The concept of gene editing has been around for thousands of years. Significant technological advancements in the last 100 years have led to a better understanding of DNA and the mutation process, enabling the recombination of DNA and the introduction of new elements.
Nicolas Kuperwasser, Director of the Genome Editing and Targeting Platform, Institute Necker Enfants Malades unpacked some of the key considerations when creating genetic models to study a certain disease. Kuperwasser commented that if one wants to introduce a mutation in the DNA it works more effectively if one creates a break in the DNA, then the natural mechanism of the cell will fix the break.
There are two key ways to create custom mutations that enhance mutation efficiency: nonhomologous end-joining and homology-directed repair (HDR). There are two main protein-based targeting methods: zinc finger nucleases or TAL-effector nucleases. Kuperwasser explained that although zinc finger nucleases are complex to engineer, they work very effectively.
However, sequence-based targeting has grown more popular over the years. CRISPR-Cas9 is a Nobel-prize-winning revolutionary gene-editing tool that allows for precise targeting and cutting of DNA. It uses a guide RNA sequence to bind and cut DNA, making it easier to manipulate and package. This technology has evolved to include various Cas families targeting both DNA and RNA. Furthermore, it is possible to use multiple guides at the same time with one Cas, allowing for several edits at the same time.
A brief evaluation of TALENs and CRISPR showed that CRISPR is more efficient. CRISPR technology can be used to create genetic models to study diseases, altering transcription, and epigenetically modifying DNA. It can also target RNA for degradation or alternative splicing, providing a comprehensive tool for model generation and functional genomics.
There are two main screening methods: pooled library screening and array screening. Pooled screening requires a large number of cells and sequence-based quantification, while arrayed screening can use fewer cells and various phenotypic readouts. Kupperwaser urged researchers to look into the advantages and disadvantages of each method depending on the research needs.
Although CRISPR has had a profoundly positive impact on the life sciences industry, key challenges in using CRISPR include off-target activity, PAM restrictions, cellular effects from double-strand breaks, and targeting non-cellular models. Recent advancements aim to address these issues by optimising the Cas9 protein and guide RNA, and developing base editors and prime editing to minimise cellular damage.
