The bacterial CRISPR/Cas9 system (Clustered Regularly Interspaced Palindromic Repeats associated protein 9 system) is among the youngest players in the gene editing playground.
The target sequence-determining CRISPR RNA (crRNA) and the auxiliary trans-activating crRNA (tracrRNA), often fused to a chimeric single guide RNA (sgRNA), work together with Cas9 to become a promising and potent tool for genome engineering.
When a new tool is developed, it often means challenging and troublesome situations with no common solutions. Below we offer some useful tips to avoid these issues as well as solutions for when they arise.
The PAM is a necessary requirement for CRISPR gene editing. In case your target sequence of choice has no adjacent NGG, consider:
Solution: Increase the tracrRNA length–there is a consistent increase in modification efficiency with increasing length of tracrRNA.
Solution: Try designing and testing 3 to 4 different DNA target sequences to increase modification efficiency for CRISPR nucleases (or nickases).
Solution: Efficiency can be increased by enriching the transfected cells via antibiotic selection and/or FAC sorting as follow-up processes to modification.