Gene Knockout: Principles, Methods and Applications

What is the Gene Knockout Technology?

Gene knockout refers to the process of inactivating or disabling specific genes in the genome of an organism. Broadly speaking, gene knockout includes the complete knockout, partial knockout, deletion of gene regulatory sequences, and deletion of segmental genome sequences. Homologous recombination is currently a widely used method for gene knockout, where the knockedout gene is used to observe phenotypic changes in organisms or cells, making it an important tool for studying gene function. With the development of gene knockout technology, in addition to homologous recombination, new principles and technologies are also gradually being applied. Successful examples include gene insertion mutations and RNA interference (RNAi), which can also achieve the purpose of gene knockout.

CRISPR-Cas9 genome editing enzyme for gene knockout.

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What is the Difference between Gene Knockout and Gene Knock-in?

Gene editing is a genetic engineering technology that can alter the genome sequence of an organism, primarily achieved through the use of artificial nucleases to target specific gene sequences in the genome for deletion, insertion, or precise modification. During gene editing, artificial nucleases can recognize and cleave the target DNA sequence, creating double-strand breaks (DSBs) in the DNA. Subsequently, cells undergo timely repair through two main pathways: non-homologous end joining (NHEJ) and homologous directed repair (HDR). In NHEJ-mediated repair of DSBs, cells often insert or delete a few base pairs (indels) at the break site, rapidly joining the DNA, resulting in changes to the gene sequence and loss of function after repair, referred to as gene knockout (KO). On the other hand, during HDR-mediated repair of DSBs, the cell repair system utilizes an additional exogenous fragment that is homologous to the target DNA on both sides of the break as a template. This template is used to synthesize gene sequences complementary to the homologous sequence at the DSB site. Since the exogenous fragment carries the desired mutation or target gene, this process allows for the precise introduction of point mutations or insertion of target genes into the genome, known as gene knock-in (KI).

Gene knockout and gene knock-in are the two fundamental applications of gene editing tools, providing powerful assistance to researchers in studying gene function, understanding genetic diseases, and developing new therapeutic methods. Additionally, researchers continue to develop and refine gene manipulation methods, aiming for significant breakthroughs in medical, agricultural, and biotechnological fields in the future.

Utilizing Homologous Recombination for Gene Knockout

Using homologous recombination for gene knockout remains the most common method for constructing animal models with gene knockout. The basic steps for constructing a gene knockout animal model using homologous recombination are as follows:

(1) Construction of the gene vector

(2) Acquisition of embryonic stem (ES) cells

(3) Homologous recombination, introducing the recombinant vector into ES cells of the same origin for homologous recombination

(4) Selection and screening of targeted cells

(5) Phenotypic studies

(6) Generation of homozygous individuals

Complete Gene Knockout

Complete gene knockout refers to the complete elimination of the activity of a target gene in cells or individual animals through homologous recombination. Complete gene knockout generally uses replacement-type or insertion-type vectors to achieve complete gene knockout in embryonic stem cells based on the principle of positive-negative dual selection. Due to the low natural occurrence rate of homologous recombination in gene transfer, even with the use of dual selection methods, it is difficult to guarantee the screening of embryonic stem cells that have truly undergone homologous recombination from numerous cells at once. Multiple molecular screening techniques such as PCR and Southern hybridization must be used to verify the cell lines in which the target gene has indeed been knocked out.

Conditional Gene Knockout

Conditional gene knockout refers to the specific time and space gene knockout achieved through a site-specific recombination system. There are four types of site-specific recombination systems: the bacteriophage Cre/LoxP system, Gin/Gix system, yeast FLP/FRT system, and R/RS system, among which the Cre/LoxP system is the most widely used site-specific recombination system. The Cre/LoxP system is a recombinase system discovered in bacteriophage P1. By utilizing the mechanism of the recombinase Cre specifically recognizing the LoxP recombination site, the system achieves precise editing or regulation of the target DNA. Cre is a recombinase that recognizes a 34bp-long DNA sequence called LoxP. LoxP consists of two 13bp palindromic sequences with an 8 bp non-palindromic sequence in the middle, giving LoxP its directional property. When two LoxP sequences are oriented in the same direction on the same DNA strand, Cre can excise the DNA segment between the two LoxP sequences and ligate the sequences flanking LoxP; when two LoxP sequences are oriented in opposite directions on the same DNA strand, Cre can cause inversion of the sequence between the LoxP sites; when two LoxP sites are located on different DNA strands or chromosomes, Cre recombinase can mediate exchange or chromosome translocation between the two DNA strands. In conditional gene knockout using the Cre/LoxP system, a positive selection marker gene such as neo is usually placed in the introns of the target gene, and LoxP sites with the same orientation are inserted into the introns flanking important functional domains of the target gene. When it is necessary to inactivate the target gene activity in the experiment, crossing with embryonic stem cells carrying the Cre recombinase gene allows Cre recombinase to excise the DNA segment between the two LoxP sites, resulting in the inactivation of the target gene. Furthermore, conditional gene knockout often utilizes the inducible characteristics of Cre expression promoter activity or Cre enzyme activity. By setting the induction time, the time-space specificity of gene mutations in animals can be artificially controlled, avoiding the problem of stillbirth due to gene mutation while increasing the recombination rate. Common inducible types include: tetracycline-inducible type; interferon-inducible type; hormone-inducible type; adenovirus-mediated type.

Utilizing Random Insertion Mutations for Gene Knockout

Gene Trapping Methodology

Gene trapping methodology is a recently developed innovative approach for gene knockout using random insertional mutagenesis. Typically, gene trapping vectors also include a reporter gene lacking a promoter, usually the neo gene. The neo gene is inserted into the chromosome of ES cells, and ES clones expressing the captured gene can be easily selected in G418-containing medium using the transcriptional regulatory elements of the captured gene. In theory, clones surviving in the selection medium should contain the target gene 100%. Information about the target gene can be obtained through screening marker gene-flanking cDNA or chromosome sequence analysis.

Gene Knockout Induced by RNAi

Due to the ability of a small amount of double-stranded RNA to block gene expression, and because this effect can be passed on to progeny cells, the RNAi reaction process can also be used for gene knockout. In recent years, an increasing number of gene knockouts have adopted RNAi, which is a simpler and more convenient method. After double-stranded RNA enters the cell, it can be cleaved into siRNA by the action of the Dicer enzyme. On the other hand, double-stranded RNA can also induce RNAi through RNA-directed RNA polymerase (RdRP), which synthesizes RNA using RNA as a template. Through this polymerase chain reaction, the intracellular siRNA is greatly increased, significantly enhancing the inhibition of gene expression. SiRNAs ranging from 21 to 23 nucleotides to several hundred nucleotides in length can induce RNAi, but the effect of long double-stranded RNA on blocking gene expression is significantly stronger than that of short double-stranded RNA.

Gene Knockout Methodology Based on Gene Editing Technology

Currently, gene editing primarily involves three methods, including Zinc Finger Nuclease (ZFN) technology, Transcription Activator-Like Effector Nuclease (TALEN) technology, and CRISPR/Cas system-mediated gene editing technology.

• Specific guide RNA (gRNA) is designed to direct the Cas9 nuclease to the specific location of the target gene.
• When the Cas9 endonuclease cleaves the double-stranded DNA, the cell initiates the more advantageous Non-Homologous End Joining (NHEJ) repair pathway. This leads to frame-shift mutations or fragment deletions at the cleavage site, resulting in gene silencing and loss of function, thus achieving gene knockout.

Prospects of Gene Knockout

Although there are still challenges in the practical application of gene knockout technology, its prospects remain very broad. Gene knockout technology plays an important role in life science research. By observing the effects of specific gene deletions, researchers can determine the role of the gene in biological development, physiology, or behavior, thereby deepening understanding of biological processes. Gene knockout technology also provides possibilities for developing new therapies for genetic diseases. By targeting, modifying, or removing defective genes that cause diseases, new avenues for disease treatment can be provided.