Month: March 2022

Different protospacers have different efficiency for producing mutations. In other words not every spacer in front of the PAM (protospacer adjacent motif) will be cut with the same efficiency as every other spacer. Software like CHOPCHOP can help you to design gene editing experiments with a much high rate of success.

Important considerations

• You want your gRNA to cut your target with high efficiency but not to cut other parts of the genome. Software helps for this.
• Mismatches in the seed region of the target sequence are more likely to prevent your gRNA from cutting than mismatches further from the PAM.

Cloning work for making plasmids can be slow and frustrating. However, making plasmids (constructs) for gene editing can be a piece of cake. Thanks to modern techniques, pipetting is pretty much the only skill you need to make CRISPR constructs to knockout genes. Here is an Excel file with a protocol for cloning gRNA protospacers. No gel purification is necessary. Efficiency is about 100%.

You will want to sequence your final CRISPR constructs. On rare occasion I have found a base pair error in the protospacer sequence. This can occur if the oligonucleotides have a small portion that are missing bases from what you have ordered. The purity of oligonucleotides is not always absolutely perfect from some suppliers. Sequencing two clones will virtually guarantee you get the final construct you want.