Synthetic biology

Synthetic biology is the design and construction of new biological parts, devices, and systems, and the re-design of existing, natural biological systems for useful purposes.

The functional aspects of this definition are rooted in molecular biology and biotechnology.

As usage of the term has expanded, synthetic biology was recently defined as the artificial design and engineering of biological systems and living organisms for purposes of improving applications for industry or biological research.

In general its purpose can be described as the design and construction of novel artificial biological pathways, organisms or devices, or the redesign of existing natural biological systems.

Synthetic biology has traditionally been divided into two different approaches. Top down synthetic biology involves using metabolic and genetic engineering techniques to impart new functions to living cells. Bottom up synthetic biology involves creating new biological systems in vitro by bringing together ‘non-living’ biomolecular components, often with the aim of constructing an artificial cell. Biological systems are thus assembled module-by-module. Cell-free protein expression systems are often employed, as are membrane-based molecular machinery. There are increasing efforts to bridge the divide between these approaches by forming hybrid living/synthetic cells, and engineering communication between living and synthetic cell populations.

Genome editing is not a new concept to the scientific community and has been around for decades. However, directing precise sequence changes at desired sites has remained a difficult and tedious challenge for researchers. Limited successes have been achieved with oligonucleotides, small molecules, or self-splicing introns, but the development of site-directed zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) has facilitated sequence-specific manipulations. Nevertheless, difficulties of protein design, synthesis, and testing have slowed adoption of these engineered nucleases for routine use in genome editing experiments. The most recent gene editing technology, the CRISPR-Cas9 system, largely overcomes these difficulties, making it an attractive method for genome editing. It is critical that we adopt a workflow and use technology that can make genome editing efficient by accurately identifying on- and off-target mutations, producing a gene-edited organism that may have fewer or no off-targeted gene side effects. Today, with the aid of gene editing tools, researchers can quickly generate model organisms that can be used to study human diseases, test efficacy of various drugs, and even create genetically modified organisms during times of an epidemic, as has been done for the Aedes aegypti mosquito, a vector for the Zika virus.

Understanding the CRISPR-Cas9 gene editing system

Clustered regularly interspaced short palindromic repeats (CRISPRs) belong to a class of repeated DNA sequences that work together with CRISPR-associated (Cas) genes to protect bacteria and archaea from invading foreign nucleotides, such as those from phages and plasmid DNA. Upon injection of phage-DNA into the bacteria, the phage-DNA is cut into small pieces and incorporated into the CRISPR locus. This locus, now containing information about the foreign phage-DNA is transcribed, this CRISPR-RNA (crRNA) along with a second non-coding RNA, trans-activating CRISPR RNA (tracrRNA), then forms a ribonucleoprotein complex with the Cas9 endonuclease and cuts the foreign DNA from this specific phage. So, a bacteria carrying information about a specific phage-DNA can have immunity towards attack from that phage. In this way, bacteria and archaea have evolved an adaptive immune response against invading DNA and can protect themselves in the future when attacked by the same phage.

This CRISPR-Cas9 system that provides adaptive immunity against foreign elements in bacteria and archaea, has been modified today to be able to edit genes in various cell types (mammalian, plant, insect, fungi, etc). Researchers combined the crRNA and tracrRNA and generated a single guide RNA (sgRNA), which recruits the Cas9 nuclease to specific genomic locations with guidance from the sgRNA. sgRNA then pairs with the complimentary genomic sequences via Watson-Crick pairing and the Cas9 nuclease induces double strand breaks (DSB). The DSB can be corrected by an error-prone mechanism called, non-homologous end joining (NHEJ), which causes insertions or deletions. If a synthetic template is present, the DSB can also be corrected by homology-directed repair (HDR), allowing the introduction of desired base-changes into the genome.

The CRISPR-Cas9 system is one of many and there are probably many such tools that have yet to be discovered. Using Sanger sequencing, performed on the Applied Biosystems™ 3130xl genetic analyzer, Ivanov et al were able to discover CRISPRs in a newly discovered Bordetella species. The Cas9 endonuclease gene in the Bordetella species is 990bp, which is smaller than that of Streptococcus pyogenesCas9 (SpyCas9) gene. The CRISPR-SpyCas9 system is the most widely studied and used, the newly discovered smaller Bordetella Cas9, which is equally functional and active as the SpyCas9 might be an attractive alternative for the widely used CRISPR-Cas9 system.

Improving the CRISPR-Cas9 workflow

We’ve provided an end-to-end integrated workflow with all the tools necessary for genome editing and downstream analysis. The Invitrogen™ GeneArt™ design tool facilitates the design and ordering of target specific gRNAs for CRISPR-mediated genome editing or TALs for TALEN mediated genome editing. Invitrogen™ transfection reagents offer several options for delivery of genome editing tools into eukaryotic cells. In addition, Invitrogen™ TOPO™ TA cloning vectors and competent cells facilitate the sequence analysis of primary transformants. Gibco™ media is available for growing the primary transformants and secondary cultures following clonal expansion. Finally, Applied Biosystems™ sequencing instruments, reagents and software (Applied Biosystems™ Minor Variant Finder Software or TIDE software) enable the determination of specific genomic editing events.

For accurate and efficient gene editing, it is critical that we confirm the sequences of the guide RNAs and synthetic templates used for DSB repair, to assure efficient targeting. Sanger sequencing can not only help in testing the sequence of the sgRNA, but also in screening cells post-gene editing, to check for on- and off target mutations and to make sure there are no unwanted insertions, deletions or mutations that may possibly lead to frameshift mutations.