title: Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system.
abstract: Despite many methodological advances that have facilitated investigation of Mycobacterium tuberculosis pathogenesis, analysis of essential gene function in this slow-growing pathogen remains difficult. Here, we describe an optimized CRISPR-based method to inhibit expression of essential genes based on the inducible expression of an enzymatically inactive Cas9 protein together with gene-specific guide RNAs (CRISPR interference). Using this system to target several essential genes of M. tuberculosis, we achieved marked inhibition of gene expression resulting in growth inhibition, changes in susceptibility to small molecule inhibitors and disruption of normal cell morphology. Analysis of expression of genes containing sequences similar to those targeted by individual guide RNAs did not reveal significant off-target effects. Advantages of this approach include the ability to compare inhibited gene expression to native levels of expression, lack of the need to alter the M. tuberculosis chromosome, the potential to titrate the extent of transcription inhibition, and the ability to avoid off-target effects. Based on the consistent inhibition of transcription and the simple cloning strategy described in this work, CRISPR interference provides an efficient approach to investigate essential gene function that may be particularly useful in characterizing genes of unknown function and potential targets for novel small molecule inhibitors.
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Atul K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Xavier
authors.initials: X
authors.lastname: Carette
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Lakshmi-Prasad
authors.initials: LP
authors.lastname: Potluri
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Jared D
authors.initials: JD
authors.lastname: Sharp
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Ranfei
authors.initials: R
authors.lastname: Xu
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Sladjana
authors.initials: S
authors.lastname: Prisic
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA robert.husson@childrens.harvard.edu.
authors.forename: Robert N
authors.identifier: http://orcid.org/0000-0001-8994-7046
authors.initials: RN
authors.lastname: Husson
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005798:Genes, Bacterial; D020043:Genes, Essential; D005822:Genetic Vectors / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000166:cytology / Q000235:genetics*; D010641:Phenotype