citations

                    CRISPR/Cas9 Ribonucleoprotein Nucleofection for Genome Editing in Primary Human Keratinocytes: Knockouts, Deletions, and Homology-Directed Repair Mutagenesis
                

Authors:

Bamundo M, Palumbo S, D'Auria L, Missero C, Di Girolamo D

In:

Source: Curr Protoc Microbiol
Publication Date: (2024)
Issue: (11): e70056

Research Area:

Cancer Research/Cell Biology

Basic Research

Molecular Biology

Regenerative medicine

Platform:

4D-Nucleofector® X-Unit

Experiment

see methods part on page 6-19.

Delivery of ribonucleoprotein complex through nucleofection (KO)
32. Prepare two independent Nucleofection Reaction mixes for cells undergoing genome editing and for control cells:

Reagent Volume
2 × 10^5 cells (in Nucleofection Solution mix; see step 31) 20 µl
RNP reaction mix (sgRNA+Cas9) or Control mix (only Cas9) (see step 21) 5 µl
Alt-RTM Cas9 Electroporation Enhancer (stock 100 µM; see step 19) 1 µl
Final volume 26 µl
The addition of Alt-RTM Cas9 Electroporation Enhancer to the Nucleofection Solution mix increases delivery efficiency.33. Carefully transfer each Nucleofection Reaction mix into a well of the 16-well nucleocuvette strip provided with the Amaxa P3 Primary Cell 4D-Nucleofector X Kit
S, avoiding bubbles. If there are air bubbles, gently tap the nucleocuvette strip and/or use a needle to pop the bubbles.
34. Switch on the Amaxa 4D-Nucleofector Core and X Units, select the 16-well nucleocuvette strip module, and choose the position of the loaded well.
35. Open the tray, transfer the 16-well nucleocuvette strip into the Amaxa 4DNucleofector machine, and select the electroporation program and the pulse code (for primary HKs, use Primary Cell P3 program and DS-138 pulse code).
Make sure that the larger gap in the strip lid is at the top of the strip to ensure perfect complementarity between the holder and the strip.
36. Press start and wait until the electroporation is completed. At the end of the run, the screen should display a “+” over the wells that were successfully electroporated.
37. Carefully remove the 16-well nucleocuvette strip from the Amaxa 4D-Nucleofector machine and incubate 10 min at room temperature.
38. Carefully add 75 µl keratinocyte growth medium to each well of the nucleocuvette strip containing the 26 µl of Nucleofection Reaction mix. Slowly pipet up and down
2 to 3 times and then transfer the entire contents to one well of the 24-well plate containing mitotically blocked 3T3-J2 cells (see step 27). Repeat this step twice total to ensure collection of all edited cells.
39. Incubate the plate overnight at 37°C, 5% CO2.

knockin:

Delivery of ribonucleoprotein complex through nucleofection
8. Prepare two independent Nucleofection Reaction mixes, for control cells and for cells undergoing genome editing. To induce site-specific mutagenesis, add resuspended
Alt-RTM HDR Donor Oligo to the mixes as follows:
Nucleofection Reaction mix: Reagent Volume
1 × 105 cells (in Nucleofection Solution mix; see step 7) 20 µl
RNP reaction mix (sgRNA+Cas9; see step 3) 5 µl
Alt-RTM Cas9 Electroporation Enhancer (stock 100 µM) 1 µl
Alt-RTM HDR Donor Oligo (100 µM; see step 1) 1.2 µl
Final volume 27.2 µl
Control Reaction mix: Reagent Volume
1 × 10^5 cells (in Nucleofection Solution mix; see step 7) 20 µl
Control mix (only Cas9; see step 3) 5 µl
Alt-RTM Cas9 Electroporation Enhancer (stock 100 µM) 1 µl
D-PBS -Ca++/Mg++ (1×) 1.2 µl
Final volume 27.2 µl
The IDT tool suggests testing both positive and negative strands of Alt-RTM HDR Donor Oligo separately to verify possible differences in editing efficiency.We observed no significant differences in efficiency or survival between positive and negative strands of Alt-RTM HDR Donor Oligos.
9. Carefully transfer each Nucleofection Reaction mix and Control Reaction mix into separate wells of the 16-well nucleocuvette strip provided with the Amaxa P3 Primary
Cell 4D-Nucleofector X Kit S, avoiding bubbles. If there are air bubbles, gently tap the nucleocuvette strip and/or use a needle to pop the bubbles.
10. Switch on the Amaxa 4D-Nucleofector Core and X Units, select the 16-well strip module, and choose the position of the loaded well.
11. Open the tray, transfer the 16-well nucleocuvette strip into the Amaxa 4DNucleofector machine, and select the electroporation program and the pulse code
(for primary HKs, use Primary Cell P3 program and DS-138 pulse code). Make sure that the larger gap in the strip lid is at the top of the strip to ensure a perfect fit between the holder and the strip.
12. Press start and wait until the electroporation is completed. At the end of the run, the screen should display a “+” over the wells that were successfully electroporated.
13. Carefully remove the nucleocuvette strip from the 4D-Nucleofector machine and incubate for 10 min at room temperature.
14. Carefully add 75 µl pre-warmed keratinocyte growth medium with 1 µM Alt-RTM HDR Enhancer V2 (see step 2) to each well of the nucleocuvette containing the 27.2 µl of Nucleofection Reaction mix or Control Reaction mix.
15. Slowly pipet up and down 2 to 3 times.
16. Divide the contents of each Nucleofection Reaction mix and Control Reaction mix into two wells of a 24-well plate containing mitotically blocked 3T3-J2 cells (see step 3) by plating 50 µl/well.

Abstract

Keratinocytes are the most abundant cell type in the human epidermis, the outermost layer of the skin. For years, primary human keratinocytes (HKs) have been used as a crucial tool for studying the pathogenesis of a wide range of skin-related diseases. To mimic the physiological and pathological behavior of human skin, organotypic 3D skin models can be generated by in vitro differentiation of HKs. However, manipulation of HKs is notoriously difficult. Liposome-mediated gene delivery often results in low transfection rates, and conventional electroporation results in high mortality, is difficult to optimize, and requires high cell numbers without necessarily achieving maximum efficiency. Additionally, HKs have a short lifespan in vitro, with a limited number of cell divisions before senescence, even when cultured on a feeder layer. Therefore, the possibility to use an efficient CRISPR/Cas9 system in HKs is not without challenge in terms of transfection technology and clonal selection. In this article, we provide detailed protocols to perform efficient gene knock-out (KO) or genomic deletion in a small number of HKs without clonal selection of edited cells. By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions. Moreover, we describe an optimized protocol for generating site-specific mutations in immortalized keratinocytes (N/TERT2G) by exploiting the homology-directed repair system combined with rapid single-clone screening. These methods can also be applied to other immortalized cells and tumoral cells of epithelial origin. Together, these protocols provide a comprehensive and powerful tool that can be used to better understand the molecular mechanisms underlying different skin diseases.

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