The rapid evolution of genome editing technologies is reshaping the landscape of life science research, and among all the available tools, CRISPR/Cas9 has undoubtedly emerged as the most transformative technology of the past decade. Compared with gene knockout, gene knock-in resembles a highly delicate “genomic surgery”，it requires not only an accurate cut but also a precisely executed integration. By inserting exogenous sequences into predetermined genomic loci, researchers can visualize endogenous proteins, construct disease-related mutation models, develop drug-screening systems, or establish stable cell line models expressing recombinant genes. These advances provide essential support for basic research, drug discovery, and even gene therapy.

At the core of the knock-in process lies the cell's intrinsic homology-directed repair (HDR) pathway. Following a CRISPR-induced double-strand break, cells must choose between two repair mechanisms: non-homologous end joining (NHEJ), which is fast but error-prone, and HDR, which is precise but inherently less efficient. For knock-in applications, HDR is the pathway of interest. With a donor template containing homologous arms, cells can seamlessly integrate the desired sequence into the genome, completing an accurate editing event.

In the following sections, Ubigene will guide you through the entire workflow of gene knock-in cell line construction—from fundamental principles to practical considerations, from optimization strategies to the unique strengths of Ubigene's technology—offering a complete, end-to-end overview of the knock-in process.

CRISPR/Cas9: The Logic Behind Precision Knock-In

Every knock-in cell line project begins with a well-designed single-guide RNA (sgRNA). Acting as a molecular “navigator,” the sgRNA directs Cas9 to a unique genomic locus, where Cas9 introduces a precise double-strand break. Once the break occurs, the donor template enters the process, providing the new DNA fragment intended for integration. Whether a knock-in succeeds depends on numerous variables—cell cycle phase, sgRNA design quality, the structure of the homology arms, transfection performance, donor format, and even the physiological state of the cells. Because so many factors intersect, knock-in experiments often evoke a mix of anticipation and apprehension among researchers.

At the heart of knock-in lies homology-directed repair (HDR), a pathway that operates most efficiently during the S and G2 phases of the cell cycle. Naturally, researchers have explored ways to keep cells within these HDR-permissive phases long enough to maximize precise repair. This is where Ubigene's EZ-HRex™ technology comes into play. By modulating cell-cycle distribution and suppressing the NHEJ pathway, EZ-HRex™ places HDR in an optimized working state, significantly enhancing homologous recombination efficiency. Using this strategy, HDR-positive genotypes can reach up to 84% within post-transfection cell populations.

In practical application, a suitable sgRNA must first be designed for the intended locus, together with a donor template that carries the desired insertion flanked by homology arms. Effective sgRNA design requires the presence of an appropriate PAM sequence near the target site (e.g., NGG for SpCas9), while minimizing similarity to other genomic regions to reduce potential off-target effects. Donor templates typically consist of double-stranded DNA containing left and right homology arms (commonly 500–1000 bp) and the insert sequence, such as a reporter gene, epitope tag, or defined mutation. In certain approaches—including long single-stranded DNA donor strategies (e.g., Easi-CRISPR)—ssDNA donors may help improve integration efficiency. In addition, alternative delivery formats, such as RNP complexes or Cas9 mRNA, can further support genome editing performance.

A Complete Workflow for Generating Gene Knock-In Cell Lines

To make the complex process easier to understand, the entire experiment is divided into two phases: the pre-experiment exploratory stage and the formal knock-in experiment.

1.Pre-Experiment: Four Key Explorations — The “Foundational Steps” Determining Project Success

(1).Antibiotic Selection Optimization
Identify the Minimal Lethal Concentration: Determine the lowest concentration of the antibiotic corresponding to your vector's resistance marker that can completely kill non-transfected cells within 2-3 days. This step is critical: if the antibiotic pressure is too low, false positives may occur; if it is too high, genuine positive clones may also be lost. The following detailed protocol may serve as a reference:

• Seed cells: Digest cells in the logarithmic growth phase to obtain a single-cell suspension. Seed cells into a 12-well plate with a total culture volume of 1 mL per well. Incubate at 37°C, 5% CO₂ for 24 hours.
• Apply antibiotic gradient: Once cells reach approximately 50% confluency, replace the medium with selection medium containing a gradient of puromycin concentrations: 0, 1, 1.5, 2, 3, and 4 μg/mL. (If the CRISPR-U™ expression plasmid carries a different antibiotic resistance gene, select the corresponding antibiotic accordingly.)
• Determine the minimal lethal dose: After 2-3 days, examine the cells under a microscope and identify the lowest concentration that results in complete cell death. This value will be used as the antibiotic selection concentration for subsequent experiments.

(2).Single-Clone Formation Assessment: Determine Whether Your Cells Can "Live Independently"
Different cell lines vary greatly in their ability to survive and proliferate as single cells: some can grow into colonies from a single cell, while others require clustering to thrive. Performing a limiting dilution assay in a 96-well plate allows you to calculate the cloning efficiency and optimize your subsequent cloning strategy. The following protocol may serve as a reference:

• Prepare single-cell suspension and count cells: Digest cells in the logarithmic growth phase to generate a single-cell suspension, then perform cell counting.
• Seed cells into 96-well plates: Dilute the suspension to the desired cell density and seed into a 96-well plate, with 100 μL of medium per well. Incubate at 37°C, 5% CO₂ under static culture conditions.
• Assess colony formation: After 7-10 days, examine the wells under a microscope to determine whether cells have proliferated and formed clusters. Record the number of single-cell-derived clones for each condition.

(3).Transfection Optimization: Identify the Most Compatible Electroporation Parameters
Digest logarithmic-phase cells into a single-cell suspension and test multiple electroporation programs. Evaluate transfection efficiency 24 hours post-transfection and document with images. Select the electroporation parameters that provide a balance of high transfection efficiency and cell viability for the formal knock-in experiment.

(4).Target Sequencing: Ensure the “Scissors” Cut in the Right Place
Before knock-in, it is essential to verify that the sgRNA targets the correct genomic locus. The workflow involves PCR amplification of the target region → sequencing → alignment with theoretical sequence. The following detailed procedure may serve as a reference:

• Genomic DNA extraction: Extract genomic DNA from blood, cells, or tissue using an appropriate genomic DNA extraction kit.
• PCR amplification of the target locus: Amplify the targeted region by PCR using the following reaction setup:

  Reagent	Volume (μL)
  2 * Taq Master Mix	25
  Forward Primer（10μM）	1
  Reverse Primer（10μM）	1
  ddH2O	22
  Template	1
  Total	50

• Agarose Gel Electrophoresis of PCR Products: Run the PCR products on an agarose gel. If the band size matches the expected size, proceed with Sanger sequencing of the PCR products.
• Sequence Verification: Align the sequencing results with the theoretical target sequence to confirm the accuracy of the gRNA target site.

Formal Experiment: Complete Workflow from Electroporation to Positive Clone Screening

(1)Electroporation of Target Cells: The Critical Starting Point of the Knock-In Experiment

• Prepare single-cell suspension and assess viability: Digest cells in the logarithmic growth phase into a single-cell suspension. Take an aliquot to determine cell count and viability.
• Centrifuge cells: Collect 5 * 10⁵ to 1 * 10⁶ cells in a sterile tube and centrifuge at 300 * g for 3 minutes.
• Resuspend cells and add plasmids: Discard the supernatant and resuspend the cell pellet in 100 μL of Buffer R. Add 5 μg of endotoxin-free CRISPR-U™ expression plasmid along with 5 μg of donor vector and mix gently.
• Prepare electroporation cuvette: Add 3 mL of Buffer E2 to the electroporation cuvette and place it into the electroporator slot.
• Load and electroporate cells: Use a 100 μL electroporation pipette tip to transfer the cell–plasmid mixture into the cuvette. Set the electroporation parameters and deliver the pulse.
• Post-electroporation culture: After electroporation, seed the cells into 6-well plates containing pre-warmed culture medium and incubate under standard conditions.
• Repeat for all samples: Repeat the above steps until all samples have been electroporated.

(2)Cell Pool Selection and Single-Clone Isolation
The cell pool provides an early indication of transfection success but cannot serve as the final assessment.

• Antibiotic selection: 24–72 hours post-transfection, examine cells under a microscope to assess electroporation efficiency and apply antibiotic selection.
• Limiting dilution for single-clone formation: Take an aliquot of cells, perform limiting dilution, and seed into a 96-well plate. Incubate at 37°C, 5% CO₂ under static culture conditions.

  Reagent	Volume (μL)
  2 * Taq Master Mix	25
  Forward Primer（10μM）	1
  Reverse Primer（10μM）	1
  ddH2O	22
  Template	1
  Total	50

  
  The PCR program is as follows:

  STEP 1	95℃	3min	Initial Denaturation
  STEP 2	95℃	15s	Cycling (30 cycles)
  	60℃	15s
  	72℃	1kb/min
  STEP 3	72℃	5min
  	4℃	Forever

• Sequencing of PCR Products: Perform sequencing of the PCR products. For sequences near the gRNA target site, positive samples often show overlapping peaks (mixed signals) at and downstream of the target site. However, if editing efficiency is low, these overlapping peaks may be subtle, making interpretation difficult. The editing efficiency observed in the cell pool can serve as a reference but does not fully reflect the actual outcome at the single-clone level.
• Seeding for clone growth: Seed cells into a 96-well plate and culture for approximately one week. Monitor clone growth and mark wells containing single-cell-derived colonies.
• Expansion of single clones: After 2-4 weeks, digest the expanded single clones and split each well into two 96-well plates for continued culture.
• Preparation for clone verification: When cell confluency reaches >60%, remove one plate for single-clone verification and genotyping.

(3)Single-Clone Screening and Verification: Identifying True "Gene Knock-In Cells"

• Cell lysis for nucleic acid release: Remove the culture medium from the 96-well plate and add 100 μL of Ubigene Rapid Nucleic Acid Release Reagent per well. Incubate at room temperature for 10 minutes.
• Centrifugation: Centrifuge at 3000 rpm for 5 minutes. The nucleic acids from the cells will be released into the supernatant.
• PCR amplification of the knock-in region: Use the supernatant as a template for PCR to amplify the target (knock-in) region. The PCR reaction setup is as follows:

  Reagent	Volume (μL)
  2 * Taq Master Mix	25
  Forward Primer（10μM）	1
  Reverse Primer（10μM）	1
  ddH2O	22
  Template	1
  Total	50

  
  The PCR program is as follows:

  STEP 1	95℃	3min	Initial Denaturation
  STEP 2	95℃	15s	Cycling (30 cycles)
  	60℃	15s
  	72℃	1kb/min
  STEP 3	72℃	5min
  	4℃	Forever

• Sequencing of PCR Products and Analysis of Knock-In Results:
  Case 1: If the sequencing results show no overlapping peaks and match the wild-type sequence, the clone is considered unmodified.
  Case 2: If the sequencing results show no overlapping peaks and match the intended knock-in sequence, the clone is considered a successful knock-in, with the same modification in all alleles.
  Case 3: If the sequencing results show overlapping peaks, the clone may be heterozygous or contain alleles with different indel patterns.

(4)Expansion and Cryopreservation of Positive Clones
For clones verified as successfully knocked-in, continue passaging to expand the cell population. Once a sufficient number of cells is obtained, cryopreserve aliquots as a backup.

Six Key Factors: “Secret Regulators” Determining Knock-In Efficiency

Gene knock-in efficiency is influenced by multiple factors, each of which can be optimized to improve outcomes:

1.HDR vs. NHEJ Repair Competition

Homology-directed repair (HDR) primarily occurs during the S/G2 phase of the cell cycle, whereas non-homologous end joining (NHEJ) can operate throughout the cell cycle. To increase HDR frequency, cell cycle synchronization is commonly employed. For example, arresting cells in the G2/M phase with specific drugs can improve precise knock-in efficiency by 3–6-fold. Concurrently, using NHEJ inhibitors (e.g., SCR7) can further enhance HDR prevalence. The CRISPR-U™ platform from Ubigene achieves higher gene cleavage and homologous recombination efficiencies, resulting in 10–20-fold improvements in genome editing efficiency.

2.Quality of sgRNA Design

Choosing an appropriate target site and a highly specific sgRNA is critical. Prefer targets with a strong PAM sequence (NGG) and unique 20-nt target sites within the genome to minimize off-target effects. Online tools can be used to evaluate potential off-target risks.

3.Donor Template Design

The length of homology arms and the form of the donor template (double-stranded DNA vs. long single-stranded DNA) significantly affect integration efficiency. Homology arms are generally recommended to be several hundred to ~1,000 bp to guide repair effectively. For small insertions, ssDNA donors can improve efficiency. Embedding selectable markers (e.g., fluorescent proteins or antibiotic resistance genes) within the donor template can facilitate rapid identification of knock-in events.

4.Transfection Efficiency

Optimizing transfection conditions—such as electroporation parameters or reagent ratios—and ensuring optimal cell health can greatly influence knock-in outcomes. Use high-quality, endotoxin-free plasmids or reagents and maintain high cell viability. Certain cell lines may require additional growth factors or ROCK inhibitors to promote single-cell survival.

5.Single-Clone Formation Conditions

Most cell lines exhibit low single-cell cloning efficiency (traditional limiting dilution often yields only 0–30%). Optimizing the culture environment can improve survival, for example, by adding extracellular matrix components to the medium, creating a “community-like” environment that supports single-cell growth and increases clonal survival.

6.High-Throughput Genotype Verification

For large numbers of clones, high-throughput sequencing or multiplex PCR can be used for rapid genotyping. Ubigene's genotype analysis tool can quickly interpret sequencing data from transfected cell pools, completing cell pool genotype analysis in under one minute. For low-cell-number samples or single clones, rapid lysis kits allow direct PCR and fast sequencing from minimal DNA.

Summary

Construction of gene knock-in cell lines relies on CRISPR/Cas9-mediated HDR. Success requires comprehensive optimization of target design, donor template construction, transfection conditions, repair pathway modulation, and clone selection. By combining chemical and biological strategies to enhance HDR with meticulous experimental workflow and efficient genotyping, knock-in efficiency can be significantly improved, yielding stable, reliably expressing cell clones.

Ubigene CRISPR-U™ Technology: Not Just Gene Knock-In, but “High-Success-Rate Knock-In”

Ubigene's CRISPR-U™ gene editing platform provides a systematic technological advantage for gene knock-in projects. The platform integrates cellular genomic characteristics for precise target design, incorporates Ubigene’s proprietary gRNA prediction algorithms, and leverages a reference database covering 10,000+ editing cases. This allows systematic evaluation of project difficulty, off-target risks, donor construction strategies, and other critical factors from the design stage, thereby enhancing success rates from the outset.

With CRISPR-U™'s accumulated expertise, Ubigene has executed over 10,000 gene editing projects across 300+ cell lines, with broad applications in basic research, functional genomics, and drug screening model development. In parallel, Ubigene's in-house genotype analysis tool and Monoclone Genotype Validation Kit(#YK-MV-100) enable high-throughput genotyping of cell pools and single clones, significantly shortening the screening timeline and improving positive clone identification efficiency.By integrating algorithms, databases, and experimental systems across the full workflow, Ubigene provides researchers with a one-stop solution for high-efficiency, high-accuracy, and highly stable gene knock-in, supporting the successful execution of complex gene editing projects.

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Reference

Wang.R, Li, WM., lV, YQ. et al. Colorectal Cancer Cells–Derived Exosomal PIK3CA Mutation DNA Promotes Tumor Metastasis by Activating Fibroblast and Affecting Tumor Metastatic Microenvironment. Advanced Science 12,27 (2025). https://doi.org/10.1002/advs.202501792