HES-KI (Important Gene Locus Knock-in) Technology Platform

HES-KI Technology Platform

HES-KI ensures high-efficiency gene knock-in through a unique selection mechanism

Gene knock-in (KI) technology holds great promise in cell therapy and industrial biomanufacturing. However, conventional knock-in strategies often suffer from low efficiency, limiting their broader application in these fields. Current knock-in methods largely depend on homology-directed repair (HDR), but cells naturally favor non-homologous end joining (NHEJ) to repair double-strand breaks (DSBs), which leads to low integration efficiency. Among the existing platforms, CRISPR/HDR combined with AAV (adeno-associated virus) donor systems is frequently used and relatively efficient. However, AAV donors are prone to tandem insertions into the genome and come with limitations such as high cost, complex production, and limited cargo capacity. These challenges further restrict the widespread application of gene knock-in technologies.
HES-KI is a novel knock-in platform that targets the C-terminal exon region of essential genes for CRISPR editing. A knock-in template is designed such that successful integration restores the function of the essential gene and inserts the desired transgene. In contrast, cells with failed knock-ins—such as those repaired via NHEJ resulting in indels—lose the function of the essential gene and cannot survive. This creates a natural selection pressure that enriches for successfully edited cells.

Design Principle

Technical Advantages

• High efficiency: The unique selection mechanism targeting essential genes enhances knock-in efficiency, avoiding non-specific insertions, with up to 68% knock-in efficiency across various cell types—significantly outperforming traditional methods.
• Stable and uniform expression: The expression of the target gene is highly stable and consistent in successfully knocked-in monoclonal cells.
• Multi-gene integration: Multiple genes can be integrated simultaneously, such as inserting multiple CAR genes to target a variety of tumor antigens, reducing tumor escape.

• Strong regulatory capability: By selecting different essential genes, the expression level of the target gene can be flexibly adjusted. AAV or plasmid donors can be chosen based on experimental requirements.

Workflow

HES-KI System Selection

Different important genes with varying expression levels can be selected as knock-in targets based on experimental needs. The appropriate CRISPR system and donor delivery method can be chosen based on the ease of cell transfection.

Important Gene	CRISPR System	CRISPR Delivery Method	Form of Donor
GAPDH TUBB KIF11 TBP ......	Cas9-sgRNA	Plasmid	Plasmid
GAPDH TUBB KIF11 TBP ......	AsCas12a-crRNA	RNP	AAV

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Case Study

Case 1 AsCas12-mediated HES-KI
Objective: Insertion of EGFP at the C-terminal of the GAPDH gene in K562 cells
Design

Result:
• Using the HES-KI technology, EGFP was inserted into the GAPDH gene in K562 cells, achieving a knock-in efficiency of 37% in multiple clones without the use of resistance selection.

• There was no significant difference in the doubling time between K562 EGFP-KI monoclonal cell lines and WT cells, and the growth of K562 cells was unaffected by the EGFP insertion.

• No significant differences were observed in EGFP mRNA expression levels among different K562 EGFP-KI monoclonal cell lines, indicating good homogeneity in the EGFP-KI monoclonal cells.

• The EGFP mRNA expression remained stable after 15 consecutive passages in the K562 EGFP-KI monoclonal cell lines.

• K562 EGFP-KI cells images

K562 EGFP-KI polyclonal cells

K562 EGFP-KI monoclonal cells

Case 2 Cas9-mediated HES-KI
Objective: Insertion of EGFP at the C-terminal of the GAPDH gene in 293T and CHO-K1 cells
Design:

Result:

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

• Images of 293T and CHO-K1 EGFP-KI polyclonal cells.

293T	CHO-K1

Case 3 HES-KI-mediated multi-gene knock-in in IPSC
Objective: Successful knock-in of GFP and mCherry genes at the C-terminal of the GAPDH gene in IPSC cells using AsCas12a-mediated HES-KI technology.
Design:

Result:

IPSC cells successfully knocked in both GFP and mCherry genes, with high fluorescence intensity and good homogeneity.

Case 4 HES-KI-mediated large fragment knock-in
Objective: Successful knock-in of a 5.2 kb large gene fragment at the C-terminal of the GAPDH gene in IPSC cells using AsCas12a-mediated HES-KI technology.
Design:

Result:

IPSC cells successfully knocked in a 5.2 kb large gene fragment. Flow cytometry analysis showed that the knock-in efficiency for GFP and mCherry genes reached 98.1%, with successful expression of the large gene fragment.