Genetic Engineering Methods

Researchers can remove genes from the genomes of organisms and replicate them in another organism. There are a few strategies for replicating a gene, a unit of heritable material that codes for a particular trait, from one organism to another. The first involves using restriction endonuclease enzymes to cut DNA and insert it into plasmids. A plasmid is a small, circular piece of bacterial DNA that replicates on its own and can be transferred between cells. Each restriction endonuclease recognizes a specific set of nucleotides. Restriction endonuclease enzymes that cut DNA at specific sequences are selected so that the target DNA sequence is flanked by cutting sites. When cut, the desired DNA sequence is separated from the rest of the plasmid. Plasmids are first isolated from bacteria, and then the plasmids and the gene that is being transferred are both cut with the same restriction endonuclease enzymes. When the plasmids and gene are cut with the same restriction endonuclease, they will have matching ends that loosely, but readily, bind together. A DNA repair enzyme permanently binds the two sections of DNA, recreating the plasmid that now contains the gene of interest.

The plasmid is now considered recombinant DNA—DNA created from more than one individual, possibly of a different species. Plasmids created this way are re-inserted into living bacteria. The bacteria are now transgenic organisms, which are organisms containing recombinant DNA from another species. The bacterium then acts as a cloning host, an organism used to replicate a DNA sequence. Each time it replicates, it will create a new copy of the plasmid and pass it on to daughter cells. Each new plasmid in a daughter cell is a DNA clone, an identical copy of a DNA molecule replicated in a population of cloning hosts, and each plasmid clone contains a copy of the original gene. When a plasmid or other DNA construct is used as a carrier of recombinant DNA, it is called a DNA vector. Another method of replicating genes involves removing and copying an expressed gene. When genes are expressed, they are transcribed to single-stranded messenger RNA (mRNA) before being translated to amino acid sequences, which become fully functional proteins. mRNA can be extracted from an organism or its cells. Then, in an initial reaction, reverse transcriptase, an enzyme that uses RNA as a template to make a DNA copy, catalyzes the synthesis of a DNA molecule with a sequence that is complementary to the mRNA template. This DNA molecule, synthesized from the single-stranded RNA template by a reverse transcriptase enzyme, is called complementary DNA (cDNA). The cDNA can be replicated with the polymerase chain reaction, which makes many copies of a target DNA sequence. Alternatively, all the messenger RNA extracted from the cells can be converted to cDNA that is then placed into vectors, which are inserted into cloning hosts.

A collection of cloning hosts that contains complementary DNA of all expressed genes from a cell or organism is a genomic library. Genomic libraries can be used for genome sequencing and genome-wide association studies. Genomic sequencing that uses a library is called top-down or map-based sequencing. With this method, relatively shorter sequences, which are faster and cheaper to sequence, can be assembled into an entire genome using a computer program. Genome-wide association studies use the libraries to identify differences in genetic code that also result in a change in phenotype. This allows scientists to specifically target genes of interest and examine how changes in the genetic code change the resulting phenotype. For example, a genome-wide association study might identify immune system genes that are important for fighting off a microbial pathogen that can then be studied further to understand the mechanisms of their action.

Clustered regularly interspaced short palindromic repeats (CRISPR) are a group of bacterial DNA sequences that is used to defend against bacteriophages, or viruses that infect bacteria. CRISPR was discovered in bacteria and archaea, and the first full described system was found in Streptococcus thermophilus bacteria. In bacteria and archaea, CRISPR serves as a defense mechanism against viral pathogens. When bacteria or archaea with the CRISPR system detect foreign DNA, they save a unique piece of the DNA called a spacer that provides an informational record of viral pathogens that previously infected the cell. Physically, in prokaryotic genomes there are short repeating sequences of DNA that are palindromic—the nucleotide codes are the same regardless of direction. Since DNA is double stranded, palindromic sequences are the same on both strands as "read" by enzymes that move along the DNA strands. Between these repeats is where the spacer entries are saved, providing the basis for the name CRISPR.

When the spacers are transcribed, CRISPR RNA (crRNA) molecules are created and form complexes with DNA cutting enzymes. The next time the cell finds a piece of DNA that complements one of the crRNAs, the enzyme complex cuts through it, effectively disabling the invading pathogen. Several enzymes with variable activities associated with CRISPR have been discovered. Each associated enzyme provides different functionalities to the CRISPR system in nature and provides researchers with additional tools in the lab. An example commonly used in laboratories is the nuclease Cas (CRISPR-associated) enzyme, which cuts through double stranded DNA using the crRNA sequence as a guide.

The CRISPR system can be altered and used by scientists to target and modify specific gene sequences. This allows rapid and precise modification of specific locations in the genome, effectively enabling researchers to edit, or engineer, genes by altering an organism's DNA. New base pairs can be introduced into genes, other base pairs can be deleted, or existing base pairs can be replaced with alternatives. A guide RNA, a single-stranded short RNA molecule used by CRISPR that is complementary to a DNA sequence, directs the precise position of gene editing to maximize the specificity and minimize off-target effects. A nuclease enzyme makes a double-strand cut through the target location. The particular nuclease enzyme used differs among applications, though variations of the CRISPR-associated (Cas) endonuclease are most common. The cell's DNA repair mechanisms are then induced to add, delete, or modify the genes before mending the break. In 2018, CRISPR was used to alter genes in mosquitoes, a common vector of the disease malaria, which is caused by the protozoa Plasmodium. This alteration disrupts the mosquitoes ability to reproduce leading to population collapse. Releasing these engineered mosquitoes into the wild is one potential method of controlling malaria.

CRISPR also enables gene expression modifications. In this version of CRISPR, the RNA guide directs the machinery to the target gene and, instead of cutting the DNA, adds a special tag that alters the gene's level of expression. CRISPR and tools that increase its functionality are developing rapidly. Researchers are constantly learning how to engineer new cell types and stabilize the engineered genes.