Genome Editing
A Brief History
Genome editing is the process of editing an organisms DNA by altering, removing or adding nucleotides to the genome. This process is accomplished through the use of engineered nucleases that can make a double stranded “cut” or a single stranded “nick” in an organism’s DNA. These sites are altered and then repaired by homologous recombination or non-homologous end joining. This type of genetic engineering is useful for a number of applications, including targeted gene mutations, chromosomal rearrangement, and the creation of transgenic animals.
Genome editing has been a heavily studied field for a number of years with an ultimate goal of specificity to limit off target effects. The first engineered nuclease technology, Zinc Finger, was presented in a 1991 publication by Pavletich and Pabo in the journal Science. Zinc Finger was the predominant genome targeting technology for over ten years, but over time drawbacks to the system emerged. Certain nucleotide triplets could not be targeted, and interactions within a zinc finger array could reduce specificity.
In 2009 when the genome targeting abilities of TAL effectors was published, they were quickly harnessed for genome editing and transcription activator-like effector nuclease, or TALEN, emerged. TALEN allowed for a larger window of potential target sites and a simple method of building TAL effector arrays was introduced in 2011. As a result of these advancements and its overall potential, TALEN was named “Method of the Year 2011” by the journal nature methods.
In 2012 clustered regularly interspaced short palindromic repeats, or CRISPR, was demonstrated as a new genome editing tool. CRISPR was discovered somewhat fortuitously by researchers studying bacterial defense mechanisms, specifically how bacteria deal with foreign plasmids and phages. Bacteria have the ability to open and alter the genomes of invading viruses with a nuclease called Cas9, inactivating them in the process. CRISPR formulates a complex containing the Cas9 nuclease and small guide RNAs (sgRNA) and can be redirected by using different sgRNAs. This property eliminates the need to construct a completely customized endonuclease for each target, something that is still required by TALEN and Zinc Finger. As a result of this the entry barrier to genome editing has been lowered significantly, allowing for more users and more innovation.
Genome Modification Services
- RNA CRISPR Genome Modification
- Genetically Modified IPSC lines
- Disease Model and Control Cell Line Development
Genome Modification Products
RNA CRISPR Platform
- Integration free genome editing
- Deep sequencing validation for each project
- Highly precise sgRNA design
- Vast reduction of off target effects
- Ability to use a limitless source of starting cells, iPSCs
RNA CRISPR Platform More Info
For a number of years Allele Biotechnology has focused on cellular reprogramming, specifically the ability to reprogram cells endogenously. This was achieved in 2012 with the publication of “Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA” in Scientific Reports. The next logical use for the powerful mRNA platform we developed was genome modification. Because of its high efficiency and the ability to use interchangeable small guiding RNA, we settled on CRISPR technology.
Our CRISPR system is completely based on validated modified messenger RNA (mRNA) technology. This approach increases CRISPR efficiency while reducing off target effects when combined with our highly precise sgRNA design. It allows for any cell type to be used as starting material, including induced pluripotent stem cells reprogrammed using our RNA platform. The resulting cell line is validated using deep sequencing to ensure only the desired changes were made.
The combination of cellular reprogramming and genome editing can serve as a powerful tool for developing assays and conducting novel research. To take advantage of this we offer both a custom service and reagents. Please fill out our contact form and someone from our technical staff will contact you to discuss your custom project. We can start with a number of different cell types and your project can start with cellular reprogramming to iPSCs, or proceed directly to genome modification. To view our reagent options proceed to the bottom of the page.