Cas12e proteins (formerly CasX) form a distinct subtype of Class II type V CRISPR-Cas effectors. Recently, it was shown that DpbCas12e from Deltaproteobacteria and PlmCas12e from Planctomycetes can introduce programmable double-stranded breaks in mammalian genomes. Thus, along with Cas9 and Cas12a Class II effectors, Cas12e could be harnessed for genome editing and engineering. The location of cleavage points in DNA targets is important for application of Cas nucleases in biotechnology. DpbCas12e was reported to produce extensive 5ʹ-overhangs at cleaved targets, which can make it superior for some applications. Here, we used high throughput sequencing to precisely map the DNA cut site positions of DpbCas12e on several DNA targets. In contrast to previous observations, our results demonstrate that DNA cleavage pattern of Cas12e is very similar to that of Cas12a: DpbCas12e predominantly cleaves DNA after nucleotide position 17–19 downstream of PAM in the non-target DNA strand, and after the 22^nd position of target strand, producing 3–5 nucleotide-long 5ʹ-overhangs. We also show that reduction of spacer sgRNA sequence from 20nt to 16nt shifts Cas12e cleavage positions on the non-target DNA strand closer to the PAM, producing longer 6–8nt 5ʹ-overhangs. Overall, these findings advance the understanding of Cas12e endonucleases and may be useful for developing of DpbCas12e-based biotechnology instruments.

Cas12e蛋白（以前称为CasX）形成II类V型CRISPR-Cas效应子的独特亚型。最近，显示了来自Deltaproteobacteria的DpbCas12e和来自Planctomycetes的PlmCas12e可以在哺乳动物基因组中引入可编程的双链断裂。因此，连同Cas9和Cas12a II类效应器，Cas12e可用于基因组编辑和工程设计。DNA靶中切割点的位置对于生物技术中Cas核酸酶的应用很重要。据报道DpbCas12e在裂解的靶标上产生大量的5′-突出端，这使其在某些应用中具有优越性。在这里，我们使用高通量测序将DpbCas12e的DNA切割位点位置精确定位在几个DNA靶标上。与以前的观察结果，我们的结果表明Cas12e的该DNA切割图案是非常类似于Cas12a的：DpbCas12e主要在非靶DNA链PAM的下游核苷酸位置17-19后裂解DNA，和22之后^第二目标链的位置，产生3–5个核苷酸长的5′-突出端。我们还表明，间隔区sgRNA序列从20nt减少到16nt，会使非目标DNA链上的Cas12e裂解位置更接近PAM，从而产生更长的6-8nt 5′-突出端。总体而言，这些发现促进了对Cas12e核酸内切酶的理解，可能对开发基于DpbCas12e的生物技术仪器很有用。