An interview with Dr. Maja Petkovic from AMS Biotechnology, ptoviding an introduction to CRISPR/Cas9 and its applications in biological research.
Please can you give a brief introduction to clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 genome editing?
RNA guided CRISPR nucleases have a great potential for genome modification in many different organisms. The nucleic acid targeted CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) system has evolved as defence mechanism in bacteria and archaea against phage and plasmids that are removed by RNA directed DNA degradation.
The Cas9 protein serves in type II CRISPR systems to locate the homologous sequence and the enzyme then cleaves DNA strands to introduce double strand break (DSB).
Nathan Devery | Shutterstock
Cas9 can be used in genome editing, since it can cleave any sequence with compatible protospacer-adjacent motif (PAM) by expressing an RNA mimic together with trans-activating chimeric RNA (tracrRNA) required for processing.
How does CRISPR/Cas9 allow for specific genome disruption and replacement?
CRISPRs can add and delete base pairs at specifically targeted DNA loci. Cas9 can be directed to cleave a specific sequence by using a compatible PAM by expressing a chimeric RNA or a spacer array together with tracrRNA that is needed for processing.
Genome editing is achieved by homologues recombination, which is activated by expression of Cas9 and a cassette that generates RNA with spacer matching the target sequence.
In what way does CRISPR/Cas9 differ from RNAi?
CRISPR can also turn off genes in a reversible fashion by targeting but not cutting a site.
In bacteria, the presence of Cas9 alone is enough to block transcription, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.
How does CRISPR/Cas9 achieve high specificity and low cell toxicity?
Target recognition by the Cas9 protein requires seed sequence within the CRISPR RNA and a conserved dinucleotide containing PAM upstream of the CRISPR RNA binding region.
Therefore the CRISPR/Cas9 system can be used to target any DNA sequence by redesigning the CRISPR RNA. Using modified Cas9 increases the frequency of homologous recombination over non-homologues end joining, which decreases the toxicity for the cells.
How long does genome editing take using CRISPR/Cas9?
First you need to design your guide RNA and clone it into vector. The cell selection can take 3-4 weeks.
After that follows the analysis and the cells can be kept growing for longer time to isolate single clones.
How successful has CRISPR/Cas9 been so far in genome editing and how much potential do you think it has?
Due to its great ease of use, Cas9 mediated genome editing is a promising genome engineering approach, especially in mammalian systems.
Do you think CRISPR/Cas9 genome editing will allow scientists to build upon the work of The Human Genome Project?
The Human Genome Project has been very successful and has also put the DNA sequences in the public domain to be freely used by researchers around the world.
The modern genome editing technologies can helps us understand the complexity of the genome in multiple tissues and cell-types.
These technologies can also be used to develop a broad range of human disease models that faithfully recapitulate predisposing or pathogenic genetics.
What implications could this have for medicine?
Biomedical field is interested in this genome editing system and there are several companies trying to develop new gene therapy treatments using this innovative gene-editing technology to snip out harmful mutations and swap in healthy DNA.
These efforts can accelerate the design of more rational targeted therapies.
Please can you outline how AMSBIO can help with CRISPR/Cas9 genome editing?
We offer cloning kits, kits for gene knockout, Cas9 expression vectors and custom cloning and design service.
What are AMSBIO’s plans for the future with regards to CRISPR/Cas9?
We plan to further develop our product portfolio and also to offer a custom service. We have also added Cas9-nickase to our product portfolio and the nickase can help reduce the off-target effect.
Where can readers find more information?
CRISPR/Cas9 Genome Editing by AMSBIO
About Maja Petkovic
Dr. Maja Petkovic has been working with lentivirus products for more than two years. She is the product manager for custom services and oversees lentivirus and adenovirus portfolio and long mRNA synthesis service. She is also managing aptamer products, immunological research products, RNA amplification and isolation products, enzyme assays, stem cell products, and lab reagents.
Dr. Petkovic has completed her PhD studies at University of Zurich, Switzerland studying DNA repair and maintenance of genomic stability. After that she has moved for post-doctoral training to Imperial College London, where she studied the role of forkhead transcription factors in breast cancer.