Since the advent of early gene editing tools like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), a more versatile and efficient generation of genome modification technologies has emerged—represented by what is now known as the Targeted Gene Editing-Pro system. This system has rapidly gained popularity among researchers as a preferred tool for precise genomic modifications.
The technology originated from a defense mechanism discovered in prokaryotic organisms, which helps them recognize and neutralize invading genetic elements. This mechanism was first characterized by scientists including Emmanuelle Charpentier and Jennifer Doudna. Later, Feng Zhang and colleagues were among the first to adapt this system for genome editing in mammalian cells.
Within the Targeted Gene Editing workflow, the system employs programmable guide components and an associated endonuclease to introduce targeted modifications at specific genomic loci, enabling efficient and customizable gene editing for a wide range of research applications.
When establishing a Targeted Gene Editing-Pro-based knockout (KO) cell line model, it is important to validate the genetic modifications which have occurred. Herein, we cover how to validate gene edited knockout (KO) cell line models developed using Targeted Gene Editing-Pro-based methods.
The Targeted Gene Editing-Pro system is composed of two essential components: a programmable endonuclease and a synthetic RNA-based guide molecule. In the initial stage of the editing process, a single guide molecule (sGuide Molecule) forms a complex with the editing enzyme to enable sequence-specific targeting. In the subsequent stage, a transcribed RNA component—analogous to the crRNA—assembles into a precursor structure that directs the enzyme to cleave the same genomic region as the sGuide Molecule.
Following this targeted cleavage, the cellular DNA repair pathway—specifically, the error-prone non-homologous end joining (NHEJ) mechanism—rejoins the broken DNA ends, often introducing small insertions or deletions (indels) that disrupt the gene’s function, thereby achieving gene knockout.
Strategies for Targeted Gene Editing-Pro Gene Knockout
There are two main strategies for using Targeted Gene Editing-Pro to achieve gene knockout, described below.
The first strategy is to knockout most of the exons for the functional domain that encodes the target gene to render it inactive. For this knockout strategy, the knockout effect can be guaranteed after the knockout, and there will be no protein residue problem, but there are some disadvantages. If the target gene fragment is too long, the difficulty of knockout will be increased. In addition, knockout fragments will dissociate in the cell and may be randomly inserted into other positions in the genome in the form of non-homologous end joining (NHEJ), which will reduce the positive rate of knockout.
The second is to knockout individual exons to cause a frameshift mutation, which achieves the purpose of target gene knockout. This knockout strategy is easier to obtain homozygous knockout cell lines at the DNA level. However, this knockout strategy requires the design of sGuide Molecule at the appropriate position. Otherwise, it will cause both frameshift and open reading frame (ORF) changes at the DNA level, but protein expression may still be found when using western Blot (WB) to detect the protein product of the targeted gene.
In addition, there are some unusual strategies to achieve gene knockout, such as introducing a donor vector. By introducing a homologous recombination (HR) vector, a stop codon is introduced into the HR vector to terminate gene translation. Another strategy is to introduce a selection marker into the HR vector, which inserts the selection marker into the coding region of the target gene, and destroys the function of the target gene.
Once a Targeted Gene Editing knockout (KO) cell line has been established, adequate validation of the specific gene edits is important. To verify whether the knockout cell line is successfully constructed, the first step is to confirm its genomic levels. Fragment knockout has the following identification strategies (shown in Figure 2). Design primers to amplify three regions by PCR, which are the region end caps (Region 1 and Region 2) and the knockout region (Region 3) of the two Guide Molecules. If a DNA band cannot be amplified in the region affected by Guide Molecule and the region 3 band that is amplified in the complete knockout region is smaller than the wild-type, it means that the cell line has been knocked out at the genomic level.
The sGuide Molecule knockout method usually produces small insertions and deletions (INDELs). When the number of generated indels is not a multiple of 3, it will cause frameshift mutation and knockout. As agarose gel only distinguishes DNA fragments with a difference of 100bp or more, it is unable to verify whether indels are generated. Therefore, it is necessary to continue to sequence and identify the amplified products. The sequencing results are compared with the wild-type sequence. If indels other than multiples of 3 occur, it means that the cell line has been knocked out at the genomic level.
In addition to verifying the genomic level of frameshift mutations, Western Blot (WB) testing can be used to confirm the effect of knockout. As shown in Figure 5, there was no expression of the target protein in the knockout cell line, indicating that the knockout cell line was constructed. Based on the experience of many cases in Cyagen, as long as a variety of factors are considered comprehensively in the planning stages of the study scheme and the sGuide Molecule is reasonably designed, ideal experimental results can be obtained whether using frameshift knockout or fragment knockout methods.
When carrying out knockout experiments, it is necessary to design a plan according to the actual situation. Factors such as the target gene, cell type, and downstream experiments will affect the design of the protocol and the final result. Does the target gene affect cell survival? Does the cell type affect the knockout efficiency? Do different import methods affect downstream experiments? If the knockout gene is related to cell cycle, proliferation, and metabolism, the knockout may be fatal, and it is difficult to obtain knockout homozygous cell lines. It is also difficult to carry out knockout experiments for cell types with slow proliferation, because sluggish cell proliferation affects the cell’s DNA repair activity and reduces editing efficiency.
There are three commonly used methods to introduce the Targeted Gene Editing-Pro system into cells: ribonucleoprotein (RNP) complex delivery, plasmid-based vectors, and viral vector-mediated delivery.
In most cases, the RNP approach is preferred—this involves synthesizing single-stranded guide molecules (sGuide Molecules), which are then incubated with the editing enzyme in vitro to form RNP complexes. These complexes are introduced into cells via electroporation. This method is both time-efficient and highly effective, enabling rapid generation of homozygous knockout monoclonal cell lines without introducing foreign genetic material into the host genome.
In contrast, plasmid and viral vector delivery methods are more suitable for cell lines with slower proliferation. However, viral delivery may result in the integration of exogenous plasmid sequences into the genome. The optimal knockout strategy should be selected based on the requirements of downstream experimental applications.
