title: Structure and variation of CRISPR and CRISPR-flanking regions in deleted-direct repeat region Mycobacterium tuberculosis complex strains.
abstract: CRISPR and CRISPR-flanking genomic regions are important for molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) strains, and potentially for adaptive immunity to phage and plasmid DNA, and endogenous roles in the bacterium. Genotyping in the Israel National Mycobacterium Reference Center Tel-Aviv of over 1500 MTBC strains from 2008-2013 showed three strains with validated negative 43-spacer spoligotypes, that is, with putatively deleted direct repeat regions (deleted-DR/CRISPR regions). Two isolates of each of three negative spoligotype MTBC (a total of 6 isolates) were subjected to Next Generation Sequencing (NGS). As positive controls, NGS was performed for three intact-DR isolates belonging to T3_Eth, the largest multiple-drug-resistant (MDR)-containing African-origin cluster in Israel. Other controls consisted of NGS reads and complete whole genome sequences from GenBank for 20 intact-DR MTBC and for 1 deleted-DR MTBC strain recognized as CAS by its defining RD deletion.
  
  
  
  NGS reads from negative spoligotype MTBC mapped to reference H37Rv NC_000962.3 suggested that the DR/CRISPR regions were completely deleted except for retention of the middle IS6110 mobile element. Clonally specific deletion of CRISPR-flanking genes also was observed, including deletion of at least cas2 and cas1 genes. Genomic RD deletions defined lineages corresponding to the major spoligotype families Beijing, EAI, and Haarlem, consistent with 24 loci MIRU-VNTR profiles. Analysis of NGS reads, and analysis of contigs obtained by manual PCR confirmed that all 43 gold standard DR/CRISPR spacers were missing in the deleted-DR genomes.
  
  
  
  Although many negative spoligotype strains are recorded as spoligotype-international-type (SIT) 2669 in the SITVIT international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least 4 different 24 loci MIRU-VNTR and RD deletion families. We report for the first time negative spoligotype-associated total loss of CRISPR region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least CRISPR-associated genes cas2 and cas1. Since cas1 deleted E.coli shows increased sensitivity to DNA damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-DR strains and Beijing family strains as both lack the cas1 gene.
authors.affiliation: National Mycobacterium Reference Center, National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel. paul.freidlin@phlta.health.gov.il.
authors.forename: Paul Jeffrey
authors.initials: PJ
authors.lastname: Freidlin
authors.affiliation: National Mycobacterium Reference Center, National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel.
authors.forename: Israel
authors.initials: I
authors.lastname: Nissan
authors.affiliation: National Mycobacterium Reference Center, National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel.|current address: Ben-Gurion University of the Negev, Beer Sheva, Israel.
authors.forename: Anna
authors.initials: A
authors.lastname: Luria
authors.affiliation: National Mycobacterium Reference Center, National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel.
authors.forename: Drora
authors.initials: D
authors.lastname: Goldblatt
authors.affiliation: Scripps Research Institute, San Diego, CA, USA.
authors.forename: Lana
authors.initials: L
authors.lastname: Schaffer
authors.affiliation: National Mycobacterium Reference Center, National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel.
authors.forename: Hasia
authors.initials: H
authors.lastname: Kaidar-Shwartz
authors.affiliation: Department of Tuberculosis and AIDS, Ministry of Health, Jerusalem, Israel.
authors.forename: Daniel
authors.initials: D
authors.lastname: Chemtob
authors.affiliation: National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel.
authors.forename: Zeev
authors.initials: Z
authors.lastname: Dveyrin
authors.affiliation: Scripps Research Institute, San Diego, CA, USA.
authors.forename: Steven Robert
authors.initials: SR
authors.lastname: Head
authors.affiliation: National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel.
authors.forename: Efrat
authors.initials: E
authors.lastname: Rorman
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D014644:Genetic Variation; D054643:INDEL Mutation; D020071:Interspersed Repetitive Sequences / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion
keywords: CRISPR-Cas; Deleted-direct repeat region deleted-DR; IS6110; MIRU-VNTR; Mycobacterium tuberculosis complex MTBC; Next Generation Sequencing NGS; Region of difference deletion RD deletion; Spacers; Spoligotype; cas1