Jul 21, 2025

Public workspaceGeneration of CRISPR/Cas9 edited mammalian cells with small epitope tags

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkrxo5v5r/v1
  • Hely Rodriguez Cruz1,
  • Michael Hanna1,
  • Pietro De Camilli1
  • 1Yale University
Michael G Hanna
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkrxo5v5r/v1
Protocol Citation: Hely Rodriguez Cruz, Michael Hanna, Pietro De Camilli 2025. Generation of CRISPR/Cas9 edited mammalian cells with small epitope tags. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkrxo5v5r/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: June 04, 2025
Last Modified: July 21, 2025
Protocol Integer ID: 219601
Keywords: ASAPCRN, generation of crispr, mammalian cells with small epitope tag, edited mammalian cell, immortalized mammalian cell culture line, small epitope tag, crispr, mammalian cell culture line, gene, cell, cas9
Abstract
This protocol explains how to endogenously edit immortalized mammalian cell culture lines to engineer an in-frame small epitope tag into a gene of interest.
Materials
  • Alt-RTM CRISPR-Cas9 crRNA (synthesized by IDT)
  • Alt-RTM CRISPR-Cas9 tracrRNA (IDT, Cat# 1075934)
  • Alt-RTM S.p. HiFi Cas9 Nuclease V3 (IDT, Cat# 1081060)
  • Nuclease-free duplex buffer (IDT, Cat# 11-01-03-01, included with Alt-R CRISPR-Cas9 tracrRNA)
  • Single stranded repair oligo (IDT)
  • PCR tube
  • Thermal cycler
  • 1xPBS
  • Trypsin-EDTA (0.25%), phenol red
  • 15 mL conical tube
  • 1.5mL microcentrifuge tubes
  • Microcentrifuge
  • Lonza 4D Nucleofector System Core Unit with X Unit
  • Lonza nucleofection solution
  • Lonza nucleofector cuvette
  • DMEM complete medium (10%FBS, 1% Pen/Strep, 0.2% Plasmocin)
  • 6-well cell culture plate
  • HDR enhancer (IDT, Cat# 10007910)
Troubleshooting
Assemble the gRNA
15m
Resuspend Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA in nuclease-free duplex buffer at 200 μM
Mix 5 μl of 200 μM crRNA and 5 μl of 200 μM tracrRNA together in a PCR tube
Heat to 95°C for 5 minutes in a thermal cycler
5m
Let cool to RT
10m
Assemble the RNP
30m
In a microcentrifuge tube, mix 1.7 μl (104 pmol) of Alt-R S.p. Cas9 nuclease together with 1.2 μl (120 pmol) Alt-R CRISPR-Cas9 crRNA/tracrRNA duplex and 2.1 μl of 1xPBS solution.
Incubate for 30 minutes at RT
30m
Prepare repair oligo
Resuspend repair oligo in 1xPBS at a concentration of 100 μM (pmol/μl).
Prepare cell & electroporation
10m
Lift A549 cells from dish using trypsin digest and collect in a 15 mL conical
5m
Count cells and pellet ~800,000 cells in a microcentrifuge tube at 300g for 3 minutes.
5m
Resuspend cell pellet in 100 µL desired Lonza nucleofection solution
Add 2 µL of repair oligo (100 μM) to 5 µL of pre-assembled RNP complex from Step 3
Add RNP complex and repair oligo to resuspended cells in nucleofector cuvette
Electroporate cells according to Lonza nucleofection kit specifications
Post electroporation
Remove cells from nuclofector cuvette and directly add to 2mL warmed complete culture media in one well of a 6-well plate
Add 3.4 µL of HDR enhancer to the 2 mL culture (optional)