RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

Abstract

A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented.

Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

“…DNA constructs. Codon-optimization of rice TIR1 for mammalian expression has been described previously 5 . To visualize mAID-vhhGFP expression in transient transfection experiments the mAID-vhhGFP sequence was cloned downstream of an IRES element of CMV-driven mCherry fused at its N terminus to the N-terminal importin β binding domain (IBB) creating IBB-mCherry-IRES-mAID-vhhGFP in a pIRESpuro backbone (Thermo Fisher Scientific).…”

“…Further, it has been reported that fusion with the AID degron can destabilize the tagged protein [3][4][5][6] . So far, the auxin system has been established in a limited number of case studies including yeast 4,7 , nematodes 8,9 , flies [1][2][3][4] and human cell lines [3][4][5][6] . However, its feasibility in a vertebrate model organism remains to be shown and large-scale application of the AID system in animal remains challenging despite the advent of CRISPR/Cas9.…”

UFMylation regulates translational homeostasis and cell cycle progression

“…UFC1 knock-out cells were generated by cotargeting the HPRT gene (Liao et al, 2015). Briefly, Flag-NLS-linker-Cas9 (Baker et al, 2016) was co-electroporated with a pAAV backbone-based plasmid (Stratagene) containing two U6-promotor-driven gRNAs targeting HPRT (GACTGTAAGTGAATTACTT), UFM1…”

Cooperative genetic networks drive a mammalian cell state transition

“…Guide RNAs (gRNAs) were designed using the computational pipeline described below, or by http://crisprscan.org. To clone the gRNAs for ESC KO of the 154 selected genes, we used RecET recombineering to insert the guide RNA protospacer encoding sequence between the U6 promoter and the universal sgRNA scaffold in pBR322-U6-cm-ccdB-sgRNA-amp, as described previously (Baker et al, 2016). We generated 331 sgRNA expressing plasmids for 149 genes (2 sgRNAs for 131genes, 3 sgRNAs for 5 genes, 4 sgRNAs for 12 gens and 6 sgRNAs for 1 gene).…”

“…In brief, the plasmid was linearized at the point of insertion by BstZ17I digestion, purified using the Invitrogen Charge Switch PCR Purification Kit and dissolved in water; 80-mer oligonucleotides containing the protospacer sequence, flanked by 30bp of homology to the target plasmid were dissolved in water. E. coli GB05 transformed with the pSC101-Prha-ETgA-tet plasmid (Baker et al, 2016), was cultured to OD600 ~0.5 in deep-well 96 well plates and then induced for 1 hour for expression of the ETgA operon with Lrhamnose. Electroporation with 200 ng (0.1 pmol) of the linearised vector and 50 pmol of oligonucleotide, using BTX 96 well electroporation plates (MOS96, 2mm gap,) and the BTX ECM630 electroporator with HT-200 adapter, was described previously (Sarov et al, 2012).…”