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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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Purification of a Cas9-Cas1-Cas2-Csn2 complexesa, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET16b vector (generating pKW07) to add an N-terminal histidyl tag to Cas9 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography. SDS-PAGE followed by Coomassie stain of the purified proteins revealed a co-purifying protein that was identified as Cas1 by mass spectrometry, representative of five technical replicates. Mass spectrometry identification of all the eluted proteins co-purifying with Cas9 is shown in Extended Data Table 2. b, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET23a vector (generating pKW06) to add an C-terminal histidyl tag to Csn2 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography followed by ion exchange chromatography. The elution fractions that constituted the peak containing the complex (Fig. 3a) were separated by SDS-PAGE and visualized by Coomassie staining, representative of three technical replicates.
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Figure 7: Purification of a Cas9-Cas1-Cas2-Csn2 complexesa, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET16b vector (generating pKW07) to add an N-terminal histidyl tag to Cas9 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography. SDS-PAGE followed by Coomassie stain of the purified proteins revealed a co-purifying protein that was identified as Cas1 by mass spectrometry, representative of five technical replicates. Mass spectrometry identification of all the eluted proteins co-purifying with Cas9 is shown in Extended Data Table 2. b, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET23a vector (generating pKW06) to add an C-terminal histidyl tag to Csn2 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography followed by ion exchange chromatography. The elution fractions that constituted the peak containing the complex (Fig. 3a) were separated by SDS-PAGE and visualized by Coomassie staining, representative of three technical replicates.

Mentions: In type I CRISPR-Cas systems, Cas1 and Cas2 form a complex13 and the dsDNA nuclease activity of Cas1 has been implicated in the initial cleavage of the invading viral DNA to generate a new spacer26. The genetic analyses presented above suggest that in the type II S. pyogenes CRISPR-Cas system, the PAM-binding function of Cas9 observed in vitro7 could specify a PAM-adjacent site of cleavage for Cas1, or other members of the spacer acquisition machinery. This would guarantee that newly acquired spacers have the correct PAM needed for Cas9 activity later in this immune pathway. This hypothesis predicts an interaction between Cas9 and Cas1, Cas2 and/or Csn2. To test this we expressed the type II Cas operon in E. coli, using a histidyl tagged version of Cas9, and looked for other proteins that co-purify. We observed an abundant co-purifying protein with an apparent molecular weight close to 33 kDa, the expected size of Cas1 (Extended Data Fig. 4a). Mass spectrometry confirmed the identity of both of these proteins as well as the presence of Cas2 and Csn2 co-purifying with Cas9 (Extended Data Table 2). This result suggested the formation of a Cas9-Cas1-Cas2-Csn2 complex and therefore we explored other purification strategies to unequivocally determine its existence. We were able to isolate a Cas9-Cas1-Cas2-Csn2 complex when the histidyl tag was added to Csn2 (Fig. 3a and b). The identity of the purified proteins was confirmed by mass spectrometry (Extended Data Table 3). This demonstrates a biochemical link between the Cas9 nuclease and the other Cas proteins that function exclusively to acquire new spacers, supporting the role of Cas9 as a PAM specificity factor in the adaptation phase of CRISPR immunity.


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)

Purification of a Cas9-Cas1-Cas2-Csn2 complexesa, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET16b vector (generating pKW07) to add an N-terminal histidyl tag to Cas9 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography. SDS-PAGE followed by Coomassie stain of the purified proteins revealed a co-purifying protein that was identified as Cas1 by mass spectrometry, representative of five technical replicates. Mass spectrometry identification of all the eluted proteins co-purifying with Cas9 is shown in Extended Data Table 2. b, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET23a vector (generating pKW06) to add an C-terminal histidyl tag to Csn2 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography followed by ion exchange chromatography. The elution fractions that constituted the peak containing the complex (Fig. 3a) were separated by SDS-PAGE and visualized by Coomassie staining, representative of three technical replicates.
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Figure 7: Purification of a Cas9-Cas1-Cas2-Csn2 complexesa, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET16b vector (generating pKW07) to add an N-terminal histidyl tag to Cas9 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography. SDS-PAGE followed by Coomassie stain of the purified proteins revealed a co-purifying protein that was identified as Cas1 by mass spectrometry, representative of five technical replicates. Mass spectrometry identification of all the eluted proteins co-purifying with Cas9 is shown in Extended Data Table 2. b, The cas9-cas1-cas2-csn2 operon of S. pyogenes SF370 was cloned into the pET23a vector (generating pKW06) to add an C-terminal histidyl tag to Csn2 and express all proteins in E. coli. Purification was performed using Ni-NTA affinity chromatography followed by ion exchange chromatography. The elution fractions that constituted the peak containing the complex (Fig. 3a) were separated by SDS-PAGE and visualized by Coomassie staining, representative of three technical replicates.
Mentions: In type I CRISPR-Cas systems, Cas1 and Cas2 form a complex13 and the dsDNA nuclease activity of Cas1 has been implicated in the initial cleavage of the invading viral DNA to generate a new spacer26. The genetic analyses presented above suggest that in the type II S. pyogenes CRISPR-Cas system, the PAM-binding function of Cas9 observed in vitro7 could specify a PAM-adjacent site of cleavage for Cas1, or other members of the spacer acquisition machinery. This would guarantee that newly acquired spacers have the correct PAM needed for Cas9 activity later in this immune pathway. This hypothesis predicts an interaction between Cas9 and Cas1, Cas2 and/or Csn2. To test this we expressed the type II Cas operon in E. coli, using a histidyl tagged version of Cas9, and looked for other proteins that co-purify. We observed an abundant co-purifying protein with an apparent molecular weight close to 33 kDa, the expected size of Cas1 (Extended Data Fig. 4a). Mass spectrometry confirmed the identity of both of these proteins as well as the presence of Cas2 and Csn2 co-purifying with Cas9 (Extended Data Table 2). This result suggested the formation of a Cas9-Cas1-Cas2-Csn2 complex and therefore we explored other purification strategies to unequivocally determine its existence. We were able to isolate a Cas9-Cas1-Cas2-Csn2 complex when the histidyl tag was added to Csn2 (Fig. 3a and b). The identity of the purified proteins was confirmed by mass spectrometry (Extended Data Table 3). This demonstrates a biochemical link between the Cas9 nuclease and the other Cas proteins that function exclusively to acquire new spacers, supporting the role of Cas9 as a PAM specificity factor in the adaptation phase of CRISPR immunity.

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