CRISPR-Cas systems have revolutionized how scientists manipulate DNA.1 Cas9 and Cas12a are two of the most well-known CRISPR-associated nucleases, both of which allow for precise gene editing. Although both Cas9 and Cas12a target DNA sequences with the help of a short single guide RNA (sgRNA), they differ in their cleavage mechanisms, making them more suitable for different genomic contexts. Despite common challenges, such as the potential for off-target effects and limited gene editing efficiency, both nucleases hold great potential for advancing research and biotechnological innovations.
Cas9: Sharp Nuclease, Blunt Cuts
Cas9, specifically the one from Streptococcus pyogenes (SpCas9), is the most widely used nuclease in CRISPR-based gene-editing.2 Once the sgRNA directs Cas9 to its target, the nuclease cleaves both strands of DNA at the same point. This creates a blunt end double-stranded break (DSB), which triggers DNA repair mechanisms, namely non-homologous end joining (NHEJ) or homology-directed repair (HDR) if a DNA template is provided. For Cas9 to function, the protospacer adjacent motif (PAM)—a two to six base pair sequence—NGG, where N is any base, must sit immediately downstream of the target on the opposite strand. While SpCas9 can only target double-stranded DNA, some Cas9 enzymes from different bacteria can cleave single-stranded DNA in a PAM-independent fashion.3
Cas9 offers fast, precise, and flexible editing in a variety of genomes, making it a favorite among researchers for targeted DNA cutting and the development of knockout models. For example, scientists are using the nuclease to tone down the bitterness of Brussels sprouts, explore the neural circuitry driving extreme weight loss seen in cancer patients, and bioengineer fungi to revolutionize the food industry.
Cas12a: A Stagger in its Step
Like Cas9, Cas12a (also called Cpf1) is an RNA-guided nuclease that also targets a short snippet of DNA, but it differs in its cutting style.4 Cas12a induces DSBs in a staggered manner that creates an overhang between the two strands.5 The overhangs promote HDR rather than NHEJ, which is prone to introducing mutations, thereby allowing for more precise insertions of genetic material. Cas12a recognizes a different PAM—TTTV, where V is an A, C, or G—that sits immediately upstream of the target site on the opposite strand. This motif requirement makes the Cas12a system good for editing organisms with AT-rich genomes.
Additionally, the ability of Cas12a to also target single-stranded DNA broadens its range of potential applications. For example, scientists have used the nuclease for molecular applications, including diagnostic facemasks.6
- Wang JY, Doudna JA. Science. 2023;379(6629):eadd8643.
- Jinek M, et al. Science. 2012;337(6096):816-821.
- Ma E, et al. Mol Cell. 2015;60(3):398-407.
- Zetsche B, et al. Cell. 2015;163(3):759-771.
- Cofsky JC, et al. eLife. 2020;9:e55143.
- Nguyen PQ, et al. Nat Biotechnol. 2021;39(11):1366-1374.