pCas9_GFP

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

Design and construction of CRISPR sgRNAs and targeting vectors
CRISPR sgRNAs were designed using the Optimized CRISPR Design tool (http://crispr.mit.edu). Predicted sgRNA off-target sites were retrospectively analysed using the WTSI Genome Editing tool (http://www.sanger.ac.uk/htgt/wge). All sgRNAs were predicted to target unique genomic sites. For the majority of those, similar sequences contained mismatches of three or more nucleotides (with at least one occurring in the PAM proximal region), and therefore off-target cleavage is unlikely (Cho et al., 2014). Sequences are provided in Table S1.

For sgRNA-encoding plasmids, single-stranded oligonucleotides (Integrated DNA Technologies) containing the guide sequence of the sgRNAs were annealed, phosphorylated and ligated into BsaI site of U6-BsaI-sgRNA backbone (kindly provided by S. Gerety, Sanger Institute, Cambridge, UK). For in vitro transcription (IVT), dsDNA templates for T7-driven transcription were generated by annealing two oligonucleotides – one containing the T7 promoter and guide RNA target sequences, and the other containing the Cas9-binding tracrRNA sequence. The annealed oligo pair was gap-filled using T4 DNA polymerase, column-purified, and then used as a template for IVT (MEGAscript T7 Transcription Kit). The RNA was purified using MEGAclear Transcription Clean-Up Kit.

Targeting vectors were constructed via Gibson assembly and Gateway cloning (Fig. S3). Briefly, linearised backbone and a Zeo/PheS bacterial selection cassette were obtained through EcoRV digestion of existing plasmids. Homology arms of ∼1 kb were amplified from genomic DNA using PCR primers with 22 bp overhangs compatible with both backbone and the Zeo/PheS double-selection cassette. Gibson reactions were performed using a standard protocol with home-made enzyme mix (Gibson et al., 2009) to create the intermediate Gateway cloning compatible intermediate vector. The Zeo/PheS cassette was replaced via LR Gateway cloning using a FRT-Ef1a-PuroR-FRT mammalian selection cassette. For AAVS1 and Rosa26 targeting vectors, the Luc-2A-GFP, Cas9-2A-GFP or rtTA expression cassettes were PCR amplified from existing plasmids (gift from M. Pule, University College London, UK) and cloned into Gateway pDONR221 using BP cloning. The cassettes were then delivery into the intermediate targeting vectors via Gateway LR cloning. Construction of the AAVS1-TRE-GFP targeting vector involved restriction digestion followed by ligation of a custom gene vector (Life Technologies) containing the TRE-GFP-2A-PuroR cassette into the digested AAVS1 intermediate targeting vector (Fig. S3). For mouse Sox2-mCherry targeting vector, 1 kb long arms were PCR amplified and tethered to mCherry sequence using Gibson reaction. The sgRNA targeting region was removed from the R-HA in targeting vector to avoid re-cutting of residual Cas9 after the homologous recombination event. For H3F3A targeting vectors, homology arms were amplified from genomic DNA and Gibson-assembled with synthetic DNA fragments (Life Technologies) containing the V5-tagged, mutant sequences of the first coding exon.


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Manufacturer protocol

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