

3’BsaIf aGGTCTCNGACG(N)x   |   3′ BsaIr tGGTCTCNCTAC(N)x

(N)x represents the arm specific sequences. Lower case bases are optional and added to insure efficient enzyme cleavage at the end of the PCR product.  The SapI or BsaI site present in the oligonucleotide is bolded.

References

Dickinson, D. J., Pani, A. M., Heppert, J. K., Higgins, C. D. & Goldstein, B. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette. Genetics 200, 1035-1049 (2015).

Dickinson, D. J., Slabodnick, M. M., Chen, A. H. & Goldstein, B. SapTrap assembly of repair templates for Cas9-triggered homologous recombination with a self-excising cassette. microPublication Biology (2018).

Nonet, M.L. Additional Landing Sites for Recombination-Mediated Cassette Exchange in C. elegans.microPublications Biology, 10.17912/micropub.biology.000503 (2021).