The Essential Principles of Constructing Knock-In Cell Lines and Key Details to Remember

Knock-in (KI) cell lines are crucial tools in biomedical research, predominantly used to model diseases, perform gene function analyses, and develop therapeutic strategies. Their construction involves integrating a specific allele or genetic alteration into a particular locus on the cell's chromosome. Creative Biogene, as a leader in cell engineering, provides reliable, affordable, and fast knock-in cell line generation service, which includes point mutation cell line generation and reporter genes knock-in cell line generation. Here, we delve into the principles governing the construction of knock-in cell lines and the details researchers should be aware of.

Understanding Knock-In Cell Lines

Unlike knockouts, where a gene of interest is wholly removed, knock-ins allow the introduction of an alteration into a gene. The alteration could be a point mutation, a large DNA sequence, a reporter tag, or an entirely different gene. The products of these modified genes can reveal a wealth of information about gene function, interactions, and disease mechanisms, serving as invaluable resources in preclinical research, pharmaceutical development, and many other scientific endeavors.

Principles of CRISPR-Cas9 Knock-in

Under the guidance of sgRNA, Cas9 endonuclease cuts the DNA double strand in a targeted manner and creates a DNA nick, and then the organism initiates the homologous recombination repair pathway, and when a highly homologous section of DNA template (Donor DNA) exists, it will repair the DNA template using Donor DNA as a template to achieve the purpose of introducing the target fragment into the genome in a targeted manner. In this process, the cutting efficiency of Cas9, the probability of repairing cellular double-stranded DNA breaks by homologous recombination, and the probability of recombination with Donor template together affect the final gene editing efficiency. This is the reason for the low rate of positive clones obtained.

Fig. 1 CRISPR-Cas9 knock-in principle.

Key Details of Constructing Knock-In Cell Lines

It is not easy to obtain a pure KI cell line. Because the success of KI cell line construction is affected by many factors, such as the length of the knock-in gene fragment, homologous recombination efficiency, cell line type, etc. can affect the editing efficiency, so this makes the technical difficulty of KI high. In the following, we will introduce the principle of constructing KI cell lines and the details that need to be paid attention to.

Design of sgRNA

Knock-in cell lines creation using the guide RNA (sgRNA) involves the introduction of specific genetic modifications into target positions in a genome. This is achieved using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 system, where sgRNA guides the Cas9 nuclease to specific genomic loci. Below are some crucial design steps to consider during their construction.

sgRNA Design and Selection: The design of the sgRNA is critical to ensure it targets the correct location on the genomic DNA. Online software is available to help with design and reduce off-target effects.
Donor DNA design: The construction of a knock-in cell line requires the development of a donor DNA construct that matches the sequence around the sgRNA cut site but includes the desired modification. This typically includes homology arms on either side of the insertion sequence.
Verification of sgRNA Activity: Before you start the knock-in procedure, it's crucial to verify the activity of the designed sgRNA using a test system.

Choice of RNP and plasmid methods

The presence of Cas9 protein and sgRNA in the cell is an important step to realize gene knock-in. Commonly used methods to express Cas9 protein and sgRNA include ribonucleoprotein (RNP) and plasmid method. These two methods have their own advantages and disadvantages. The plasmid method is relatively simple, short cycle and low cost, but it cannot stably express Cas9 protein. The RNP method has low off-target probability, no DNA integration risk and promoter compatibility issues, and the cutting efficiency test can be completed in 5-6 days, which significantly shortens the experimental cycle. Gene editing occurs rapidly after 12-24h of transfection, and plasmid vectors generally take 72h, so the RNP method has higher efficiency than plasmid expression vectors. And it does not introduce redundant expression elements or consequent cytotoxicity.

Design of Donor templates

Donor templates have an important impact on the efficiency of homologous recombination. The following factors need to be considered during the experiment:

Distance of the insertion site from Double Strand Breaks (DSBs).
Length of the knock-in fragment.
Length of homology arm.
Selection of homology arm template.

Cell type and growth status

Before transient transfer, attention should be paid to the appropriateness of the cell type, such as focusing on the proliferation rate of the cells; cells that proliferate too slowly also undergo recombination inefficiently. It is also important to ensure that the cells are in a good growth state, in the logarithmic growth phase, at a suitable growth density, and free of mycoplasma contamination.

Transient voltage

The appropriate transient voltage can affect the editing efficiency and cell survival; too low a voltage will affect the editing efficiency and too high a voltage will cause cell death. Therefore, it is necessary to find out the appropriate transient voltage for different cell types. Generally speaking, the transient voltage is related to the diameter of the cell, and the larger the diameter, the lower the voltage. It is recommended to choose the optimal transient voltage by combining with the pre-experiment.

* For research use only. Not intended for any clinical use.

Quick Inquiry

The Essential Principles of Constructing Knock-In Cell Lines and Key Details to Remember

Understanding Knock-In Cell Lines

Principles of CRISPR-Cas9 Knock-in

Key Details of Constructing Knock-In Cell Lines

Related Products and Services at Creative Biogene