Part:BBa_K3791019

Efficient gRNA Spectinomycin

This part is an improved version of the spectinomycin gRNA: BBa_K3791009. Thus, its function is the same as the one mentioned there: to target a specific spectinomycin-resistance gene fragment. However, its efficiency was increased by using the architecture that will be now further explained. Our idea originated when becoming aware of Cas12a ability to process its own CRISPR RNAs (crRNAs). This means that, once the succession of direct repeat (DR) and spacer sequences are transcribed, the resulting transcript (named pre-crRNA) can be processed into mature gRNAs as a result of the dual RNase/DNase activity of Cas12a [1]. For that event to happen, the spacer sequence (necessary for target DNA recognition) should be followed and preceded by a DR. Specifically, it is known that Cas12 cuts the pre-crRNA 4 nucleotides upstream of the hairpin structures formed by the DR [2]. That was taken into account when designing all these efficient gRNAs (BBa_K3791015, BBa_K3791016, BBa_K3791017 and BBa_K3791018), in order not to lose key nucleotides after Cas12a processing. From this knowledge, the following sequence structure emerged: repeat + spacer + 4 nucleotides + repeat.

Usage, Biology and Characterization

As this part is a simplification of our final construct, usage, biology and characterization is explained there: BBa_K3791024

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

References

[1] Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D., & Platt, R. J. (2019). Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nature Methods, 16(9), 887–893. https://doi.org/10.1038/s41592-019-0508-6
[2] Paul, B., & Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. Biomedical Journal, 43(1), 8–17. https://doi.org/10.1016/j.bj.2019.10.005