CRISPR-Cas12 and Cas13: the lesser known siblings of CRISPR-Cas9

Abstract

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) technology has become a keystone in genetic engineering as an invaluable tool to understand precise and molecular mechanisms of disease development, drug resistance and single-cell biology (Fang and Wang 2016; Wang and Wang 2017; Wang et al. 2018). CRISPR is divided into class I and class II, with 6 divisions and finally over 30 different subtypes (Koonin et al. 2017). Of those, type II-ACRISPR-Cas9 (Csn1) was found to cut DNAwith a guide RNACas12a (Cpf1) from the bacterium Francisella novicida was and differentiated itself from Cas9 by relying on a “T-rich” PAM sequence, requiring CRISPR RNA (crRNA), and “stagger” cutting when targeting DNA, making it ideal for multiplexed genome editing as one vector can carry additional crRNAs. Furthermore, it can cleave DNA multiple times as the recognition sequence is not affected by post-repair. Other emerging systems, Cas13a (C2c2) and Cas13b (C2c6) are specialised at RNA interference (Shmakov et al. 2017). It is important to recognise key differences of functions between Cas12 and Cas13 and hurdles to be overcome prior to therapeutic application. One of obstacles before clinical application is to ascertain the mechanisms of type V effectors regarding target recognition and cleaving. Cas12 and Cas9 share similar structural shapes with minor deviations except for the RuvC domain which is superimposable. A T-rich PAM sequence is required such as 5′-TTN-3′ when DNA is cleaved. A distinct difference of Cas12a property is the presence of a unique fold domain due to lack of an HNH domain, similar to the RuvC domain. The positively charged central channel of a nuclease (NUC) lobe may determine the cleaving of the target strand after catalytic residue mutations in the RuvC domain of Cas12a in the bacterium Acidaminococcus sp. inhibited the cleaving in target and non-target strands (Yamano et al. 2016). Although Cas12b lacks an HNH domain and possess an NUC domain that determines target strand cleavage activity similar to Cas12a, the structure of the NUC domain of Cas12b is distinctly different from a Cas12a NUC domain. Both Cas12a and Cas12b use the RuvClike domain to cleave DNA strands after significant conformational changes related to the initial cleaving of the non-target strand. Mutations in the NUC domain in Francisella novicida resulted in incomplete inactivation of the target strand cleaving. This highlights the possibility that DNA cleavage activity was determined by the RuvC-like domain instead of the NUC domain that was initially proposed. The NUC domain participates in guide target binding (Swarts et al. 2017). The size of Cas12b is significantly smaller than those of commonly used https://doi.org/10.1007/s10565-019-09489-1