CRISPR (CRISPR/Cas9) on Zageno
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)](https://zageno.com/catalog/gene-editing/crispr?q=gRNA "Guide RNA Kits")
- 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.
Click on the comparison below for a clearer view!
This example highlights how every kit has positive and negative aspects, and that only side-by-side comparison can allow you to correctly judge which is the right kit for you.
For instance, this comparison shows:
- that the Sureguide gRNA synthesis kit is the cheapest per reaction / preparation
- that the Guide-it Complete sgRNA Screening System is the only kit able to both produce gRNA as well as test its efficacy
- that the Geneart Precision gRNA synthesis kit is the only kit able to produce knock-in as well as knock-out
The ZAGENO comparison engine does not show the best kit, but instead helps you, the researcher, choose the most sutiable kit for your experiment.
Go to our How it Works page for a guide to using the ZAGENO comparison engine.
Video: Bozeman Science/YouTube