Limits...
Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation.

Li M, Wang R, Xiang H - Nucleic Acids Res. (2014)

Bottom Line: Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation.Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences.We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China.

Show MeSH
Adaptation priming tolerates 23 PAM sequences. (A) Spacer acquisition assay performed to target plasmids with protospacer1 preceded by 62 different PAM sequences (TTC and TTG not included). For each plasmid, three independent transformant colonies were tested, and a representative result is shown. The wild-type CRISPR generates a ∼200-bp PCR product, and larger-sized PCR products indicate that new spacers have been acquired causing expanded CRISPRs. (B) Scheme showing the provirus-derived sequence (protospacer13v, framed) that is partially matched by spacer13. (C) Spacer acquisition assay performed to target plasmids (pNNN13v) containing protospacer13v that is preceded by seven different PAM sequences, including TTC and TTG.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4066796&req=5

Figure 2: Adaptation priming tolerates 23 PAM sequences. (A) Spacer acquisition assay performed to target plasmids with protospacer1 preceded by 62 different PAM sequences (TTC and TTG not included). For each plasmid, three independent transformant colonies were tested, and a representative result is shown. The wild-type CRISPR generates a ∼200-bp PCR product, and larger-sized PCR products indicate that new spacers have been acquired causing expanded CRISPRs. (B) Scheme showing the provirus-derived sequence (protospacer13v, framed) that is partially matched by spacer13. (C) Spacer acquisition assay performed to target plasmids (pNNN13v) containing protospacer13v that is preceded by seven different PAM sequences, including TTC and TTG.

Mentions: The majority of PAM mutations should block target interference because of the strict PAM selectivity. However, priming adaptation can counter these escape mutations by acquiring new spacers from the target-bearing DNA (17). Whether all types of PAM mutations can be tolerated to prime adaptation remains unknown. Therefore, for the 60 plasmids that escaped CRISPR interference, a spacer acquisition assay was subsequently performed against their transformants after a 5-day cultivation. Specific primers were used to amplify the CRISPR leader end, and arrays with new spacers incorporated were expected to produce larger-sized PCR products (20). Strikingly, CRISPR adaptation was observed for nearly one third of these escape plasmids (Figure 2A), revealing 19 different PAM sequences that are recognized to prime adaptation, which we described as PAP (priming adaptation permissive). Correspondingly, the remaining 41 tri-nucleotides were referred as PAIN (priming adaptation and interference non-permissive) sequences. Notably, PAP sequences are not all equally favoured, because faint expanded bands were observed for pTAA1, pCAG1, pCCG1 and pCGC1, whereas for most of the other plasmids, evident CRISPR expansion was observed (Figure 2A).


Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation.

Li M, Wang R, Xiang H - Nucleic Acids Res. (2014)

Adaptation priming tolerates 23 PAM sequences. (A) Spacer acquisition assay performed to target plasmids with protospacer1 preceded by 62 different PAM sequences (TTC and TTG not included). For each plasmid, three independent transformant colonies were tested, and a representative result is shown. The wild-type CRISPR generates a ∼200-bp PCR product, and larger-sized PCR products indicate that new spacers have been acquired causing expanded CRISPRs. (B) Scheme showing the provirus-derived sequence (protospacer13v, framed) that is partially matched by spacer13. (C) Spacer acquisition assay performed to target plasmids (pNNN13v) containing protospacer13v that is preceded by seven different PAM sequences, including TTC and TTG.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066796&req=5

Figure 2: Adaptation priming tolerates 23 PAM sequences. (A) Spacer acquisition assay performed to target plasmids with protospacer1 preceded by 62 different PAM sequences (TTC and TTG not included). For each plasmid, three independent transformant colonies were tested, and a representative result is shown. The wild-type CRISPR generates a ∼200-bp PCR product, and larger-sized PCR products indicate that new spacers have been acquired causing expanded CRISPRs. (B) Scheme showing the provirus-derived sequence (protospacer13v, framed) that is partially matched by spacer13. (C) Spacer acquisition assay performed to target plasmids (pNNN13v) containing protospacer13v that is preceded by seven different PAM sequences, including TTC and TTG.
Mentions: The majority of PAM mutations should block target interference because of the strict PAM selectivity. However, priming adaptation can counter these escape mutations by acquiring new spacers from the target-bearing DNA (17). Whether all types of PAM mutations can be tolerated to prime adaptation remains unknown. Therefore, for the 60 plasmids that escaped CRISPR interference, a spacer acquisition assay was subsequently performed against their transformants after a 5-day cultivation. Specific primers were used to amplify the CRISPR leader end, and arrays with new spacers incorporated were expected to produce larger-sized PCR products (20). Strikingly, CRISPR adaptation was observed for nearly one third of these escape plasmids (Figure 2A), revealing 19 different PAM sequences that are recognized to prime adaptation, which we described as PAP (priming adaptation permissive). Correspondingly, the remaining 41 tri-nucleotides were referred as PAIN (priming adaptation and interference non-permissive) sequences. Notably, PAP sequences are not all equally favoured, because faint expanded bands were observed for pTAA1, pCAG1, pCCG1 and pCGC1, whereas for most of the other plasmids, evident CRISPR expansion was observed (Figure 2A).

Bottom Line: Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation.Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences.We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China.

Show MeSH