Introduction to CRISPR:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a method of gene editing that utilizes the Cas9 protein and specific guide RNAs to either disrupt host genes or insert sequences of interest. Initially used in bacteria as an adaptive immunity response, CRISPR has been since utilized in the biological field as a new alternative to genome engineering. Furthermore, CRISPR provides a cheaper alternative to other gene editing techniques such as zinc fingers, and is quickly being adopted as the technique of choice.
Cas9 (CRISPR associated protein 9) is a naturally occurring enzyme found in some bacteria that is used for immunity. Cas9 works by using guide RNA with short sequences complimentary to potential foreign DNA, combating infection. This mechanism has similarities to RNA interference found in many eukaryotes. Because of its capabilities, Cas9 has been used recently in experiments to serve as a genome editing tool.
How CRISPR works:
- Cas9 with designed guide RNA[s] are injected into developing embryos.
- Once inside the cell body, Cas9 searches through the cell’s for a DNA sequence that corresponds to the sequence in the designed guide RNA.
- Once the sequence is found, Cas 9 is able to unwind the DNA helix.
- After DNA has been unwound, Cas9 creates a double stranded break in the cell’s DNA.
- Repair enzymes repair the double-stranded break while integrating new experimental DNA.
Advantages to CRISPR over ZNF or TALEN:
- The sequence of the guide RNA that is a part of the Cas9 protein can be easily and relatively cheaply designed.
- Efficacy is high and much faster with CRISPR due to the Cas9protein being able to be directly injected into developing embryos rather than traditional homologous recombination techniques.
Due to the above advantages, CRISPR can effectively introduce mutations in multiple genes in one step, which allows for rapid turnaround in genome engineering. Changes that previously were introduced one at a time over multiple generations can now be introduced simultaneously in one generation, greatly increasing the ability to study the effects of the changes and develop hypotheses.
If you would like a more detailed introduction, MilliporeSigma has provided a webinar that explains the basics and MilliporeSigma’s specific product offerings.