For future iGEM teams

Design of biosensors using IN Dual-gRNA


    Cas12a recognizes and cleaves the target sequence of dsDNA depending on the target sequence and the T-rich PAM sequence. At this time, Cas12a exhibits collateral cleavage activity that indiscriminately cleaves the surrounding ssDNA [1]. CRISPR-AIOD assay, in which two different gRNAs are added, can improve the sensitivity [2]. The improvement of sensitivity by adding two different gRNAs has also been confirmed by assay using Cas13a [3].

    We have appended these documentations from the paper to the Parts Registry page of BBa_K2872019. In addition, the contents described here are adapted to the following parts.
    CRISPR Cas12a : BBa_K3602014, BBa_K2872019, BBa_K2927005
    CRISPR Cas13a : BBa_K3602001, BBa_K2323010, BBa_K2926000, BBa_K2926001


Generally, in assays using a single gRNA, Cas is activated by the binding of a single Cas-gRNA complex to a single target DNA molecule. On the other hand, when two different gRNAs are introduced, two Cas-gRNA complexes bind to a single target DNA molecule, resulting in the activation of two Cas molecules. Since the number of Cas molecules activated per target molecule is doubled, sensitivity is expected to be improved [2], [3].

Figure1. Mechanism of CRISPR Cas12a

Figure2. Mechanism of CRISPR Cas13a


In the Cas12a-based CRISPR-AIOD assay, two target sequences are first set up on the DNA to be detected as targets. Different from the usual gRNA design, these two sequences are not restricted by the PAM sequence, but they must be close to each other. Next, two types of gRNAs are designed based on the set sequences, and primers that can bind upstream and downstream of the two target sites are designed. Finally, by adding the designed two types of gRNAs and primers together with recombinase polymerase amplification (RPA) reagent for target DNA, one-pot and highly sensitive detection become possible. As a result of introducing two types of gRNAs, it has been reported that a lower copy of DNA can be detected than when a single gRNA is added [2].

When designing a gRNA with Cas13a, two target sequences on the RNA to be detected are set in the same way. In contrast to Cas12a, which does not require PAM to be taken into account when designing, in Cas13a, both gRNAs to be designed are restricted by the protospacer-flanking site (PFS). Studies have shown that simultaneous introduction of the two designed gRNAs and Cas13a is possible even with low-copy RNAs, and the amount of signal detected per time has been improved [3].

In our team

Since the research theme of iGEM Gifu 2021 was the quantification of Human herpesvirus-6 using Cas12a, the detection of DNA by dual-gRNA was very attractive. It will be possible to detect even mild fatigue as a signal, when the detection sensitivity is improved and even low-copy DNA can be detected. On the other hand, as the number of target sequences increases, the risk of off-target effects also increases, so it is necessary to investigate whether there are enough off-target sites. Due to the impact of the spread of the COVID-19 infection(From our Description), we decided to abandon the dual-gRNA design because we judged that it would increase the time spent on design and validation experiments compared to the design of conventional gRNAs.


1) Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky and Jennifer A. Doudna. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360, 436-439 (2018)
2) Xiong Ding, Kun Yin, Ziyue Li, Rajesh V. Lalla, Enrique Ballesteros, Maroun M. Sfeir and Changchun Liu. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nature Communications, 11, 4711(2020)
3) Parinaz Fozouni, Sungmin Son, María Díaz de León Derby, Gavin J Knott, Carley N Gray, Michael V D'Ambrosio, Chunyu Zhao, Neil A Switz, G. Renuka Kumar, Stephanie I Stephens et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell 184, 323–333(2021)