CRISPR (Clustered regularly-interspaced short palindromic repeats) was originally discovered in E. coli during the 1980s. It was only in 2007 that it was understood to be a bacterial defense mechanism against viruses and foreign DNA, as part of the CRISPR / Cas9 system. Since then it has been modified by scientists to be used for genome engineering purposes and so reviewed as the scientific discovery of the century. Understanding and use of system has progressed so far that CRISPR human trials are now being initiated in order to test their safety.
What is CRISPR?
CRISPR consists of repeated sequences interspaced with unique sequences (CRISPR RNA [crRNA]) that correspond to viral DNA segments; specifically, viruses that the bacteria was prone to become infected by, often so-called bacteriophages. The CRISPR system enables the bacterium to recognize and defend against specific infections. Upstream of these repeats are the genes which encode Cas (CRISPR-associated proteins), which are a group of enzymes able to unwind (helicases) and cut (nucleases) the DNA. The enzymes are hence able to target and break up invading DNA, by using the crRNA as a guide.
Our Gene Drives: The Solution to Mosquitos, Malaria and Zika? article expands on future uses of CRISPR in the future.
The CRISPR-associated enzyme 9 (Cas9) has then been developed from this bacterial defense system, whereby the crRNA is replaced with RNA complementary to your target gene of choice. Double strand breaks are then induced at the target gene due to the endonuclease activity of Cas9. The ability of CRISPR Cas9 to specifically edit the genome is then utilized to cut and replace genes of interest.
The specificity comes from being able to tailor the 20 base crRNA to your needs while genes can be replaced by injecting the replacement DNA into the cell.
Thus, there is a huge and ever-growing number of uses for the CRISPR Cas9 system. Applications range from CRISPR genome editing using CRISPR knockout, to germline engineering where, besides knock-outs, CRISPR knock-ins can be performed.
One critical thing to consider is the protospacer adjacent motif (PAM). For Cas9 (from Streptococcus pyogenes), this is the short sequence NGG which is required downstream of the 20 nt target site.
Check out our CRISPR-Cas9 Troubleshoot, to get a better understanding of the ideal experiment workflow.
Design of a CRISPR system includes
construction of the target sequence (crRNA) or guide sequence (gRNA)
combining the target sequence with a CRISPR DNA template
choosing a method of delivery into the cell
Product searches you undertake at ZAGENO may differ if you intend to induce double strand or single strand breaks on your target site or vary depending on the desired effect on the gene of interest:
You also need to take into account which cell type you are targeting when choosing your kit. There are general kits which can be used on different types of eukaryotes or animals in particular, and specialty kits specific to mammals or particular species (like humans).
Compare gRNA CRISPR Kits!
To assist you in choosing the correct CRISPR product for your research, ZAGENO has developed an easy-to-use comparison feature, aligning the important attributes for each kit in a simple table.
The ZAGENO comparison engine does not show the best kit, but instead helps you, the researcher, choose the most suitable kit for your experiment.
Go to our How it Works page for a guide to using the ZAGENO comparison engine.
Video: Bozeman Science/YouTube