Overview
Protospacer adjacent motif (PAM) is important for Cas12a to recognize its target. However, current Cas12a can only recognize TTTV as PAM, which greatly limits its detection ability. If Cas12a can recognize more PAMs, users will have larger range to find a perfect protospacer. It would be fabulous if we can achieve this! After paying great efforts in literature and doing thousands of testing, we finally produced Cas12a-RVRR, the advanced Cas12a that can recognize more PAMs!
First part: Introduce point mutations to WT AsCas12a
AsCas12a cleaves the target DNAs when the PAM sequences are identified. However, the widespread use of AsCas12a nucleases is limited by their requirement for a rather long TTTV PAM sequence (V can be A, C, or G), which rarely occurs in mammalian or bacteria genomes1. Thus, operators have very limited choices when selecting protospacers. To address this limitation, we aimed to engineer variants of AsCas12a that can recognize alternative PAM sequences in order to increase its targeting range.
Previous work has reported several PAM binding sites on Cas12a and some of which showed positive effects on PAM identification. Changing of these amino acids can influence Cas12a’s binding ability with PAMs and protospacers. Among these sites, V548, R542, N552 and E174 have been widely reported2. Therefore, we speculated that variants with certain mutations at these sites might enhance the binding activity of AsCas12a on targets with larger compatibility of PAM sequences.
We got our wide-type AsCas12a protein from BBa_K2965021. For higher expression yield in E. coli, we did codon optimization on the original coding sequence. Although the DNA sequences was changed, the amino acid sequence remained the same, so the protein was not changed after codon optimization. Then we introduced the selected mutations into WT plasmid through circular PCR reaction in the order of S542R, K548V, N552R and E174R. As a result, we successfully extracted three mutant proteins —— AsCas12a-R, AsCas12a-RVR and AsCas12a-RVRR.
Figure 1. AsCas12a mutant sequences
Second part:experimental verification of improved abilities
After obtaining mutated AsCas12a proteins, we analyzed the improved activities of the mutant Cas12a comparing to the corresponding WT AsCas12a. Firstly, we evaluated the recognition and cleavage activities of the three AsCas12a variants. We used target DNA with original PAM TTTC for testing. The results indicated that the three mutants did improve Cas12a’s activity in binding and cleavage. The fluorescence signal rises the most rapidly in Cas12a-RVRR group, and reached much higher point within 40min. The intensity value, which was almost 10000, even reached the upper limit of our instrument (Figure 2A). The Percentage of increased binding efficiency was introduced to represent to what extent the mutations can promotes Cas12a’s activity. Percentage of increased binding efficiency is calculated as following: \begin{equation} Percentage\ of\ increased\ binding\ efficiency = {{Intensity(variants)-Intensity(WT)}\over {Intensity(WT)}} \end{equation} We can see that, taking WT as standard, the three variants made ~50%, ~280% and ~480% increases in binding activity. And we can see that the activity promotion rises with the number of introduced mutations increasing (Figure 2B). This also proved that these amino acid residues are all involved in Cas12a binding targets.
Then we selected six candidate PAMs with one, two, and three bases different from the original PAM TTTV 3 4. We used the same protospacer with different PAMs as target DNAs in this test. Among all the six candidates, we found three PAMs, which do not go well with WT Cas12a, were effectively recognized by Cas12a variants. Figure 3A shows the fluorescent signal of WT Cas12a and Cas12a-RVRR binding PAM TTCA. We can know that RVRR mutations dramatically increased the binding ability of Cas12a recognizing strange PAMs. Then we also calculated Percentage of increased binding efficiency of the three variants with three strange PAMs, to see the promotion levels.
According to Figure 3B-D, we can see that the binding efficiency of AsCas12a variants were greatly improved compared to that of WT AsCas12a. Taking Cas12a-RVRR as example, the binding activity increased for almost 60% with TTCC and CTCC. With TTCA, the binding activity even rises by almost 300%. And other variants also performed very well when binding with these strange PAMs, much better than WT. According to these data, we can announce that the binding activity and PAM compatibility was indeed improved by us. Although we only chose 6 PAMs for testing, we believe the enlarged compatibility is far beyond these. Future teams can use our Cas12a variants and explore more strange PAMs that may be recognized by Cas12a-R, Cas12a-RVR and Cas12a-RVRR. We created an optimized Cas12a for the future iGEMers to use. We are really proud of this achievement.
Reference
1 Tóth E, Varga É, Kulcsár PI, et al. Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases. Nucleic Acids Res. 2020;48(7):3722-3733. doi:10.1093/nar/gkaa110
2 Gao L, Cox DBT, Yan WX, et al. Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol. 2017;35(8):789-792. doi:10.1038/nbt.3900
3 Maule G, Casini A, Montagna C, et al. Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing. Nat Commun. 2019;10(1):3556. Published 2019 Aug 7. doi:10.1038/s41467-019-11454-9
4 Collingwood MA, Vakulskas CA, Zhang L, et al. Engineered AsCas12a Variants with Enhanced Activity and Broadened PAM Compatibility. J Biomol Tech. 2019;30(Suppl):S49.
