Limits...
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: 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.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

Cas9 determines the PAM sequence of acquired spacersa, c, Genetic composition of the CRISPR-Cas loci tested for spacer during phage infection (a), or in the absence of infection, with the experimental set up shown in Fig. S4 (c). b, d, Sequence logos obtained after the alignment of the 3’ flanking sequences of the protospacers matched by the newly acquired spacers in panels a and c, respectively. Numbers indicate the positions of the flanking nucleotides downstream from the spacer. n; number of sequences used in each alignment.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4385744&req=5

Figure 2: Cas9 determines the PAM sequence of acquired spacersa, c, Genetic composition of the CRISPR-Cas loci tested for spacer during phage infection (a), or in the absence of infection, with the experimental set up shown in Fig. S4 (c). b, d, Sequence logos obtained after the alignment of the 3’ flanking sequences of the protospacers matched by the newly acquired spacers in panels a and c, respectively. Numbers indicate the positions of the flanking nucleotides downstream from the spacer. n; number of sequences used in each alignment.

Mentions: Given this newfound requirement in the CRISPR adaptation process and the well-established PAM recognition function of Cas9 during the surveillance and destruction of viral target sequences, we hypothesized that this nuclease could participate in the selection of PAM sequences during spacer acquisition. To test this we exchanged the cas9 genes of S. pyogenes (Sp) and S. thermophilus (St) CRISPR-Cas systems to create two chimeric CRISPR loci: tracrRNASp-cas9St-cas1Sp-cas2Sp-csn2Sp and tracrRNASt-cas9Sp-cas1St-cas2St-csn2St (Fig. 2a). We chose the type II-A CRISPR-Cas system of S. thermophilus (also known as CRISPR323) because it is an ortholog of the S. pyogenes system24. While the PAM sequence for the Sp CRISPR-Cas system is NGG, the PAM sequence for the St system is NGGNG23 (Fig. 2b and Extended Data Table 1). We infected each naïve strain with phage ϕNM4γ4, sequenced the newly acquired spacers, and obtained the PAM of the matching protospacers using WebLogo25. We found that each chimeric system acquired spacers with PAMs that correlated with the cas9, but not the tracrRNA, cas1, cas2 or csn2, allele present (Fig. 2b and Extended Data Table 1). To rule out the possibility that non-functional spacers are negatively selected during phage infection, i.e. they are acquired randomly and only those cells containing spacers with a correct PAM for Cas9 cleavage provide immunity and allow cell survival, we sequenced the PAMs of spacers acquired in the absence of phage infection (Fig. 1c and 2c). Either Cas9Sp or Cas9St were produced in cells overexpressing Cas1Sp, Cas2Sp and Csn2Sp. In this experiment, as explained above, spacers matching chromosomal or plasmid sequences will be acquired. The PCR products containing new spacers were cloned into a commercial vector from which they were sequenced (Extended Data Table 1). Expression of Cas9Sp led to the incorporation of spacers matching protospacers with an NGG PAM sequence, whereas the expression of Cas9St in the same cells shifted the composition of the PAM to NGGNG (Fig. 2d). These results demonstrate that Cas9 specifies PAM sequences to ensure the acquisition of functional spacers during CRISPR adaptation.


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)

Cas9 determines the PAM sequence of acquired spacersa, c, Genetic composition of the CRISPR-Cas loci tested for spacer during phage infection (a), or in the absence of infection, with the experimental set up shown in Fig. S4 (c). b, d, Sequence logos obtained after the alignment of the 3’ flanking sequences of the protospacers matched by the newly acquired spacers in panels a and c, respectively. Numbers indicate the positions of the flanking nucleotides downstream from the spacer. n; number of sequences used in each alignment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4385744&req=5

Figure 2: Cas9 determines the PAM sequence of acquired spacersa, c, Genetic composition of the CRISPR-Cas loci tested for spacer during phage infection (a), or in the absence of infection, with the experimental set up shown in Fig. S4 (c). b, d, Sequence logos obtained after the alignment of the 3’ flanking sequences of the protospacers matched by the newly acquired spacers in panels a and c, respectively. Numbers indicate the positions of the flanking nucleotides downstream from the spacer. n; number of sequences used in each alignment.
Mentions: Given this newfound requirement in the CRISPR adaptation process and the well-established PAM recognition function of Cas9 during the surveillance and destruction of viral target sequences, we hypothesized that this nuclease could participate in the selection of PAM sequences during spacer acquisition. To test this we exchanged the cas9 genes of S. pyogenes (Sp) and S. thermophilus (St) CRISPR-Cas systems to create two chimeric CRISPR loci: tracrRNASp-cas9St-cas1Sp-cas2Sp-csn2Sp and tracrRNASt-cas9Sp-cas1St-cas2St-csn2St (Fig. 2a). We chose the type II-A CRISPR-Cas system of S. thermophilus (also known as CRISPR323) because it is an ortholog of the S. pyogenes system24. While the PAM sequence for the Sp CRISPR-Cas system is NGG, the PAM sequence for the St system is NGGNG23 (Fig. 2b and Extended Data Table 1). We infected each naïve strain with phage ϕNM4γ4, sequenced the newly acquired spacers, and obtained the PAM of the matching protospacers using WebLogo25. We found that each chimeric system acquired spacers with PAMs that correlated with the cas9, but not the tracrRNA, cas1, cas2 or csn2, allele present (Fig. 2b and Extended Data Table 1). To rule out the possibility that non-functional spacers are negatively selected during phage infection, i.e. they are acquired randomly and only those cells containing spacers with a correct PAM for Cas9 cleavage provide immunity and allow cell survival, we sequenced the PAMs of spacers acquired in the absence of phage infection (Fig. 1c and 2c). Either Cas9Sp or Cas9St were produced in cells overexpressing Cas1Sp, Cas2Sp and Csn2Sp. In this experiment, as explained above, spacers matching chromosomal or plasmid sequences will be acquired. The PCR products containing new spacers were cloned into a commercial vector from which they were sequenced (Extended Data Table 1). Expression of Cas9Sp led to the incorporation of spacers matching protospacers with an NGG PAM sequence, whereas the expression of Cas9St in the same cells shifted the composition of the PAM to NGGNG (Fig. 2d). These results demonstrate that Cas9 specifies PAM sequences to ensure the acquisition of functional spacers during CRISPR adaptation.

Bottom Line: 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.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