whereas the 5′-CCA- , 5′-CCG- , and 5′-CTT - PAM - flanked protospacer was cleaved with just ~10 % . Taken together , these results clearly demonstrate that RbCas12a requires a PAM defined as 5′-YYN for cleavage , at least in a pure in vitro system . It should be noted that 5′-ССV and 5′-СTW ( where V represents C , A , or G ; and W represents A or T ) were not able to cleave DNA templates with efficiencies of more than 25 % under the conditions of the experiment .   Recently , we demonstrated that the cleavage activity of at least three V - A nucleases ( AsCas12 , LbCas12a , and FnCas12a ) persists after a substantial reduction in the crRNA scaffold length , up to its complete removal , at high concentrations of crRNA . Furthermore , we showed that the trans - addition of a 20 - base scaffold RNA rescued the cleavage activity of these enzymes . Thus , we proposed the concept of split crRNA , which , in a complex with V - A nucleases , was comparable to cleavage activity observed with full - sized crRNA [ 17 ] . Here , we expand our observations and investigate the ability of RbCas12 to be programed with split crRNAs . As shown in Figure 6A , in reactions containing both full - sized and split crRNAs , the target DNA was cleaved by~90 % under the conditions of the experiment . We previously showed that increasing the spacer - only crRNA concentration could increase the cleavage efficacy of AsCas12a [ 17 ] . The data presented in Figure 6B demonstrate that increasing the concentration of spacer RNA from 0.5 to 5 µM causes dose - dependent DNA substrate cleavage by RbCas12a . It is possible to cleave the DNA template reasonably efficiently ( ~50 % after a 20 - min incubation ) at a protein : RNA ratio of more than 1:500 . Efficient cleavage of cognate targets was also observed when split crRNAs with unrelated spacer sequences were tested ( Figure S2 ) .