Knock-In Cell Line
Gene knock-in (KI) is a precise gene editing technique used to insert exogenous gene sequences into specific sites of the genome, allowing stable expression in cells.
▍ Technical Principle:
DNA double-strand break (DSB) is the key step in gene knock-in. This break triggers two main DNA repair pathways in cells: non-homologous end joining (NHEJ) and homology-directed repair (HDR).
The core of the knock-in process involves using a gRNA or crRNA to guide the Cas enzyme to cut the DNA at a target site. The cell then uses an externally provided donor template to repair the break. Depending on the mechanism—HDR or NHEJ—the desired gene sequence is precisely inserted into the genome.

Service | Efficiency | Advantages | Applicable Scenarios |
---|---|---|---|
CRISPR/HDR | 5-30% | Highly versatile – ideal for introducing point mutations or inserting DNA fragments | Actively dividing cells |
CRISPaint | 20-50% | Works independently of the cell cycle | Cells with slow or infrequent division |
Bingo™ | 30-80% | No need for double-strand breaks or donor templates – low off-target risk | Precise small-fragment knock-ins or single-base corrections |
HES-KI | 40%-90% | Built-in screening system ensures efficient knock-in | C-terminal tagging at essential gene loci |
● Industrial Production: Delivers high stability and uniformity of target gene expression, ensuring consistent yield and quality in large-scale manufacturing. ● Gene Therapy: Enables stable integration of therapeutic genes in cell-based treatments. ● Stem Cell Engineering: Achieves efficient transgene knock-in in iPSCs. ● Functional Genomics: Facilitates screening of essential gene domains and regulatory elements. ● Disease Modeling: Generates genetically modified cell lines that reflect physiologically relevant expression levels.
Service Details
Cell Types |
Tumor cells, stem cells, and various other lines. Click to view the full cell line list. |
---|---|
Service Options | Targeted knock-in of fluorescent proteins, tag proteins, or at custom loci |
Delivery Standard | One monoclonal stable cell line (2 cryovials, 1×10⁶ cells per vial) |
Turnaround/Price |
|
HES-KI Technology
HES-KI Technology Overview
HES-KI (Hotspot-Exon-Selection Knock-In) is a novel knock-in strategy targeting the C-terminal exon region of essential genes using CRISPR editing. A custom knock-in template is designed so that successful integration restores the function of the essential gene while incorporating the desired transgene. In contrast, cells with failed knock-in—such as those with indels caused by NHEJ—lose essential gene function and cannot survive. This enables effective enrichment of correctly edited cells through a built-in positive selection mechanism.
HES-KI Advantages
High Efficiency
Stable and Uniform Expression
Multiplex Gene Integration
Tunable Expression
HES-KI Case Study

• Using the HES-KI technology, EGFP was successfully knocked into the GAPDH locus in K562 cells. Without any antibiotic selection, the polyclonal knock-in efficiency reached 37%, demonstrating the high efficiency of the platform even under non-selective conditions.

• No significant difference in doubling time was observed between EGFP-KI K562 clones and wild-type K562 clones, indicating that EGFP insertion does not affect cell growth.



K562 EGFP-KI polyclonal cells | |
![]() |
![]() |
K562 EGFP-KI monoclonal cells | |
![]() |
![]() |

• Using the HES-KI technique, the knock-in efficiency of EGFP reached 68% in 293T polyclonal cells and 55% in CHO-K1 polyclonal cells.

• Images of 293T and CHO-K1 EGFP-KI polyclonal cells
293T | CHO-K1 |
![]() |
![]() |
![]() |
![]() |
CRISPaint Gene Knock-in
CRISPaint Technology Overview
CRISPaint is a gene knock-in technique based on the CRISPR-Cas9 system. It leverages the cell's own non-homologous end joining (NHEJ) repair mechanism to insert exogenous DNA fragments at the specified C-terminal position of a target gene, without relying on homologous recombination (HDR). The CRISPaint design is highly flexible, making it suitable for various gene function studies and genome editing applications.Core Technical Principles
1. CRISPR-Cas9-Mediated Double-Strand Break (DSB)
The CRISPR-Cas9 system induces a double-strand break at the target genomic location, activating the cell's DNA repair mechanisms.
2. Universal Donor Vector Design
CRISPaintCRISPaint uses a universal donor DNA template to implement a flexible knock-in strategy through three key components:
Target sgRNA plasmid: Cuts at the specific location in the gene.
Frame sgRNA plasmid: Cuts the donor plasmid to linearize it within the cell. Three types of frame sgRNAs are available to ensure correct open reading frame (ORF) expression of the inserted gene.
Universal Donor plasmid: Contains the gene sequence to be inserted (e.g., a fluorescent protein) along with the target sequences for the three types of Donor sgRNAs.
3. Non-Homologous End Joining (NHEJ)
The target gene forms a double-strand break (DSB), and the donor is cut. The cell uses the NHEJ repair mechanism to integrate the linearized donor template into the DSB site, enabling efficient gene knock-in.

CRISPaint Technical Advantages
High Efficiency
Wide Applicability
Flexibility
CRISPaint Experimental Workflow
CRISPaint Case Study


• Sanger sequencing confirmed the precise insertion of the Neo gene at the C-terminus of gene A in H9 cells.

Bingo™-Mediated Gene Knock-In
Bingo™ Technology Overview
Prime Editing (PE) is a gene editing technique designed for precise insertion of small DNA fragment, without introducing double-strand breaks (DSBs). The method uses a Cas9 nickase fused to a reverse transcriptase (Cas9n-RT), guided by a prime editing guide RNA (pegRNA) to the target site. Cas9n creates a single-strand nick, and the reverse transcriptase uses the pegRNA template to synthesize the desired DNA sequence at that site, achieving accurate gene insertion.
Bingo™ Technical Advantages
Safer
Highly precise
Versatile
Bingo™ Experimental Workflow
Bingo™ Case Study
Cell Name | HCT116 |
---|---|
Gene | EGFR(Gene ID: 1956) |
Mutation Site | c.2319_2320 ins CAC |
SpacerGCCCAGCAGGCGGCACACGTG
Bingo™ pegRNA | Spacer | GCCCAGCAGGCGGCACACGTG |
---|---|---|
Extension | GTGGACAACCCCCACcacGTGTGCCGC | |
Bingo™ gRNA | GCACcacGTGTGCCGCCTGCT |
• Polyclonal sequencing results of EGFR gene point mutation (c.2319_2320 ins CAC) in HCT116 cell pool.


CRISPR/HDR Gene Knock-In
CRISPR/HDR Technology Overview
CRISPR/HDR is a gene editing approach that integrates foreign DNA through the CRISPR system and the cell’s homology-directed repair (HDR) pathway. The sgRNA guides Cas9 to a specific target site, where it introduces a double-strand break (DSB). A donor DNA template containing homology arms is then precisely inserted at the break site through HDR, enabling accurate gene knock-in.
CRISPR/HDR Technical Advantages
Highly flexible
Highly accurate
CRISPR/HDR Workflow
Advantage and Characteristic

Optimazied Strategy

Optimazied Strategy

Optimazied Strategy

Optimazied Strategy
Reference Materials
Enhancing CRISPR-mediated homology-directed repair (HDR) efficiency through cell cycle synchronization
This study explores a method to enhance CRISPR-mediated HDR efficiency by synchronizing the cell cycle. Using small molecules to modulate the cell cycle, researchers achieved a 1.2- to 1.5-fold increase in knock-in efficiency across various cell lines. The study also demonstrated this approach's application in animal embryos, significantly increasing knock-in frequency in pig embryos. This technique improves knock-in success by guiding cells to an HDR-favorable cycle stage, offering a new optimization strategy for CRISPR gene editing.
Knock-in strategy based on CLASH technology
The CLASH (Cas9-Linked Adaptor Synthesis for Homology-directed repair) technology enables efficient large-scale gene knock-in for cell engineering. This method combines the Cas9 protein and adaptor synthesis, allowing parallel knock-in across various cell types. By providing specific adaptors during the DNA repair process, it significantly enhances homology-directed repair (HDR) efficiency, thereby increasing knock-in success rates. This technology shows great potential in cell engineering and gene editing, especially for complex bioengineering applications requiring multi-gene modifications.