Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

Abstract

The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9_Sth1), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9_Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9_Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.

Figure 1. In vivo screen for functional Cas9 orthologues in mycobacteria

(A) Schematic of CRISPRi-mediated transcriptional repression (adapted from reference). Anhydrotetracycline (ATc)-inducible (P_Tet) dcas9 is directed to specific DNA targets by ATc-inducible or constitutively (P_con) expressed sgRNA, which prevents transcription initiation or elongation. (B) Phylogenetic tree of selected Cas9 orthologues. Listed are the Cas9 name abbreviations, size (amino acids), and sub-type classification. (C) dCas9 orthologues show variable efficacy in mycobacteria. The Renilla reporter 5’UTR was engineered such that the optimal PAM for each dCas9 protein could be tested with an identical sgRNA targeting sequence (UTR1) for all dCas9 proteins. The x-axis denotes each dCas9 protein tested; the y-axis shows the ratio of the luminescence repression observed with the UTR1 targeting sgRNA normalized to the luminescence repression observed with a non-targeting control sgRNA. The fold inhibition is shown in white type for each column. (D) sgRNAs targeting NGG PAMs were co-expressed with dCas9_Spy (+ATc) and the effects on the Renilla target measured by luciferase assay. Shown above the graph is a schematic depicting the Renilla gene (yellow arrow) and transcriptional start site (black arrow) and the relative targeting position of each sgRNA (P = promoter; UTR = 5’ untranslated region, NT = Renilla non-template strand). The ΔRL promoter sample depicts basal levels of luminescence. (E) sgRNAs targeting NNAGAAW PAMs were co-expressed with dCas9_Sth1 (the cas9 allele derived from the CRISPR1 locus; +ATc) and luminescence repression measured as in Figure 1D. Fewer sgRNAs were tested for dCas9_Sth1 because of the less frequent PAM occurrence in the Renilla target. (F) sgRNAs targeting NGGNG PAMs were co-expressed with dCas9_Sth3 (the cas9 allele derived from the CRISPR3 locus; +ATc) and luminescence repression measured as in Figure 1D. (G) sgRNAs targeting NNGTGA PAMs were co-expressed with dCas9_Spa (+ATc) and luminescence repression measured as in Figure 1D. Fewer sgRNAs were tested for dCas9_Spa because of the less frequent PAM occurrence in the Renilla target. Error bars are standard deviations of three technical replicates (C–G).

Figure 2. dCas9_Spy sensitizes M. smegmatis to sub-lethal drug treatment

(A) dCas9_Spy can mediate high-level knockdown of endogenous targets in M. smegmatis. sgRNAs targeting dnaE1 (Ms3178) were co-expressed with dCas9_Spy or dCas9_Sth1 (+ATc). Gene knockdown was quantified by qRT-PCR; the consequences of dnaE1 knockdown were monitored by spotting dilutions of each culture on the indicated media. Error bars are 95% confidence intervals of three technical replicates. (B) sgRNAs targeting pptT (Ms2648) in M. smegmatis were co-expressed with the indicated dCas9 protein (+ATc) and plated as in Figure 2A. (C) The indicated dCas9 proteins were HA-tagged, co-expressed with a non-targeting control sgRNA (+ATc), and monitored by western blot. RpoB protein levels are shown as loading controls. *: background α-HA cross-reactive band. (D) dCas9_Spy sensitizes M. smegmatis to sub-minimum inhibitory concentration (sub-MIC) drug treatment. Non-targeting control sgRNAs were co-expressed with dCas9_Spy or dCas9_Sth1 (+ATc); the consequences of dCas9 expression in the presence of a sub-MIC concentrations of the indicated drugs were monitored by spotting serial dilutions of each culture as in Figure 2A. Approximately 5,000 cells are deposited in the first spot and each subsequent spot is a 10-fold serial dilution. AMK: amikacin; NAT: nourseothricin; STR: streptomycin; RIF: rifampicin. Data shown in B–D are representative of three independent experiments.

Figure 3. In vivo identification of permissive PAM position variants for dCas9_Sth1

The Renilla 5’UTR from Figure 1C was modified to contain the indicated PAM sequences listed below the x-axis. Each PAM contains a single-base mutation within the context of the consensus NNAGAAT dCas9_Sth1 PAM. dCas9_Sth1 was co-expressed with the UTR1 sgRNA and luminescence repression measured as in Figure 1C.

Figure 4. dCas9_Sth1 CRISPRi is highly active against endogenous genes in mycobacteria

(A–E) dCas9_Sth1 achieves high-level knockdown of endogenous targets in M. smegmatis. sgRNAs targeting the indicated genes were co-expressed with dCas9_Sth1 (+ATc). Gene knockdown was quantified and visualized as in Figure 2A. (F) dCas9_Sth1-mediated target knockdown is specific. Three sgRNAs targeting M. smegmatis dnaE1 (Ms3178) were co-expressed with dCas9_Sth1 (+ATc) in wild-type M. smegmatis strains or strains complemented with M. tuberculosis dnaE1 (Rv1547). All three M. smegmatis dnaE1 targeting sgRNAs have perfect PAM matches to M. tuberculosis dnaE1 and either one, two, or four mismatches between the sgRNA targeting sequence and M. tuberculosis dnaE1, as indicated to the right of each strain. Positions of the sgRNA-M. tuberculosis dnaE1 mismatches (position 1 is closest to the target PAM): sgRNA1 (5), sgRNA2 (6,21), sgRNA3 (7,9,15,19). Strains were spotted as in Figure 2D. (G) dCas9_Sth1 achieves high-level knockdown of endogenous targets in M. tuberculosis. sgRNAs targeting the indicated genes were co-expressed with dCas9_Sth1 (+ATc). Gene knockdown was quantified as in Figure 2A (fold knockdown +/− 95% confidence interval is shown beneath the +ATc plates). The consequences of gene knockdown were monitored by plating dilutions of each culture on the indicated media. (H) dCas9_Sth1 achieves robust target knockdown far from the transcriptional start site (TSS) in M. smegmatis. sgRNAs targeting the pptT operon were co-expressed with dCas9_Sth1. Gene knockdown of pptT was quantified as in Figure 2A. The first two genes of the pptT gene operon are depicted at scale below the graph; the black bar beneath the pptT gene marks the site of the qPCR amplicon. (I) dCas9_Sth1 achieves robust target knockdown far from the TSS in M. tuberculosis. sgRNAs targeting the groES-groEL1 operon were co-expressed with dCas9_Sth1. Gene knockdown of groEL1 was quantified as in Figure 2A. The groES-groEL1 operon is depicted at scale below the graph; the black bar beneath the groEL1 gene marks the site of the qPCR amplicon. Data shown in A–G are representative of three independent experiments.

Figure 5. Functional profiling of the mycobacterial folate synthesis pathway

(A) Schematic of mycobacterial folate metabolism. The targeted genes (folP1 Ms6103, folC-incorrectly annotated as pseudogene in M. smegmatis, and folA Ms2671) and the inhibitors of those genes (SMX = sulfamethoxazole, TMP = trimethoprim) are shown colored. (B) dCas9_Sth1-induced growth inhibition due to knockdown of folate pathway targets. sgRNAs targeting the indicated genes were co-expressed with dCas9_Sth1 (+ATc) and spotted as in Figure 2D. (C) Multiplexed targeting reveals synthetic lethal interactions in the folate pathway. The consequences of partial knockdown of folate pathway targets, individually or in combination, were monitored by co-expressing “hypomorphic” sgRNAs targeting the indicated genes and dCas9_Sth1 (+ATc). Strains were spotted as in Figure 2D. (D) Partial knockdown of folate pathway targets sensitizes cells to TMP and SMX. sgRNAs from Figure 5C were co-expressed with dCas9_Sth1 (+ATc) and spotted on plates containing sub-MIC concentrations of TMP or SMX. Data shown in B–D are representative of three independent experiments.