Jun 01, 2023

Public workspaceTransient CRISPR-Cas9 Coupled with Electroporation Protocol

DOI
dx.doi.org/10.17504/protocols.io.bp2l64x2kvqe/v1
  • 1Duke University
Amy Gladfelter
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DOI: dx.doi.org/10.17504/protocols.io.bp2l64x2kvqe/v1
Protocol Citation: Amy Gladfelter 2023. Transient CRISPR-Cas9 Coupled with Electroporation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l64x2kvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 03, 2018
Last Modified: June 01, 2023
Protocol Integer ID: 17448
Abstract
Transient CRISPR-Cas9 transformation of Cryptococcus neoformans.
Attachments
Icon for CRISPR Electroporation Protocol Cryptococcus.pdf
CRISPR Electroporati...
70KB
Guidelines
References

1. Lin, X., et al., Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation. Med Mycol, 2015. 53(3): p. 225-234.

2. Fan, Y. and X. Lin, Multiple applications of a transient CRISPR-Cas9 coupled with electroporation (TRACE) system in the Cryptococcus neoformans species complex. Genetics, 2018.
PCR amplification of CAS9, sgRNA, your construct.
  • CAS9: Use plasmid pXL1-CAS9-HYG as template with primers CAS9-F and CAS9-R. (6985 bp)

  • sgRNA: U6P and sgRNA scaffold
  1. U6pPromoteris PCR amplified using serotype Dgenomic DNA astemplate with primers U6P-F and GOI-sgRNA-R. ~295 bp
  2. SgRNA scaffold is PCR amplified using pYF515 astemplate with primers GOI- sgRNA-F and sgRNA-R. ~108 bp
  3. sgRNA construct is PCR amplified using above two PCR product sastemplate with primers U6P-F and sgRNA-R. ~383 bp
MixAmount2 µg your construct DNA, 100 ng sgRNA, and 170 ng CAS9 in an Eppendorf tube.

Vacuum dry the DNA and elute inAmount5 µL DNAse/RNAse free water.
Note
Notes: use the combination of 2 μg construct DNA, 1 μg CAS9 DNA, and 700 ng sgRNA can increase the transformation efficiency, but low dose is sufficient to obtain transformants.


Inoculate recipient strain in Amount5 mL YPD liquid medium, culture overnight at Temperature30 °C with shaking at 250 rpm.


Use the overnight culture to inoculateAmount100 mL fresh YPD medium at an initial inoculum of OD600=0.2. Grow the cells for additionalDuration04:00:00 toDuration05:00:00 until the cell density reached OD600 between 0.6-1.0. From this step on, everything on ice and centrifugation at Temperature4 °C




Collect cells by centrifugation at 3200g forDuration00:05:00 at Temperature4 °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 Amount10 mL ice-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 (Duration01:00:00 ).
Note
(30 to 60 mins in 2015 paper)



(Optional) Wash cells with Amount10 mL ice-cold EB buffer once (2015 paper).

Collect the cells by centrifugation and resuspend inAmount250 µL EB buffer.

Mix Amount45 µL cells with Amount5 µL 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 Amount1 mL of YPD medium and culture at Temperature30 °C for Duration02:00:00 (Duration01:30:00 in 2015 paper) before plating onto the appropriate selective agar medium.