Lentiviral strategies for functional genomics applications

The triumphal sequencing of the Human Genome Project offered new insights into the fundamental inner workings of humans, promising a big step toward curing humankind of most diseases. More than 15 years after its completion, biologists continue to struggle with this vast amount of genomic sequence encoding tens of thousands of genes of unknown function. Sequencing the genome was an incredible challenge but, in broader perspective, was only the first small step. The most difficult challenge lies ahead; deciphering the cryptic meaning of the 3.3 billion base pairs of DNA, by assigning functions to the tens of thousands of genes, and determining how they work together to make us human. This is the grand biological promise yet to be fulfilled, and with the recent development of new biotechnological tools, the biggest discoveries are yet to come. 

The gold standard for deciphering gene function is to disrupt normal gene expression and study the resulting phenotypes. Such loss-of-function experiments have been performed for more than 100 years, beginning with the work of Thomas Morgan who discovered that genes are on chromosomes and carry mutations responsible for phenotypes. With this knowledge, generations of scientists have worked hard to comprehensively mutate genes, using chemicals, radiation, and random virus integration to painstakingly map each phenotype to a specific mutant gene. This forward genetics approach has taken decades, as artisan techniques fraught with numerous technical challenges complicate the attempts to map random and seemingly minute lesions in a sea of genomic DNA. The sequencing of the human genome offered a map by which gene function could be deciphered but lacked the means to selectively disrupt a specific gene in a nonrandom manner.

The discovery of RNAi by Fire and Mello promised a magic bullet to target any gene, provided the investigator knows the DNA sequence (reverse genetics). The timing of the discovery could not have been more perfect, as the method was published shortly after the human genome sequence became freely available. The work of Fire and Mello astounded the scientific world by showing that simple injection of double-stranded RNA into C. elegans could potently silence any gene sequence and produce phenotypes that revealed gene function. As the mechanisms for RNAi were established, it soon found use in human cells to inhibit specific genes. Its ease of use made RNAi the method of choice for deciphering gene function. However, RNAi produces hypomorphic phenotypes, which do not always mirror the complete loss-of-function that can occur with genetic mutation. This and other practical caveats have encouraged scientists to develop new tools for reverse genetics.

Complete loss-of-function reverse genetics approaches became available with the discovery of zinc-finger nucleases (ZFNs) and later, transcription activator-like effector nucleases (TALENs). These approaches utilize customizable DNA-binding domains (DBDs) that are engineered to recognize specific target DNA sequences. Fused to nucleases, DBDs can be used to introduce double-strand breaks (DSBs) and subsequent frameshift mutations into genes, which can lead to their knockout.

A more recent and incredibly powerful addition to the genome editing toolbox is the CRISPR/Cas system. Its involvement in bacterial resistance against viral infections was initially described in Streptococcus thermophilus. Currently, the Type II CRISPR/Cas9 system from Streptococcus pyogenes is the most widely used CRISPR system and was successfully applied to edit human genomes. The full history of the discovery and development of the CRISPR/Cas system has been excellently reviewed previously. Together, these powerful tools offer a new promise to rapidly and efficiently decipher any gene’s function.  The above text describes functional genomics approaches and is taken from a review by Boettcher and McManus.

 With the increasing variety of molecular tools available for loss-of-function experiments, many researchers have difficulty with selecting the most appropriate system. The ViraCore stands ready to help you develop technologies and approaches to address your needs.