Journal: Nucleic Acids Research

Article Title: Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

doi: 10.1093/nar/gkaa272

Figure Lengend Snippet: Phylogenetically diverse Cas12a nucleases exhibit varying apparent targeting activities and can process and utilize each other's gRNAs. ( A ) Phylogenetic tree of the Cas12a nucleases analyzed in this work (red) and other Cas12a nucleases that are commonly used or were reported in our previous work (gray). ( B ) Direct repeat sequences of the Cas12a nucleases represented in the phylogenetic tree. Red text indicates non-conserved bases of the gRNA. Note that Fn3Cas12a shares the same gRNA as FnCas12a. ( C ) Representative figure showing the GFP reporter assay in TXTL. A GFP reporter plasmid, Cas12a plasmid, and a targeting or non-targeting gRNA are added to a TXTL reaction. At the beginning of the reactions, each reaction expresses GFP, Cas12a, and the gRNA. The reaction containing the non-targeting gRNA will continue to express GFP as the gRNA will be unable to direct the Cas12a to cleave the GFP reporter plasmid, while the reaction containing the targeting gRNA will halt GFP production cleavage of the remaining reporter plasmid. ( D ) Time-courses for the different Cas12a nucleases in the GFP reporter assay using a targeting (red) or non-targeting (gray) gRNA. In all cases, the target was flanked by a canonical 5′ TTTC PAM and was immediately upstream of the −35 element of the promoter driving GFP expression. Each solid line and shaded region represent the mean and standard deviation from three separate TXTL reactions. ( E ) Fold-reduction in GFP levels for each Cas12a nuclease targeting two different protospacers flanked by ATTTC PAMs. For both target sequences, each Cas12a target and cleave the DNA using each other's full-length (bottom row) and mature (top row) gRNAs. Note that the Adurb193 and HkCas12a share the same processed gRNA sequence. The fold-reduction was calculated using the GFP fluorescence data from the 16 h time-point from the reactions containing the targeting non-targeting gRNA. The error bars represent the standard deviation from three separate TXTL reactions. See for the associated time-courses.

Article Snippet: The empty gRNA expression plasmids were constructed using a previously built plasmid expressing an empty Acidaminococcus sp. BV3L6 Cas12a gRNA (Addgene, Cat: 78956).

Techniques: Reporter Assay, Plasmid Preparation, Expressing, Standard Deviation, Sequencing, Fluorescence

Journal: Nucleic Acids Research

Article Title: Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

doi: 10.1093/nar/gkaa272

Figure Lengend Snippet: The characterized Cas12a nucleases are associated with various apparent cleavage activities and PAM sequences. ( A ) Representative figure showing the TXTL-based PAM screen. A 5N PAM library, Cas12a, and a targeting or non-targeting gRNA are added to a TXTL reaction. After a 16 h incubation at 29°C, the library members containing a recognizable PAMs are cleaved, while the non-PAMs are left remaining in the reaction. The non-PAM members are PCR amplified and prepared for NGS. ( B ) Fold-change plots and PAM wheels representing the depleted motifs from the 5N PAM library for each Cas12a tested. For the fold-change plots, note the inverted y-axis. See for versions of the fold-change plots rescaled to maximize separation between nts. As part of the screen for Fn3Cas12a, 72 out of the 1024 PAM sequences were absent from both libraries or from the targeted library, preventing their inclusion in the PAM wheel. See for the complete list of these sequences.

Article Snippet: The empty gRNA expression plasmids were constructed using a previously built plasmid expressing an empty Acidaminococcus sp. BV3L6 Cas12a gRNA (Addgene, Cat: 78956).

Techniques: Incubation, Amplification

Journal: Nucleic Acids Research

Article Title: Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

doi: 10.1093/nar/gkaa272

Figure Lengend Snippet: Some of the Cas12a nucleases can accommodate a T at the –1 position within the canonical TTTV PAM. Fold-reduction of GFP expression for each Cas12a and motif indicated. The fold-reduction was calculated using the GFP fluorescence data from the 16 h time-point from the TXTL reactions containing the targeting and non-targeting gRNA. The error bars represent the standard deviation from three separate TXTL reactions. See for the associated time-courses.

Article Snippet: The empty gRNA expression plasmids were constructed using a previously built plasmid expressing an empty Acidaminococcus sp. BV3L6 Cas12a gRNA (Addgene, Cat: 78956).

Techniques: Expressing, Fluorescence, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

doi: 10.1093/nar/gkaa272

Figure Lengend Snippet: Altering residues in the PAM domain between PdCas12a and PiCas12a modifies PAM recognition of PiCas12a. ( A ) Sequence alignment of Pd and PiCas12a. These nucleases share 95.7% amino-acid identity, with most of the variations stemming from the 3′ end of the sequence. The black/bold/underlined sequences indicate matching sequences, while regular black text indicates unmatched residues. The red/bolded sequence indicate the residues investigated in this work. The asterisks indicate residues shown to alter PAM specificity when aligned with AsCas12a ( , ) (see Document S1 for alignment with AsCas12a and other Cas12a nucleases). The sequences were aligned using MUSCLE with default settings. ( B ) Fold-reduction of GFP expression for each PiCas12a variant and motif. The fold-reduction was calculated using the GFP fluorescence data from the 16 h time-point from the reactions containing the targeting non-targeting gRNA. Error bars represent the standard deviation from three separate TXTL reactions. See for the associated time-courses.

Article Snippet: The empty gRNA expression plasmids were constructed using a previously built plasmid expressing an empty Acidaminococcus sp. BV3L6 Cas12a gRNA (Addgene, Cat: 78956).

Techniques: Sequencing, Expressing, Variant Assay, Fluorescence, Standard Deviation