The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, several practical aspects should be carefully considered in order to achieve the best results from this system. Such considerations include: which promoter to use, whether to opt for wild-type of double nickase, how to achieve multiplexing and, perhaps most importantly, which vector to use.
For example, the chosen promoter may influence the range of target sites available (54). Using the U6 or T7 promoters requires a G or GG, respectively, at the 5´ end. Generating gRNAs with mismatches to the first two bases, or simply adding two guanines to the 5´ end, can reduce such restrictions.
Undoubtedly, the most important decision is to decide which vector to use. A variety of vectors have been validated for different cells and model organisms, and final application, from cutting or nicking to activating genes and screening libraries. Several groups have provided repositories of these plasmids, which are available through Addgene.
Examples of NEB products that can be used to support CRISPR workflows are shown below.
Quick High Yield RNA Synthesis Kit