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. 2021 Jun;16(6):e2100040.
doi: 10.1002/biot.202100040. Epub 2021 Mar 9.

Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA

Qingzhou Meng  1   2 , Xinjie Wang  1   3   4 , Yanqun Wang  1 , Lu Dang  2 , Xinyi Liu  5 , Xiaodong Ma  4 , Tian Chi  6 , Xian Wang  3   6 , Qin Zhao  7 , Guang Yang  3 , Ming Liu  1 , Xingxu Huang  1   6 , Peixiang Ma  1   3
Affiliations

Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA

Qingzhou Meng et al. Biotechnol J. 2021 Jun.

Abstract

Detection of pathogens with single-nucleotide variations is indispensable for the disease tracing, but remains technically challenging. The D614G mutation in the SARS-CoV-2 spike protein is known to markedly enhance viral infectivity but is difficult to detect. Here, we report an effective approach called "synthetic mismatch integrated crRNA guided Cas12a detection" (symRNA-Cas12a) to detect the D614 and G614 variants effectively. Using this method, we systemically screened a pool of crRNAs that contain all the possible nucleotide substitutions covering the -2 to +2 positions around the mutation and identify one crRNA that can efficiently increase the detection specificity by 13-fold over the ancestral crRNA. With this selected crRNA, the symRNA-Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and the results are confirmed to be accurate by Sanger sequencing. Overall, we have developed the symRNA-Cas12a method to specifically, sensitively and rapidly detect the SARS-CoV-2 D614G mutation.

Keywords: COVID-19; CRISPR-based detection; CRISPR/Cas12a; D614G; SARS-CoV-2.

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References

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