ADVANCES IN GENE KNOCKOUT TECHNOLOGY
METHODS
How Do Gene Knockouts Work?
There are a variety of methods that can be used to complete gene knockouts. Listed below are a few of these methods:
In Vivo Homologous Recombination-Mediated Genetic Engineering
Also known as recombineering, in vivo homologous recombination-mediated genetic engineering has been one of the first systems used to conduct traditional gene knockouts in organisms. Recombineering works by introducing a linear DNA into cells that express the phage-encoded recombination enzymes. These enzymes incorporated their linear DNA into the target, yielding recombinant molecules. This method can be used to complete gene knockouts without the use of a drug marker or other alterations. A selection cassette is used to insert a cassette a resistance marker, and then the cassette is removed using a counter-selection, usually resulting in a clean gene deletion. (5.2)
In Vivo Homologous Recombination-Mediated Genetic Engineering
Also known as recombineering, in vivo homologous recombination-mediated genetic engineering has been one of the first systems used to conduct traditional gene knockouts in organisms. Recombineering works by introducing a linear DNA into cells that express the phage-encoded recombination enzymes. These enzymes incorporated their linear DNA into the target, yielding recombinant molecules. This method can be used to complete gene knockouts without the use of a drug marker or other alterations. A selection cassette is used to insert a cassette a resistance marker, and then the cassette is removed using a counter-selection, usually resulting in a clean gene deletion. (5.2)
Transposon Mutagenesis
"Transposon mutagenesis is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting the function of an extant gene on the chromosome." In this method, a genomic fragment of interest is cloned into a vector that contains unique enzyme sites. First, the enzyme sites at the genomic fragment of interest are destroyed. Once they are destroyed, a transposition will lead the random integration of the transposon into a genetic clone. The genomic fragments that contain enzyme sites will destroy the sites of interest in the genomic DNA after being cloned, which will complete the gene knockout. (5.3)
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)
"CRISPRs are DNA loci containing short repetitions of base sequences that are present within prokaryotes and function as a primitive immune system cleaving foreign DNA (from invading viruses)." When CRISPRs are paired with Cas9, an RNA-guided DNA endonuclease enzyme, they can cleave genomic DNA at a specific site for knocking out gene expression. Since guide RNAs are assigned to specific genomic sequences, they will direct Cas9 to knockout certain genes. (5.1)
Watch this video to learn more about how gene knockout is conducted using CRISPR technology.