Transient CRISPR-Cas9 Coupled with Electroporation Protocol
Amy Gladfelter
Abstract
Transient CRISPR-Cas9 transformation of Cryptococcus neoformans.
Before start
Attachments
Steps
PCR amplification of CAS9, sgRNA, your construct.
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CAS9: Use plasmid pXL1-CAS9-HYG as template with primers CAS9-F and CAS9-R. (6985 bp)
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sgRNA: U6P and sgRNA scaffold
- U6pPromoteris PCR amplified using serotype Dgenomic DNA astemplate with primers U6P-F and GOI-sgRNA-R. ~295 bp
- SgRNA scaffold is PCR amplified using pYF515 astemplate with primers GOI- sgRNA-F and sgRNA-R. ~108 bp
- sgRNA construct is PCR amplified using above two PCR product sastemplate with primers U6P-F and sgRNA-R. ~383 bp
Mix2µg your construct DNA, 100 ng sgRNA, and 170 ng CAS9 in an Eppendorf tube.
Vacuum dry the DNA and elute in5µLDNAse/RNAse free water.
Inoculate recipient strain in 5mL YPD liquid medium, culture overnight at 30°C with shaking at 250 rpm.
Use the overnight culture to inoculate100mLfresh YPD medium at an initial inoculum of OD600=0.2. Grow the cells for additional4h 0m 0sto5h 0m 0s until the cell density reached OD600 between 0.6-1.0.
From this step on, everything on ice and centrifugation at 4°C
Collect cells by centrifugation at 3200g for0h 5m 0sat 4°C.
Wash cells with ice-cold water (EB Buffer instead of water in 2015 paper). (wash 1/2)
Wash cells with ice-cold water (EB Buffer instead of water in 2015 paper). (wash 2/2)
Suspend cells in 10mLice-cold EB buffer (10 mM Tris-HCl, pH 7.5, 1mM MgCl2, 270 mM Sucrose) with 1mM DTT.
Incubate the cells on ice for an hour (1h 0m 0s).
(Optional) Wash cells with 10mLice-cold EB buffer once (2015 paper).
Collect the cells by centrifugation and resuspend in250µL EB buffer.
Mix 45µL cells with 5µL DNA mix from step 2 in a pre-cooled 2 mm gap electroporation cuvette.
Transform the DNA by electroporation using the BioRad gene pulser with settings of 0.45 kV, 125 μF, 600 Ω. (If using an Eppendorf multiporator, use the bacterial mode with V=2 kV with τ optimized for 5 ms)
Suspend electroporated cells in 1mLof YPD medium and culture at 30°C for 2h 0m 0s (1h 30m 0sin 2015 paper) before plating onto the appropriate selective agar medium.
