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Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA |
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| Authors: | Qingzhou Meng Xinjie Wang Yanqun Wang Lu Dang Xinyi Liu Xiaodong Ma Tian Chi Xian Wang Qin Zhao Guang Yang Ming Liu Xingxu Huang Peixiang Ma |
| Institution: | 1. Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China;2. Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China Guangdong Key Laboratory of Mental Health and Cognitive Science, Institute for Brain Research and Rehabilitation, Centre for Studies of Psychological Application, South China Normal University, Guangzhou, China;3. Guangzhou Laboratory, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China;4. Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, China;5. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China;6. Guangdong Key Laboratory of Mental Health and Cognitive Science, Institute for Brain Research and Rehabilitation, Centre for Studies of Psychological Application, South China Normal University, Guangzhou, China;7. School of Life Science and Technology, ShanghaiTech University, Shanghai, China;8. Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China School of Life Science and Technology, ShanghaiTech University, Shanghai, China;9. Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, China;10. Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China |
| 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/Cas12a CRISPR-based detection D614G SARS-CoV-2 |