LAUSR

Dear Editor, The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR–Cas) system has exhibited powerful abilities to manipulate genomes of animals and plants (Knott and Doudna, 2018). Up to now, numerous Cas nucleases have been harnessed for genome editing in human cells, such as Cas9, Cas12a (also known as Cpf1), and Cas12b (also termed C2c1). Cas12b, a Class 2 type V-B CRISPR system, generates staggered double-strand breaks (DSBs) in the target DNA (Stella et al., 2017) and recognizes a distal 5′-T-rich protospacer adjacent motif (PAM) sequence (Shmakov et al., 2015), making it a complement to Cas9 (recognizing 5′-NGG-3′ PAM) in genome editing. Three Cas12b nucleases have been engineered for targeted genome editing in mammals or plants: BhCas12b v4 (Strecker et al., 2019), BvCas12b (Strecker et al., 2019), and AaCas12b (Teng et al., 2018). However, they have not been compared parallelly with each other. In this study, we compared the three Cas12b proteins for genome editing in mammalian cells. It has been reported that BhCas12b v4, BvCas12b, and AaCas12b recognize 5′-DTTN-3′ (D = A, T, or G; N = A, T, G, or C), 5′-ATTN-3′, and 5′-TTN-3′ as PAM sequences, respectively (Supplementary Figure S1). To compare their editing efficiencies, eight target sites with qualified PAMs were selected in HEK293T cells to test the endogenous editing ac-