Genetic Engineering of Esophageal Organoids: CRISPR-Based Knock-in for Cell Lineage Tracing

Abstract

Esophageal organoids serve as powerful systems to study epithelial lineage hierarchies and cancer biology. Gene manipulation in these organoids has traditionally involved overexpression or knockout strategies(1–5). However, CRISPR/Cas9-based knock-in (KI) approaches now enable precise cell lineage tracing and live imaging. Here, we describe protocols to generate fluorescent KI organoids from murine esophageal epithelium by tagging Krt13 (BFP) and Sox2 (mNeon). These dual-reporter organoids allow direct monitoring of growth dynamics and differentiation trajectories. We outline CRISPR/Cas9 design, donor construction using homology-independent approaches (CRISPaint), delivery into organoid cells, enrichment and single-clone isolation, and validation by fluorescence. For organoids, homology-directed repair (HDR) can be relatively inefficient to deliver the reporter frame. Thus, we highlight the practical advantages of non-homologous end joining (NHEJ)-based methods, which enable robust, frame-accurate KI with minimal cloning. The methods outlined here can be applied broadly for cell lineage tracing, damage-response studies, and cancer modeling.

1. Introduction

CRISPR-mediated knock-in enables precise lineage marking and live imaging in epithelial organoids. For basal-to-differentiated trajectories, Krt13 is a valuable marker of differentiation, whereas Sox2 labels progenitor/stem compartments(6–8). Tagging these loci with fluorescent reporters allows prospective isolation and dynamic analysis without overexpression artifacts.

CRISPR–Cas9 creates a targeted double-strand break (DSB) that cells repair mainly by NHEJ or HDR(9). NHEJ frequently generates insertions and deletions (indels) suitable for knockouts, whereas HDR copies information from a donor to install precise edits (including tags). However, NHEJ typically occurs at low frequency and is restricted to dividing cells. In organoids, HDR efficiency is often a bottleneck and requires donor plasmids with gene-specific homology arms. In contrast, NHEJ-based targeted insertion of exogenous DNA has been utilized. Additionally, it has been demonstrated that this approach outperforms HDR for KI in epithelial organoids (10–12).

Herein, we provide a step-by-step framework applicable to mouse esophageal organoids for tagging cells expressing Krt13, a suprabasal/differentiated cell marker encoded by the Krt13 gene, and Sox2, a basal/stem/progenitor cell marker, to visualize epithelial differentiation and stem cell dynamics.

2. Materials

Prepare all solutions with sterile, nuclease-free water. Follow institutional biosafety approvals for genome editing and animal-derived materials.

2.1. Organoids culture

1. Murine esophageal organoids in standard expansion medium (EMEOM) and Matrigel (3, 4)

2. Dissociation reagents (0.5% Trypsin- EDTA)

3. PBS (Ca²⁺/Mg²⁺-free)

4. ROCK inhibitor (Y-27632)

5. Complete medium (DMEM supplemented with 10% FBS)

6. Opti-MEM medium

2.1. Reagents for cloning

1. Designed oligos

2. BbsI-HF restriction enzyme (NEB, #R3539S)

3. rCutSmart buffer (NEB)

4. FASTAP phosphatase (Thermofisher, #EF0641)

5. T4 Polynucleotide Kinase (PNK) (NEB, #M0201S)

6. T4 DNA ligase buffer (NEB, #B0202S)

7. T4 DNA ligase (NEB, #M0202S)

8. GeneJET Gel Extraction Kit (Thermofisher, #K0691)

2.2. CRISPR/Cas9 and Donor Components

1. sgRNA plasmids (pSPgRNA) targeting Krt13 and Sox2 near the stop codon (10–20 bp upstream for C-terminal tagging)

2. Donor reporter plasmid

   • CRISPaint-TagBFP-PuroR (addgene, #80969)

   • CRISPaint-TagmNEON-PuroR (addgene, #174090)

3. Frame-selector plasmids with Cas9 ORF

   • pCAS9-mCherry-Frame +0 (addgene, #66939)

   • pCAS9-mCherry-Frame +1 (addgene, #66940)

   • pCAS9-mCherry-Frame +2 (addgene, #66941)

2.3. Equipment

1. Electroporator (NEPA21) and cuvettes (2 mm gap).

2. Fluorescence microscope for reporter verification.

3. Methods

Critical points are highlighted in Notes. Carry out all steps at room temperature, following safety guidelines, unless noted (Note 1).

3.1. sgRNA and Plasmids preparation (Timing: 1–2 days)

1. Select cut sites within ~10–20 bp of the stop codon in the last exon of Krt13 and Sox2 (Note 2).

2. Clone sgRNA oligos into pSPgRNA

   1. Oligo pairs preparation

      Forward oligo (100 μM)	1 μl
      Reverse oligo (100 μM)	1 μl
      10X T4 DNA ligation buffer	1 μl
      T4 PNK	0.5 μl
      D.W.	6.5 μl
      total	10 μl

      • 37°C 30 min

      • 95°C 5 min

      • Ramp down to 25°C at 5°C/min

      • Dilute oligo pair product 1:200 in D.W.

   2. pSPgRNA enzyme digestion

      pSPgRNA plasmid	x μl (=5 μg)
      BbsI-HF	3 μl
      FASTAP	3 μl
      rCutSmart buffer	6 μl
      D.W.	up to 60 μl
      total	60 μl

      • 37 °C 2 hr

      • 65 °C 20 min (heat inactivation)

      • Load the product on the agarose gel and extract DNA using the Gel extraction kit

   3. Ligation

      Gel extracted enzyme cut plasmid	x μl (=50 ng)
      Diluted oligo pair prepared from step a	1 μl
      10X T4 DNA ligase buffer	2 μl
      T4 DNA ligase	1 μl
      D.W.	up to 20 μl
      total	20 μl

      • Incubate at RT for more than 15 min

      • Perform transformation into competent cells and purify plasmid using Midi- or Maxi-prep. kit

3. Choose reporter strategy:

   • Krt13: BFP plasmid (pCRISPaint-TagBFP-PuroR) to detect differentiated cells.

   • Sox2: mNeon plasmid (pCRISPaint-mNeon-PuroR ) to detect basal cells.

4. Prepare three frame-selector plasmids.

   • This plasmid expresses Cas9 protein with sgRNA that cleaves the pCRISPaint plasmid

   • Each frame selector plasmid cuts the pCRISPaint plasmid in a different frame (0, +1, or +2), allowing the reporter to be incorporated into the target gene (Krt13 or Sox2) with all available frames.

   • This plasmid encodes mCherry to validate transfection

3.2. Prepare Single Cells from Organoids (Timing: 30–60 min)

Organoid handling methods are described previously(4).

Organoids need to be adapted to the medium and Matrigel through passages if they are freshly extracted from esophageal tissue. In the EMEOM medium, organoids can usually be maintained for 10 days before passaging. However, 5–7-day-old organoids show better viability after electroporation.

1. Replace medium 24h before transfection.

2. Harvest organoids and wash in ice-cold Medium and PBS (Note 3).

3. Editing efficiency increases when cells are in a proliferative state; avoid overly differentiated cultures.

4. Dissociate to single cells with 0.5% Trypsin-EDTA (10 min).

5. Inactivate Trypsin with complete medium.

6. Filter (40 μm cell strainer), count, and keep cells on ice.

7. Centrifuge at RT, 1000 RPM for 4 min and remove supernatant

8. Resuspend cells with 600 μl of Opti-MEM.

9. Count the cell number. 1 × 10⁵ cells - 1 × 10⁶ are optimally required for each electroporation (Note 4).

3.3. Electroporation (Timing: 1–2 h)

Optimal electroporation conditions can vary. This protocol is based on the esophageal organoid electroporation conditions as previously described(1).

1. Prepare cells in 150 μl Opti-MEM.

2. Mix cells with plasmids with the following recipe.

   Sample	Frame selector +0	Frame selector +1	Frame selector +2	CRISPaint-TagBFP-PuroR	pSPgRNA-Krt13
   1	3 μg			3 μg	4 μg
   2		3 μg		3 μg	4 μg
   3			3 μg	3 μg	4 μg

3. Mix the cell suspension + DNA by pipetting up and down, and transfer the solution to a cuvette immediately.

4. Electroporate using the following settings.

   	Poring pulse	Transfer pulse
   Voltage	175 V	20 V
   Number of pulses	2	5
   Duration	5 msec	50 msec
   Interval	50 msec	50 msec
   Decay rate	10 %	10 %
   Polarity	+	+/−

5. Combine samples 1, 2, and 3 in a new EP tube and add 600 μl Opti-MEM.

6. Incubate at RT for 30 min.

7. Centrifuge at 600 g for 3 min.

8. Remove the supernatant and resuspend the cells in EMEOM medium and Matrigel for a seeding ratio of 2 to 3 (EMEOM : Matrigel).

9. Seed cells on a 48-well plate (20 μl/well).

10. After gel solidification, add EMEOM medium (500 μl/well) with Y-27632 (10 μM) for 24–48 h recovery.

3.4. Recovery, Clonal selection, and amplification (Timing: 1–3 weeks)

1. Culture for 5–10 days to allow reporter expression (Note 5).

   Frame selector makers can be an indicator of successful transfection at first. However, it gradually disappears unless it is stably incorporated into the genome.

2. For manual selection, expansion of organoids is required. Expand cells until the 3^rd passaging point and select the positive organoid clones (Fig. 2) (Note 6).

3. Transfer all the organoids to a round-bottom 96-well plate with a dilution to have 10–20 organoids in each well.

4. Using a fluorescence microscope, mark wells with positive organoids and transfer them to a new EP tube.

5. Seed positive organoid with Matrigel and grow in EMEOM medium.

6. Expand candidate clones for genotyping and banking (Note 7).

Figure 2.

Fluorescent images of the Organoid after transfection. (A) BFP and mCherry from Frame selector are visible at day 4 of transfection and the next passage.

3.5. Second round of electroporation and clone isolation (Timing: 1–3 weeks)

1. Conduct the same experimental procedures with CRISPaint-TagmNEON-PuroR in the Krt13-BFP organoids established from Steps 3.2–3.4.

2. Prepare three samples from a single established and amplified organoid line to conduct electroporation. Electroporation conditions are the same as those used previously (Step 3.3.4).

   Sample	Frame selector +0	Frame selector +1	Frame selector +2	CRISPaint-TagmNEON-PuroR	pSPgRNA-Krt13
   1	3 μg			3 μg	4 μg
   2		3 μg		3 μg	4 μg
   3			3 μg	3 μg	4 μg

3. Select the double-positive organoids manually (Fig. 3) (Note 8, 9).

4. Expand candidate clones for genotyping and banking (Note 10).

Figure 3.

Fluorescent images of Krt13-BFP; Sox2-mNEON organoids. (A) Fluorescence signals of double-positive (BFP and mNEON) organoids at different days after transfection (top panels) and the next passage with selected organoids (bottom panels).

4. Notes

1. Safety: Use appropriate containment for gene editing; follow local IBC/IACUC requirements for mouse-derived tissues.

2. Check off-targets in the mouse genome; prefer high on-target scores with minimal predicted off-targets. Ensure that the PAM sequence is not included in the designed oligos.

3. Approximately 8 wells of a 48-well plate are sufficient for a single electroporation.

4. As few as 1 × 10⁴ cells/reaction if cells are not enough. However, the chances of finding transfected cells will be decreased.

5. Organoids develop differentiated cell layers at a later stage of growth. Therefore, selecting Krt13-BFP+ organoids can be available at later days (after day 7) of each passage.

6. For manual selection, do not dissociate organoids using Trypsin. Alternatively, FACs-based sorting can be used depending on the characteristics of genes. In this protocol, organoids were selected manually since the BFP signal is more visible in mature organoids than in single cells.

7. Do not pool multiple organoids for amplification. Rather than that, make multiple clones. Bulk-edited pools are heterogeneous, which complicates their interpretation.

8. Overall, organoid growth can be decreased after electroporation. The organoids can be damaged after selection due to the process of serial dilution and manual picking (Fig. 3, Top right panel). Viability and the organoid growth are enhanced from the next passage (Fig. 3, bottom panel). It may require additional passages.

9. Signal expectations: Fluorescence-based selection can be limited by the reporter genes used. In this protocol, Krt13 and Sox2 are clearly visible and distinguishable because they are known to be expressed in differentiated and basal cells, respectively. Additional validation might be required if genes and their expression are not well described previously.

10. Archiving: Cryobank early passages of each verified clone and maintain a record of sequence-validated junctions.

Figure 1.

sgRNA design for knock-in. (A) Design of sgRNA targeting sites in Krt13 and Sox2 genes. (B) Oligomers for cloning into the pSPgRNA plasmid. The BbsI enzyme cut-overhang is considered in this protocol. PAM sequences are not included in the oligomer.

At-a-Glance Timeline.

• Design & cloning: 1–2 weeks

• Organoid culture and electroporation: 1 week

• Selection of clones and amplification: 4–5 weeks (for one reporter)