1. Volume 62, Issue 1, Pages 137-147 (April 2016)
Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems 
Ryan T. Leenay, Kenneth R. Maksimchuk, Rebecca A. Slotkowski, Roma N. Agrawal, Ahmed A. Gomaa, Alexandra E. Briner, Rodolphe Barrangou, Chase L. Beisel 
Molecular Cell 
Volume 62, Issue 1, Pages (April 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 62, 137-147DOI: (10.1016/j.molcel.2016.02.031)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Overview of the PAM Screen Achieved by NOT-Gate Repression, PAM-SCANR
(A) The screening platform consists of a library of potential PAM sequences cloned upstream of the lacI promoter. Immediately downstream of lacI is the LacI-dependent lacZ promoter controlling expression of GFP. A catalytically dead CRISPR-Cas system is targeted to a protospacer within the lacI promoter, resulting in GFP fluorescence only in the presence of a functional PAM.
(B) Cells harboring a functional PAM can be isolated by fluorescence-activated cell sorting (FACS). The screen can identify functional PAMs comprehensively or individually.
(C) Tuning the stringency of the screen with IPTG. The presence of IPTG reduces the fraction of LacI proteins capable of gene repression, thereby facilitating the upregulation of GFP. More IPTG lowers the threshold for GFP upregulation, in turn relaxing the stringency of the screen. Related to Figures S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Representing PAMs and Their Enrichment with the PAM Wheel
(A–C) The sequence logos and PAM wheels are shown for prior high-throughput depletion screens conducted with the wild-type type II-A Cas9 from S. pyogenes (A), the VQR variant of the S. pyogenes Cas9 (B), and the type II-A Cas9 from S. aureus (C) (Kleinstiver et al., 2015; Ran et al., 2015). The raw sequencing reads were processed to generate the standard sequence logo and the PAM wheel. For the PAM wheel, sequences from the inner to outer circle match the PAM read moving away from the protospacer. Colors correspond to the relative frequency of the innermost nucleotide. For a given sequence, the area of the sector in the PAM wheel is directly proportional to the relative enrichment in the library. For the S. aureus Cas9, the +1 and +2 positions of the PAM exhibited similar enrichments and therefore were treated as a 2-nt gap, as seen by the smaller circle in (C). Related to Krona S1–S4.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Comprehensive Screening of the Type I-E CRISPR-Cas System in E. coli
(A) PAM wheel and sequence logo for comprehensive screening of the E. coli I-E Cascade with a 4-nt PAM library. See Figure 2 for more information about the PAM wheel. Comparisons between the two library replicates of the screen are shown. Related to Figure S3 and Krona S5.
(B) Correlating library enrichment and PAM-SCANR fluorescence for individually cloned PAMs.
(C) Validating representative PAMs by gene repression. The PAMs were cloned upstream of the −35 element of the lacZ promoter controlling expression of GFP. The fold repression is in relation to a non-targeting RNA.
(D) Validating representative PAMs by cell killing. Representative PAMs and the PAM-SCANR protospacer were recombineered into E. coli’s genome, and the cells were transformed with a plasmid encoding cas3. The fold reduction in the transformation efficiency of the guide RNA plasmid over the non-targeting plasmid is shown. Values represent the mean and SEM from experiments starting with at least three independent colonies.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Stringency Tuning and Screening for the S. pyogenes and S. thermophilus Type II-A Cas9s
(A) Stringency tuning of PAM-SCANR constructs with the catalytically dead Cas9 (dCas9). A sub-saturating concentration of IPTG reveals the NAG weak PAM for the S. pyogenes Cas9.
(B) Individual screening of the S. thermophilus CRISPR1 Cas9. A 5-nt PAM library with a 2-nt gap was used to identify individual PAM sequences. Sequences identified multiple times are specified out of 38 sequencing runs total, with the number of appearances in parentheses. The mean, single-cell fluorescence is shown for post-sorted cultures harboring the indicated PAM sequence.
(C) Comprehensive screening of the S. thermophilus CRISPR1 Cas9. The PAM wheel and sequence logo are shown for the same replicate subjected to individual sequencing. See Figure 2 for more information about the PAM wheel. Related to Figures S2–S4 and Krona S6.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Comprehensive Screening of the Type V-A Cpf1 Protein from F. novicida
(A) PAM wheel and sequence logo for comprehensive screening of the V-A catalytically dead Cpf1 (dCpf1) with a 4-nt PAM library. See Figure 2 for more information about the PAM wheel. Comparisons between the two biological replicates of the screen are shown. Related to Figure S3 and Krona S7.
(B) Validating representative PAMs by gene repression. See Figure 3C for detailed information. Values represent the mean and SEM from experiments starting with at least three independent colonies.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Comprehensive Screening of the Uncharacterized Type I-C System from B. halodurans
(A) PAM wheel and sequence logo for comprehensive screening with a 4-nt PAM library. See Figure 2 for more information about the PAM wheel. Comparisons between the two biological replicates of the screen are shown. Related to Figure S3 and Krona S8.
(B) Validating representative PAMs by gene repression. See Figure 3D for detailed information. Values represent the mean and SEM from experiments starting with at least three independent colonies.
Molecular Cell  , DOI: ( /j.molcel )
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