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The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system.

Semenova E, Kuznedelov K, Datsenko KA, Boudry PM, Savitskaya EE, Medvedeva S, Beloglazova N, Logacheva M, Yakunin AF, Severinov K - Nucleic Acids Res. (2015)

Bottom Line: Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA.Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e.The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.

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Primed CRISPR adaptation in cells producing unit-sized crRNA transcripts with or without Cas6e. (A) Results of primed adaptation experiment (see ‘Materials and Methods’ section) with indicated cells are shown. The leader-proximal end of CRISPR cassette was amplified using an appropriate primer pair, amplification products were separated by agarose gel electrophoresis and revealed by ethidium bromide staining. (B) DNA fragments corresponding to expanded CRISPR cassettes shown in (A) were subjected to Illumina sequencing. Statistics for reads corresponding to spacers derived from priming protospacer-containing plasmid is presented. ‘Derived spacers’ refers to spacers that originate from recognition of protospacers with correct AAG PAM but are either excised from target DNA with short shifts in the upstream or downstream direction or are inverted during acquisition in CRISPR array. (C) Mapping of spacers acquired in (A) on the priming protospacer plasmid is schematically shown. The position of the priming protospacer (purple triangle) as well as known features of the plasmid are shown. The heights of red bars indicate the efficiency (number of times) a spacer arising from a protospacer in this position was observed. Red bars protruding inside and outside of circles indicating the protospacer plasmid show spacers derived from different strands of DNA.
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Figure 6: Primed CRISPR adaptation in cells producing unit-sized crRNA transcripts with or without Cas6e. (A) Results of primed adaptation experiment (see ‘Materials and Methods’ section) with indicated cells are shown. The leader-proximal end of CRISPR cassette was amplified using an appropriate primer pair, amplification products were separated by agarose gel electrophoresis and revealed by ethidium bromide staining. (B) DNA fragments corresponding to expanded CRISPR cassettes shown in (A) were subjected to Illumina sequencing. Statistics for reads corresponding to spacers derived from priming protospacer-containing plasmid is presented. ‘Derived spacers’ refers to spacers that originate from recognition of protospacers with correct AAG PAM but are either excised from target DNA with short shifts in the upstream or downstream direction or are inverted during acquisition in CRISPR array. (C) Mapping of spacers acquired in (A) on the priming protospacer plasmid is schematically shown. The position of the priming protospacer (purple triangle) as well as known features of the plasmid are shown. The heights of red bars indicate the efficiency (number of times) a spacer arising from a protospacer in this position was observed. Red bars protruding inside and outside of circles indicating the protospacer plasmid show spacers derived from different strands of DNA.

Mentions: Cells carrying a cas6eH20A allele and expressing crRNA from natural CRISPR arrays are incapable of primed adaptation (18). We followed CRISPR adaptation in cells harboring pG8_crRNA transformed with compatible plasmid containing protospacer. Individual transformants were grown at conditions of induction of cas genes in the absence of ampicillin in the medium, thus allowing the protospacer plasmid loss. After overnight growth, culture aliquots were analyzed by PCR to monitor CRISPR array expansion. KD263 cells transformed with protospacer plasmid were used as a control. The results are presented in Figure 6A. As expected, adaptation was observed in KD263 cells (18,32). Similar levels of adaptation were also observed in KD390 and H20A cells transformed with protospacer plasmid. A somewhat reduced adaptation level was detected in Δcas6e cultures. No adaptation was observed in cells harboring a plasmid without a protospacer. Therefore, unit-sized crRNA generated without Cas6e processing appears to support primed adaptation and the endoribonucleolytic activity of Cas6e (and in fact the Cas6e protein itself) is dispensable in this context.


The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system.

Semenova E, Kuznedelov K, Datsenko KA, Boudry PM, Savitskaya EE, Medvedeva S, Beloglazova N, Logacheva M, Yakunin AF, Severinov K - Nucleic Acids Res. (2015)

Primed CRISPR adaptation in cells producing unit-sized crRNA transcripts with or without Cas6e. (A) Results of primed adaptation experiment (see ‘Materials and Methods’ section) with indicated cells are shown. The leader-proximal end of CRISPR cassette was amplified using an appropriate primer pair, amplification products were separated by agarose gel electrophoresis and revealed by ethidium bromide staining. (B) DNA fragments corresponding to expanded CRISPR cassettes shown in (A) were subjected to Illumina sequencing. Statistics for reads corresponding to spacers derived from priming protospacer-containing plasmid is presented. ‘Derived spacers’ refers to spacers that originate from recognition of protospacers with correct AAG PAM but are either excised from target DNA with short shifts in the upstream or downstream direction or are inverted during acquisition in CRISPR array. (C) Mapping of spacers acquired in (A) on the priming protospacer plasmid is schematically shown. The position of the priming protospacer (purple triangle) as well as known features of the plasmid are shown. The heights of red bars indicate the efficiency (number of times) a spacer arising from a protospacer in this position was observed. Red bars protruding inside and outside of circles indicating the protospacer plasmid show spacers derived from different strands of DNA.
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Related In: Results  -  Collection

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Figure 6: Primed CRISPR adaptation in cells producing unit-sized crRNA transcripts with or without Cas6e. (A) Results of primed adaptation experiment (see ‘Materials and Methods’ section) with indicated cells are shown. The leader-proximal end of CRISPR cassette was amplified using an appropriate primer pair, amplification products were separated by agarose gel electrophoresis and revealed by ethidium bromide staining. (B) DNA fragments corresponding to expanded CRISPR cassettes shown in (A) were subjected to Illumina sequencing. Statistics for reads corresponding to spacers derived from priming protospacer-containing plasmid is presented. ‘Derived spacers’ refers to spacers that originate from recognition of protospacers with correct AAG PAM but are either excised from target DNA with short shifts in the upstream or downstream direction or are inverted during acquisition in CRISPR array. (C) Mapping of spacers acquired in (A) on the priming protospacer plasmid is schematically shown. The position of the priming protospacer (purple triangle) as well as known features of the plasmid are shown. The heights of red bars indicate the efficiency (number of times) a spacer arising from a protospacer in this position was observed. Red bars protruding inside and outside of circles indicating the protospacer plasmid show spacers derived from different strands of DNA.
Mentions: Cells carrying a cas6eH20A allele and expressing crRNA from natural CRISPR arrays are incapable of primed adaptation (18). We followed CRISPR adaptation in cells harboring pG8_crRNA transformed with compatible plasmid containing protospacer. Individual transformants were grown at conditions of induction of cas genes in the absence of ampicillin in the medium, thus allowing the protospacer plasmid loss. After overnight growth, culture aliquots were analyzed by PCR to monitor CRISPR array expansion. KD263 cells transformed with protospacer plasmid were used as a control. The results are presented in Figure 6A. As expected, adaptation was observed in KD263 cells (18,32). Similar levels of adaptation were also observed in KD390 and H20A cells transformed with protospacer plasmid. A somewhat reduced adaptation level was detected in Δcas6e cultures. No adaptation was observed in cells harboring a plasmid without a protospacer. Therefore, unit-sized crRNA generated without Cas6e processing appears to support primed adaptation and the endoribonucleolytic activity of Cas6e (and in fact the Cas6e protein itself) is dispensable in this context.

Bottom Line: Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA.Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e.The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.

Show MeSH
Related in: MedlinePlus