CRISPR-Cas9 vs CRISPR-Cas12a
The first step in your experimental design is to select a suitable CRISPR-Cas system. There are many Cas enzymes found in bacteria, but here we focus on Cas9 and Cas12a, which are part of the two Cas systems most commonly used for genome editing. The Cas9 enzyme is an endonuclease that is typically derived from S. pyogenes. The Cas12a enzyme, also known as Cpf1, is often derived from Acidaminococcus or Lachnospiraceae for genome editing purposes.
To choose between Cas9 and Cas12a, you need to look at the genome and the specific gene you are going to edit, and decide whether the target is an AT-rich or GC-rich region. Typically, if you merely want to knock out a gene, you have a lot of design space to examine. In this case, either Cas9 or Cas12a can likely be used, unless the region is very GC-rich or AT-rich. However, if you want to perform HDR, the region to examine is approximately ±10 bp around your desired cut site. A GC-rich sequence is more likely to contain an NGG PAM (N = any base), which can be targeted with Cas9. An AT-rich sequence is more likely to contain a TTTV PAM (V = A, C, or G), which can be targeted with Alt-R™ Cas12a Ultra, a mutant form of Cas12a that works extremely well with TTTV PAMs. The following table compares the Cas9 and Cas12a systems in more detail.
| Feature | Cas9 system | Cas12a (Cpf1) system |
|---|---|---|
| Applications | General genome editing | General genome editing |
| Guide RNA options | crRNA and tracrRNA | crRNA |
| Guide RNA lengths (native) | crRNA: 42 nt | crRNA: 42–44 nt |
| CRISPR enzyme | Class 2, Cas type II | Class 2, Cas type V |
| Enzyme variants | Wild-type | Wild-type |
| DNA cleavage | Wild-type and high fidelity: the blunt-ended cut is 3 bases upstream of the target protospacer sequence. Nickase: introduces a nick in one strand. dCas9: no cleavage. | A staggered cut is created. The 5′ overhang is near the 5′ end of the target protospacer sequence |
| PAM sequence* | NGG | TTTV for Cas12a V3 |
* N = any base; V = A, C, or G
Have you made your choice? Click below to discover the products and tools for the specific CRISPR-Cas system.
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