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Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Heler R, Samai P, Modell JW, Weiner C, Goldberg GW, Bikard D, Marraffini LA - Nature (2015)

Bottom Line: The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively.Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process.These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.

ABSTRACT
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

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The S. pyogenes type II CRISPR-Cas system displays a strong bias for the acquisition of spacers matching viral protospacers with NGG PAMsa, Analysis of bacteriophage-insensitive mutant colonies using PCR and agarose gel electrophoresis, representative of five technical replicates. Bacteria and phage were mixed in top agar and incubated overnight. DNA was isolated from individual colonies resistant to phage infection and used as template for a PCR reaction with primers (arrows) H182 and H183 (Extended Data Table 2), which amplify the 5’ end of the S. pyogenes CRISPR array. The size of the PCR band indicates the number of new spacers (shown at the top of the gel). Cells without additional spacers resist infection by a CRISPR-independent mechanisms, presumably envelope resistance. b, Analysis of acquired spacers during phage infection of a population of bacteria carrying the S. pyogenes type II CRISPR-Cas system. Liquid cultures of bacteria were infected with phage, surviving cells were collected at the end of the infection, DNA extracted and used as template for a PCR reaction as described above. Amplification products were separated by agarose gel electrophoresis and the DNA of the bands corresponding to products with additional spacers was extracted and sent for Mi-Seq next generation sequencing. Reads corresponding to newly acquired spacers were plotted according to their position in the phage ϕNM4γ4 genome (x-axis) and their abundance (y-axis). Each dot represents a unique spacer sequence; blue and red dots indicate a corresponding protospacer with an NGG or non-NGG PAM. Top and bottom plots indicate protospacers in the top and bottom strands of the ϕNM4γ4 DNA. The map as well as the different functions of the phage genes are indicated in between the plots. The raw data used to make this graph is in the Supplementary file. c, Weblogo showing the conservation of the 5’ flanking sequences of 10,000 protospacers randomly selected from the experiment shown in b. Absolute conservation of the NGG PAM was observed.
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Figure 4: The S. pyogenes type II CRISPR-Cas system displays a strong bias for the acquisition of spacers matching viral protospacers with NGG PAMsa, Analysis of bacteriophage-insensitive mutant colonies using PCR and agarose gel electrophoresis, representative of five technical replicates. Bacteria and phage were mixed in top agar and incubated overnight. DNA was isolated from individual colonies resistant to phage infection and used as template for a PCR reaction with primers (arrows) H182 and H183 (Extended Data Table 2), which amplify the 5’ end of the S. pyogenes CRISPR array. The size of the PCR band indicates the number of new spacers (shown at the top of the gel). Cells without additional spacers resist infection by a CRISPR-independent mechanisms, presumably envelope resistance. b, Analysis of acquired spacers during phage infection of a population of bacteria carrying the S. pyogenes type II CRISPR-Cas system. Liquid cultures of bacteria were infected with phage, surviving cells were collected at the end of the infection, DNA extracted and used as template for a PCR reaction as described above. Amplification products were separated by agarose gel electrophoresis and the DNA of the bands corresponding to products with additional spacers was extracted and sent for Mi-Seq next generation sequencing. Reads corresponding to newly acquired spacers were plotted according to their position in the phage ϕNM4γ4 genome (x-axis) and their abundance (y-axis). Each dot represents a unique spacer sequence; blue and red dots indicate a corresponding protospacer with an NGG or non-NGG PAM. Top and bottom plots indicate protospacers in the top and bottom strands of the ϕNM4γ4 DNA. The map as well as the different functions of the phage genes are indicated in between the plots. The raw data used to make this graph is in the Supplementary file. c, Weblogo showing the conservation of the 5’ flanking sequences of 10,000 protospacers randomly selected from the experiment shown in b. Absolute conservation of the NGG PAM was observed.

Mentions: To investigate the mechanisms of recognition of PAM-adjacent protospacers during spacer acquisition, we cloned the type II-A CRISPR-Cas locus of S. pyogenes (Fig. 1a) into the staphylococcal vector pC19416 and introduced the resulting plasmid [pWJ40 (ref.17)] into Staphylococcus aureus RN422018, a strain lacking CRISPR-Cas loci. We chose this experimental system because it facilitates the genetic manipulation of the S. pyogenes CRISPR-Cas system. We first tested the ability of the cells to mount adaptive CRISPR immunity by infecting them with the staphylococcal phage ϕNM4γ4, a lytic variant of ϕNM419 (see Methods for a description of ϕNM4γ4 isolation). Plate-based assays performed by mixing bacteria and phage in top agar allowed the selection of phage-resistant colonies that were checked by PCR to look for the expansion of the CRISPR array (Extended Data Fig. 1a). On average 50 % of the colonies acquired one or more spacers (8/13, 5/11 and 7/16 in three independent experiments), whereas the rest of the resistant colonies survived phage infection by a non-CRISPR mechanism, most likely including phage receptor mutations (Extended Data Fig. 2a). To maximize the capture of new spacer sequences, we performed the same assay in liquid and recovered surviving bacteria at the end of the phage challenge. These were analyzed by PCR of the CRISPR array and the amplification products of expanded loci were subjected to Illumina MiSeq sequencing to determine the extent of spacer acquisition. Analysis of 2.96 million reads detected protospacers adjacent to 2083 out of 2687 NGG sequences present in the viral genome, although with variation in the frequency of acquisition of each sequence (Extended Data Fig. 1b). The data revealed a prominent selection of spacers matching protospacers with downstream NGG PAM sequences (99.97 %, Extended Data Fig. 1c). The acquisition of new spacers by cells in liquid culture proved to be simple and highly efficient, providing the possibility to look at millions of new spacers in a single step. It was therefore implemented in the rest of our studies.


Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Heler R, Samai P, Modell JW, Weiner C, Goldberg GW, Bikard D, Marraffini LA - Nature (2015)

The S. pyogenes type II CRISPR-Cas system displays a strong bias for the acquisition of spacers matching viral protospacers with NGG PAMsa, Analysis of bacteriophage-insensitive mutant colonies using PCR and agarose gel electrophoresis, representative of five technical replicates. Bacteria and phage were mixed in top agar and incubated overnight. DNA was isolated from individual colonies resistant to phage infection and used as template for a PCR reaction with primers (arrows) H182 and H183 (Extended Data Table 2), which amplify the 5’ end of the S. pyogenes CRISPR array. The size of the PCR band indicates the number of new spacers (shown at the top of the gel). Cells without additional spacers resist infection by a CRISPR-independent mechanisms, presumably envelope resistance. b, Analysis of acquired spacers during phage infection of a population of bacteria carrying the S. pyogenes type II CRISPR-Cas system. Liquid cultures of bacteria were infected with phage, surviving cells were collected at the end of the infection, DNA extracted and used as template for a PCR reaction as described above. Amplification products were separated by agarose gel electrophoresis and the DNA of the bands corresponding to products with additional spacers was extracted and sent for Mi-Seq next generation sequencing. Reads corresponding to newly acquired spacers were plotted according to their position in the phage ϕNM4γ4 genome (x-axis) and their abundance (y-axis). Each dot represents a unique spacer sequence; blue and red dots indicate a corresponding protospacer with an NGG or non-NGG PAM. Top and bottom plots indicate protospacers in the top and bottom strands of the ϕNM4γ4 DNA. The map as well as the different functions of the phage genes are indicated in between the plots. The raw data used to make this graph is in the Supplementary file. c, Weblogo showing the conservation of the 5’ flanking sequences of 10,000 protospacers randomly selected from the experiment shown in b. Absolute conservation of the NGG PAM was observed.
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Figure 4: The S. pyogenes type II CRISPR-Cas system displays a strong bias for the acquisition of spacers matching viral protospacers with NGG PAMsa, Analysis of bacteriophage-insensitive mutant colonies using PCR and agarose gel electrophoresis, representative of five technical replicates. Bacteria and phage were mixed in top agar and incubated overnight. DNA was isolated from individual colonies resistant to phage infection and used as template for a PCR reaction with primers (arrows) H182 and H183 (Extended Data Table 2), which amplify the 5’ end of the S. pyogenes CRISPR array. The size of the PCR band indicates the number of new spacers (shown at the top of the gel). Cells without additional spacers resist infection by a CRISPR-independent mechanisms, presumably envelope resistance. b, Analysis of acquired spacers during phage infection of a population of bacteria carrying the S. pyogenes type II CRISPR-Cas system. Liquid cultures of bacteria were infected with phage, surviving cells were collected at the end of the infection, DNA extracted and used as template for a PCR reaction as described above. Amplification products were separated by agarose gel electrophoresis and the DNA of the bands corresponding to products with additional spacers was extracted and sent for Mi-Seq next generation sequencing. Reads corresponding to newly acquired spacers were plotted according to their position in the phage ϕNM4γ4 genome (x-axis) and their abundance (y-axis). Each dot represents a unique spacer sequence; blue and red dots indicate a corresponding protospacer with an NGG or non-NGG PAM. Top and bottom plots indicate protospacers in the top and bottom strands of the ϕNM4γ4 DNA. The map as well as the different functions of the phage genes are indicated in between the plots. The raw data used to make this graph is in the Supplementary file. c, Weblogo showing the conservation of the 5’ flanking sequences of 10,000 protospacers randomly selected from the experiment shown in b. Absolute conservation of the NGG PAM was observed.
Mentions: To investigate the mechanisms of recognition of PAM-adjacent protospacers during spacer acquisition, we cloned the type II-A CRISPR-Cas locus of S. pyogenes (Fig. 1a) into the staphylococcal vector pC19416 and introduced the resulting plasmid [pWJ40 (ref.17)] into Staphylococcus aureus RN422018, a strain lacking CRISPR-Cas loci. We chose this experimental system because it facilitates the genetic manipulation of the S. pyogenes CRISPR-Cas system. We first tested the ability of the cells to mount adaptive CRISPR immunity by infecting them with the staphylococcal phage ϕNM4γ4, a lytic variant of ϕNM419 (see Methods for a description of ϕNM4γ4 isolation). Plate-based assays performed by mixing bacteria and phage in top agar allowed the selection of phage-resistant colonies that were checked by PCR to look for the expansion of the CRISPR array (Extended Data Fig. 1a). On average 50 % of the colonies acquired one or more spacers (8/13, 5/11 and 7/16 in three independent experiments), whereas the rest of the resistant colonies survived phage infection by a non-CRISPR mechanism, most likely including phage receptor mutations (Extended Data Fig. 2a). To maximize the capture of new spacer sequences, we performed the same assay in liquid and recovered surviving bacteria at the end of the phage challenge. These were analyzed by PCR of the CRISPR array and the amplification products of expanded loci were subjected to Illumina MiSeq sequencing to determine the extent of spacer acquisition. Analysis of 2.96 million reads detected protospacers adjacent to 2083 out of 2687 NGG sequences present in the viral genome, although with variation in the frequency of acquisition of each sequence (Extended Data Fig. 1b). The data revealed a prominent selection of spacers matching protospacers with downstream NGG PAM sequences (99.97 %, Extended Data Fig. 1c). The acquisition of new spacers by cells in liquid culture proved to be simple and highly efficient, providing the possibility to look at millions of new spacers in a single step. It was therefore implemented in the rest of our studies.

Bottom Line: The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively.Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process.These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.

ABSTRACT
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

Show MeSH
Related in: MedlinePlus