A CRISPR-guided mutagenic DNA polymerase strategy for the detection of antibiotic-resistant mutations in M. tuberculosis

Siyuan Feng, Lujie Liang, Cong Shen, Daixi Lin, Jiachen Li, Lingxuan Lyu, Wanfei Liang, Lan lan Zhong, Gregory M. Cook, Yohei Doi, Cha Chen, Guo bao Tian

Research output: Contribution to journalArticlepeer-review

Abstract

A sharp increase in multidrug-resistant tuberculosis (MDR-TB) threatens human health. Spontaneous mutation in essential gene confers an ability of Mycobacterium tuberculosis resistance to anti-TB drugs. However, conventional laboratory strategies for identification and prediction of the mutations in this slowly growing species remain challenging. Here, by combining XCas9 nickase and the error-prone DNA polymerase A from M. tuberculosis, we constructed a CRISPR-guided DNA polymerase system, CAMPER, for effective site-directed mutagenesis of drug-target genes in mycobacteria. CAMPER was able to generate mutagenesis of all nucleotides at user-defined loci, and its bidirectional mutagenesis at nick sites allowed editing windows with lengths up to 80 nucleotides. Mutagenesis of drug-targeted genes in Mycobacterium smegmatis and M. tuberculosis with this system significantly increased the fraction of the antibiotic-resistant bacterial population to a level approximately 60- to 120-fold higher than that in unedited cells. Moreover, this strategy could facilitate the discovery of the mutation conferring antibiotic resistance and enable a rapid verification of the growth phenotype-mutation genotype association. Our data demonstrate that CAMPER facilitates targeted mutagenesis of genomic loci and thus may be useful for broad functions such as resistance prediction and development of novel TB therapies.

Original languageEnglish
Pages (from-to)354-367
Number of pages14
JournalMolecular Therapy - Nucleic Acids
Volume29
DOIs
Publication statusPublished - 13-09-2022

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Drug Discovery

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