Standard detection cassette for Cas activity
Since detecting the entire antibiotic-resistance gene (ARGs) is relatively dangerous, developing a safe and efficient characterization platform is quite essential. By designing the target DNA part, we provided a feasible and systematic pipeline by which other teams could easily design an in vitro detection target for CRISPR/Cas system. Our design contains two primers, by which the target can be amplified. There are also four unique restriction sites at each end of the fragment and between the primers with the protospacer, which came originally from ARGs and is designed for easily changing sequences on the whole fragment. The stop codon at the end of the protospacer is designed for safety reason, which ensures that no functional protein will be expressed even if unexpected leakage occurred. This template is useful to all the future teams who are engaged in in vitro DNA detection, especially dealing with dangerous genes.
On Existing Parts
The coding sequence of AsCas12a (link: BBa_K2965021 ) has already been in the part collection of iGEM Registry. To pave the way for purification, we introduced two tags, His tag (link:BBa_M0021) and MBP tag (link:BBa_K3454045), into the coding sequence of AsCas12a(link: BBa_K2965037, BBa_K2965038, BBa_K2965039, BBa_K2965040 ) . This made it more convenient for protein extraction.
Improvement of AsCas12a
Now AsCas12a can only recognize three PAM sequences, TTTV PAM sequence (V can be A, C, or G), which limits its detection ability for perfect protospacer is rare. We introduced four point mutations (S542R, K548V, N552R and E174R) based on two latest articles, aiming to improve its compatibility of PAM and its cleavage activity1,2,3. Consequently, Cas12a can now recognize six additional PAM sequences, three of whom has really high efficiency (Table 1). Thus, the most recent breakthroughs concerning Cas12a were brought into the collection of standard biological parts and the future teams can use the optimized Cas12a.
The optimized Cas12a was named as Cas12a-RVRR. We did characterization and proved the optimized ability of Cas12a-RVRR, which you can refer to Improvement for details. These mutants have been submitted to parts registry, please refer to BBa_K3454011, BBa_K3454012, BBa_K3454013 for details.
Table 1. The original PAM sequences and altered sequences that can be recognized by AsCas12a. The yellow cells refers to the PAM that can improve AsCas12a’s cleavage efficiency.
RNA in vitro synthesis template
crRNA is required for Cas12a’s recognition. Here we provided parts collection with several templates that needed for RNA in vitro synthesis. These templates share the same T7 promoter and scaffold RNA sequence. Future teams can use it by simply changing the spacer sequence.
We further enhanced the programmability of Cas12a detection by adding aptamer as an essential part. In the meantime, we summarized a brand-new designing norm of locked activator and modifying aptamer based on the simplest base complementation pairing rule, which can be used as a reference for subsequent teams. This method features an additional sequence at the front and end of the aptamer, which can then lock the activator DNA that is picked relatively randomly (Figure 2). Besides, a well-studied aptamer for kanamycin was modified and firstly introduced to the standard parts collection.
Figure 2.The design norm of locked activator and modifying aptamer.
Recombinase Polymerase Amplification (RPA)
Moreover, we developed a method for RPA efficiency test to measure its extension rate, which is constructing the forward and the reverse primer back to back into a small plasmid. By using RPA amplifying this plasmid, with all the other parameters the same, we can tell the amplification efficiency by reading the products’ concentrations. This is unprecedented and can be useful to all the future teams dealing with RPA.
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