The main goal of biotechnology and genetic engineering is making changes to an organism's genome to make useful products. Biotechnology is often used in agriculture, food production, and medicine. Genetic engineering takes on many forms, including breeding to enhance selected traits and the insertion of genes from a related or unrelated organism.
An important tool in biotechnology is the process of genome editing, where a strand of DNA is inserted, replaced, or removed from a genome using specially designed nucleases, which break the phosphodiester bonds between the nucleotides of DNA. These nucleases are designed to create double-stranded breaks at a desired location within the genome, triggering the cell's natural DNA repair mechanisms, known as homologous recombination (HR) and non-homologous end joining (NHEJ), which can be harnessed to make targeted gene insertions or deletions (indels).
Streptococcus pyogenes, known as Cas9, is a popular tool in genome editing as an RNA-guided DNA endonuclease that cleaves complementary DNA strands.
CRISPRs provide a kind of record of immunity in bacterial genomes. Infection with viruses caused the evolution of an adaptive method for silencing viral genes. CRISPRs are short foreign DNA fragments that are incorporated into the host genome. Processing of these fragments results in CRISPR-RNAs (crRNA) that form endonuclease-RNA complexes to cleave foreign DNA from invaders, acting like an early immune system. Cas9 is one of these nucleases, creating double-stranded breaks in target DNA. Recognizing a specific target DNA, called the protospacer adjacent motif (PAM), is the key to Cas9 activity.
The Cas9 nucleases are simple and monomeric, but have high frequencies of off-target indel mutations, making their use in human therapeutic applications suboptimal. Enhancing their specificity is crucial to future applications of the CRISPR/Cas system. There has been a significant amount of work done on dimerization of Cas nucleases to improve specificity. One such study, published recently in Nature Biotechnology, describes RNA-guided FokI nucleases (RFN) for which dimerization is necessary for genome editing:
This dimerization enhances the specificity of nuclease activity because it depends on the binding of the two guide RNA (gRNA) strands to the DNA with a defined orientation and spacing. This reduces the likelihood of non-specific cutting because it is unlikely that there are multiple regions within the genome that contain both complementary sequences.
The improved specificity of the Cas9 nuclease was achieved by fusion of the dimerization-dependent FokI nuclease domain to the inactive Cas9 protein with a five amino acid linker, then co-expressing this system with plasmids containing pairs of gRNA. This improved the RNA targeting range, and also provided the tools for expressing gRNA from RNA polymerase promoters, thus allowing the opportunity for cell-type specific and/or inducible control of genome editing. The authors also found that non-specific indels coming from partial mismatches or off-site targeting of the gDNA is negligible when using this method. The longer a gRNA molecule, the lower the probability of finding an identical sequence somewhere else in the genome. For example, the authors suggest the use of a 45 base-pair long gRNA in human cells, targeting a specific site in the genome. There is a low probability of having the same 45 base-pair combination somewhere else in the genome. Using dimerization and two gRNAs decreases this likelihood even more. Furthermore, the authors found that PAM-orientation was also key to specificity, with the PAM oriented outward. This reduces the risk of having non-specific indels made in the genome and improves the use of Cas9 for genome editing in human cells.
Now why would we want to use genome editing in human cells? Relax, it's not about creating superhumans (though that could be fun!). Rather, genome editing is used in human cells to target HIV infection and for studying disease genotypes in stem cells, to name a few.
The authors also created a program for scanning the genome of interest to find appropriate RFN target sites. They have made it freely available here.