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iPSC Cas9 RNP + donor DNA transfectionWard LabReagents neededSynthego synthetic sgRNAs CLYBL safe harbor sgRNA sequence (junction-spanning): ATGTTGGAAGGATGAGGAAATIGRE safe harbor sgRNA sequence (junction-spanning): GCATCTCTCAAGGGCAGTCAAlt-R HiFi Cas9 Nuclease (IDT)Lipofectamine Stem (Life Technologies)E8 media + Rock InhibitorAccutaseMatrigel-coated platescrRNA/donor DNA designCLYBL and TIGRE safe-harbor integration vectors are available from the Ward lab or AddgeneFor other integration sites, we use the IDT crRNA designer, and design 1-2 sgRNAs per gene for each project: work best within 15nt of your cut site; even better is if the crRNA spans the left and right homology arms, so that it won’t cut your donor DNA. If not, ensure to engineer silent mutations into the donor homology arm when you clone it.We typically use 750-1000 nt left and right homology arms. If using WTC11 iPSCs, you can design the homology arms based on specific genomic sequence of this line: primer blast to design efficient and specific primers to generate these homology arms from iPSC genomic DNA via PCR. c-term tags, ensure that you delete the native stop codon and ensure that your tag is in-frame with the gene of interest.iPSCsIt’s important to grow in iPSCs in E8 (not E8 flex), and ensure that iPSCs are very healthy and not overgrown on the day of transfectionAccutase-dissociate iPSCs in the morning of transfection and re-plate at the appropriate densitySince Lipofectamine stem transfection efficiencies are inhibited by residual accutase, it is important to change the media on the cells using pre-warmed E8 + RI ~1-2 hours prior to transfectionRNP Transfection ProtocoliPSC platingAccutase-dissociate iPSCs on the morning of transfection; cells don’t need to be fully individualizedplate 250,000 iPSCs per well in E8/RI onto a Matrigel-coated 24-well plate1-2 hours prior to transfection, change media on each well with fresh, pre-warmed E8/RI (0.5 mL/well)sgRNA preparationResuspend crRNA (2nmol) in 20 uL of RNAse-free TE buffer (from Synthego) to make a 100 uM stock solution. (if not already done).Dilute required amount of sgRNA in TE for transfections to 1 uM (final concentration)RNP complex formationFlick tube of HiFi Cas9 enzyme to mix and briefly spin to bottom of tubeDilute required amount of HiFi Cas9 (62 uM stock solution) to 1uM in room-temp Optimeme.g. 0.5 uL of 62 uM HiFi Cas9 in 30.5 uL Optimemnote that 1 uL of 1 uM Cas9 = 162 ngleftover diluted Cas9 can be stored at -30 deg CProduce RNP complexFor transfecting a single well of a 24 well dish:Tube 1 19 uL OptiMEM I3uL 1uM HiFi Cas93uL cr/trRNA complexMix by pipettingFor difficult integrations, can scale up to 12-well (2x scale) or 6-well (4x scale)Incubate for 5 minutes at room temp to form RNP complexesSetup transfection mixFor transfecting a single well of a 24 well dish:Tube 1 (above RNP complex)Add 500 ng donor DNA to RNP complex and mix by pipettingAdd 100 uL pCE-mp53DD (dominant-negative p53; addgene)Tube 225 uL OptiMEM 1 1 uL Lipofectamine StemFlick tube to mixAdd Tube 2 solution to tube 1 and flick tube to mixIncubate mixture for 10 minutes at room tempAdd mixture to iPSCs and immediately swirl plate to mixThe day following transfection, individualize cells with accutase and plate in 6 wells of a 6-well dish via 50% serial dilutions for clonal outgrowthIf antibiotic resistance of transgene is present, can treat with 1ug/mL puromycin (or 25-150 ug/mL blasticidin, if this is the selection marker) 2-3 days post-transfection,We find that co-transfection with a dominant-negative p53 plasmid (episomal) dramatically improves survival of iPSCs that undergo Cas9 dsDNA breaks, consistent with the literatureNon-clonal antibiotic-resistant stable cell populations are often sufficient for downstream experimentsFloxed selection cassettes can be efficiently removed via treatment with tat-Cre (Millipore) ................
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