Structured Review

GenScript cas13b
HEPN domains mediate RNA cleavage by <t>Cas13b,</t> whose activity is repressed by Csx27 (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic (top) and results (bottom). A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region on the opposite strand of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Schematic (top) and denaturing gel (bottom) showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. Gel lane containing RNA ladder not shown. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5′ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum .
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Images

1) Product Images from "Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28"

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28

Journal: Molecular cell

doi: 10.1016/j.molcel.2016.12.023

HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27 (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic (top) and results (bottom). A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region on the opposite strand of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Schematic (top) and denaturing gel (bottom) showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. Gel lane containing RNA ladder not shown. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5′ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum .
Figure Legend Snippet: HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27 (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic (top) and results (bottom). A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region on the opposite strand of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Schematic (top) and denaturing gel (bottom) showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. Gel lane containing RNA ladder not shown. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5′ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum .

Techniques Used: Activity Assay, Sequencing, Plasmid Preparation, Transformation Assay, Selection, Mutagenesis, Incubation, Electrophoretic Mobility Shift Assay, Labeling

Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR containing or long-DR containing CRISPR arrays from the B. zoohelcum .
Figure Legend Snippet: Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR containing or long-DR containing CRISPR arrays from the B. zoohelcum .

Techniques Used: CRISPR, RNA Sequencing Assay, In Vitro, Synthesized

Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5′ PFS (D) and 3′ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli . (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum .
Figure Legend Snippet: Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5′ PFS (D) and 3′ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli . (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum .

Techniques Used: Expressing, Sequencing

Cas13b is a programmable single-stranded RNase with collateral activity (A) Schematic showing the RNA secondary structure of the cleavage target in complex with a targeting 30-nt spacer connected to short direct repeat (top). Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (bottom). Reactions were incubated for 10 minutes. The ssRNA target is 5′ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5′ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5′ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5′ to the 3′ end of the target. Gel lane containing RNA ladder not shown. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the orange bars indicate the 5′ PFS and 3′ PFS sequences. The orange letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity after 60 minutes of incubation, with the 5′ PFS tested as A, and the 3′ PFS tested as ANN (bottom). The orange 3′ PFS letters represent the RNA bases at the second and third 3′ PFS position within each target ssRNA. Gel lane containing RNA ladder not shown. Dashed line indicates two separate gels shown side by side. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 5 ). Gel lane containing RNA ladder not shown. (E) .
Figure Legend Snippet: Cas13b is a programmable single-stranded RNase with collateral activity (A) Schematic showing the RNA secondary structure of the cleavage target in complex with a targeting 30-nt spacer connected to short direct repeat (top). Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (bottom). Reactions were incubated for 10 minutes. The ssRNA target is 5′ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5′ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5′ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5′ to the 3′ end of the target. Gel lane containing RNA ladder not shown. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the orange bars indicate the 5′ PFS and 3′ PFS sequences. The orange letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity after 60 minutes of incubation, with the 5′ PFS tested as A, and the 3′ PFS tested as ANN (bottom). The orange 3′ PFS letters represent the RNA bases at the second and third 3′ PFS position within each target ssRNA. Gel lane containing RNA ladder not shown. Dashed line indicates two separate gels shown side by side. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 5 ). Gel lane containing RNA ladder not shown. (E) .

Techniques Used: Activity Assay, Incubation, Labeling

Efficient RNA targeting by Cas13b is correlated with local RNA accessibility (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b ) against 10 6 Monte Carlo simulations: empirical P -values from left to right of 3e–6, 1e–6, 8.7e–3, 6e–6. (D) Empirical cumulative distribution function of safely depleted B. zoohelcum spacers over all genes from 5′ UTR into gene and from 3′ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum .
Figure Legend Snippet: Efficient RNA targeting by Cas13b is correlated with local RNA accessibility (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b ) against 10 6 Monte Carlo simulations: empirical P -values from left to right of 3e–6, 1e–6, 8.7e–3, 6e–6. (D) Empirical cumulative distribution function of safely depleted B. zoohelcum spacers over all genes from 5′ UTR into gene and from 3′ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum .

Techniques Used:

Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. ( B) .
Figure Legend Snippet: Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. ( B) .

Techniques Used: CRISPR

2) Product Images from "Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28"

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

Journal: bioRxiv

doi: 10.1101/092577

Cas13b is a programmable single-stranded RNase with collateral activity. (A) Schematics showing the RNA secondary structure of the cleavage target alone (left top), cleavage target in complex with a targeting 30nt spacer (left middle), and crRNAs with either the short or long direct repeat (left bottom). For the cleavage target alone, the dark bases show the spacer-targeted region. Numbers along the targets represent nucleotide distance from the 5’ labeled end. Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (right). Reactions were incubated for 10 minutes. The short and long direct repeat crRNAs cleave ssRNA with similar efficiency. The ssRNA target is 5’ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5’ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5’ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5’ to the 3’ end of the target. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the 5’ PFS and 3’ PFS sequences are indicated by the gold bars. The gold letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the preference for the NAN or NNA 3’ PFS after 60 minutes of incubation (bottom). The 5’ PFS is tested as A, and the 3’ PFS is tested as ANN. The gold 3’ PFS letters represent the RNA bases at the second and third 3’ PFS position within each target ssRNA. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 7 ) is substituted with seven monomers of the variable loop base in the gel (top). Denaturing gel showing cleavage bands by variable loop base (bottom). The targets were incubated for 30 minutes. (E) Denaturing gel showing BzCas13b collateral cleavage activity after 30 minutes of incubation, with schematic of cleavage experiment to the right. Two crRNAs (A and B) target substrate 1 (1A and 1B) or substrate 2 (2A and 2B).
Figure Legend Snippet: Cas13b is a programmable single-stranded RNase with collateral activity. (A) Schematics showing the RNA secondary structure of the cleavage target alone (left top), cleavage target in complex with a targeting 30nt spacer (left middle), and crRNAs with either the short or long direct repeat (left bottom). For the cleavage target alone, the dark bases show the spacer-targeted region. Numbers along the targets represent nucleotide distance from the 5’ labeled end. Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (right). Reactions were incubated for 10 minutes. The short and long direct repeat crRNAs cleave ssRNA with similar efficiency. The ssRNA target is 5’ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5’ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5’ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5’ to the 3’ end of the target. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the 5’ PFS and 3’ PFS sequences are indicated by the gold bars. The gold letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the preference for the NAN or NNA 3’ PFS after 60 minutes of incubation (bottom). The 5’ PFS is tested as A, and the 3’ PFS is tested as ANN. The gold 3’ PFS letters represent the RNA bases at the second and third 3’ PFS position within each target ssRNA. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 7 ) is substituted with seven monomers of the variable loop base in the gel (top). Denaturing gel showing cleavage bands by variable loop base (bottom). The targets were incubated for 30 minutes. (E) Denaturing gel showing BzCas13b collateral cleavage activity after 30 minutes of incubation, with schematic of cleavage experiment to the right. Two crRNAs (A and B) target substrate 1 (1A and 1B) or substrate 2 (2A and 2B).

Techniques Used: Activity Assay, Labeling, Incubation

HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27. (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic and results. A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Denaturing gel showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5’ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum wildtype and R116A/H121A/R1177A/H1182A Cas13b.
Figure Legend Snippet: HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27. (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic and results. A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Denaturing gel showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5’ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum wildtype and R116A/H121A/R1177A/H1182A Cas13b.

Techniques Used: Activity Assay, Sequencing, Plasmid Preparation, Transformation Assay, Selection, Mutagenesis, Incubation, Electrophoretic Mobility Shift Assay, Labeling

Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants. (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR-or long-DR-containing CRISPR arrays from the B. zoohelcum genome by either wildtype or HEPN mutant BzCas13b (D1, R116A/H121A; D2, R1177A/H1182A; Q, R116A/H121A/R1177A/ H1182A). The schematic shows fragment lengths of a cleaved CRISPR array.
Figure Legend Snippet: Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants. (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR-or long-DR-containing CRISPR arrays from the B. zoohelcum genome by either wildtype or HEPN mutant BzCas13b (D1, R116A/H121A; D2, R1177A/H1182A; Q, R116A/H121A/R1177A/ H1182A). The schematic shows fragment lengths of a cleaved CRISPR array.

Techniques Used: CRISPR, RNA Sequencing Assay, In Vitro, Synthesized, Mutagenesis

Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS. (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5’ PFS (D) and 3’ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli. (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum spacers, with abundances colored by type of spacer.
Figure Legend Snippet: Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS. (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5’ PFS (D) and 3’ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli. (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum spacers, with abundances colored by type of spacer.

Techniques Used: Expressing, Sequencing

Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b. (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. (B) A schematic phylogenetic tree of the subtype VI-B loci. Loci with csx27 (brown) comprise variant VI-B1; loci with csx28 (gold) comprise variant VI-B2.
Figure Legend Snippet: Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b. (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. (B) A schematic phylogenetic tree of the subtype VI-B loci. Loci with csx27 (brown) comprise variant VI-B1; loci with csx28 (gold) comprise variant VI-B2.

Techniques Used: CRISPR, Variant Assay

Efficient RNA targeting by Cas13b is correlated with local RNA accessibility. (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b) against 10 6 Monte Carlo simulations: empirical P-values from left to right of 3e-6, 1e- 6, 8.7e-3, 6e-6. (D) Empirical cumulative distribution of safely depleted B. zoohelcum spacers over all genes from 5’ UTR into gene and from 3’ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum spacers.
Figure Legend Snippet: Efficient RNA targeting by Cas13b is correlated with local RNA accessibility. (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b) against 10 6 Monte Carlo simulations: empirical P-values from left to right of 3e-6, 1e- 6, 8.7e-3, 6e-6. (D) Empirical cumulative distribution of safely depleted B. zoohelcum spacers over all genes from 5’ UTR into gene and from 3’ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum spacers.

Techniques Used:

Related Articles

Clone Assay:

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct ( ) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct (Table S1) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Positron Emission Tomography:

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct ( ) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct (Table S1) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Synthesized:

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct ( ) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct (Table S1) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Protein Purification:

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct (Table S1) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Expressing:

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct ( ) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct (Table S1) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Plasmid Preparation:

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct ( ) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28
Article Snippet: .. BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs). .. The BzCas13b expression construct (Table S1) was transformed into One Shot® BL21(DE3)pLysE (Invitrogen) cells.

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    GenScript cas13b
    HEPN domains mediate RNA cleavage by <t>Cas13b,</t> whose activity is repressed by Csx27 (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic (top) and results (bottom). A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region on the opposite strand of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Schematic (top) and denaturing gel (bottom) showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. Gel lane containing RNA ladder not shown. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5′ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum .
    Cas13b, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27 (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic (top) and results (bottom). A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region on the opposite strand of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Schematic (top) and denaturing gel (bottom) showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. Gel lane containing RNA ladder not shown. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5′ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum .

    Journal: Molecular cell

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1016/j.molcel.2016.12.023

    Figure Lengend Snippet: HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27 (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic (top) and results (bottom). A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region on the opposite strand of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Schematic (top) and denaturing gel (bottom) showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. Gel lane containing RNA ladder not shown. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5′ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum .

    Article Snippet: The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: Activity Assay, Sequencing, Plasmid Preparation, Transformation Assay, Selection, Mutagenesis, Incubation, Electrophoretic Mobility Shift Assay, Labeling

    Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR containing or long-DR containing CRISPR arrays from the B. zoohelcum .

    Journal: Molecular cell

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1016/j.molcel.2016.12.023

    Figure Lengend Snippet: Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR containing or long-DR containing CRISPR arrays from the B. zoohelcum .

    Article Snippet: The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: CRISPR, RNA Sequencing Assay, In Vitro, Synthesized

    Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5′ PFS (D) and 3′ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli . (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum .

    Journal: Molecular cell

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1016/j.molcel.2016.12.023

    Figure Lengend Snippet: Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5′ PFS (D) and 3′ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli . (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum .

    Article Snippet: The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: Expressing, Sequencing

    Cas13b is a programmable single-stranded RNase with collateral activity (A) Schematic showing the RNA secondary structure of the cleavage target in complex with a targeting 30-nt spacer connected to short direct repeat (top). Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (bottom). Reactions were incubated for 10 minutes. The ssRNA target is 5′ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5′ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5′ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5′ to the 3′ end of the target. Gel lane containing RNA ladder not shown. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the orange bars indicate the 5′ PFS and 3′ PFS sequences. The orange letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity after 60 minutes of incubation, with the 5′ PFS tested as A, and the 3′ PFS tested as ANN (bottom). The orange 3′ PFS letters represent the RNA bases at the second and third 3′ PFS position within each target ssRNA. Gel lane containing RNA ladder not shown. Dashed line indicates two separate gels shown side by side. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 5 ). Gel lane containing RNA ladder not shown. (E) .

    Journal: Molecular cell

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1016/j.molcel.2016.12.023

    Figure Lengend Snippet: Cas13b is a programmable single-stranded RNase with collateral activity (A) Schematic showing the RNA secondary structure of the cleavage target in complex with a targeting 30-nt spacer connected to short direct repeat (top). Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (bottom). Reactions were incubated for 10 minutes. The ssRNA target is 5′ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5′ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5′ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5′ to the 3′ end of the target. Gel lane containing RNA ladder not shown. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the orange bars indicate the 5′ PFS and 3′ PFS sequences. The orange letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity after 60 minutes of incubation, with the 5′ PFS tested as A, and the 3′ PFS tested as ANN (bottom). The orange 3′ PFS letters represent the RNA bases at the second and third 3′ PFS position within each target ssRNA. Gel lane containing RNA ladder not shown. Dashed line indicates two separate gels shown side by side. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 5 ). Gel lane containing RNA ladder not shown. (E) .

    Article Snippet: The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: Activity Assay, Incubation, Labeling

    Efficient RNA targeting by Cas13b is correlated with local RNA accessibility (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b ) against 10 6 Monte Carlo simulations: empirical P -values from left to right of 3e–6, 1e–6, 8.7e–3, 6e–6. (D) Empirical cumulative distribution function of safely depleted B. zoohelcum spacers over all genes from 5′ UTR into gene and from 3′ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum .

    Journal: Molecular cell

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1016/j.molcel.2016.12.023

    Figure Lengend Snippet: Efficient RNA targeting by Cas13b is correlated with local RNA accessibility (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b ) against 10 6 Monte Carlo simulations: empirical P -values from left to right of 3e–6, 1e–6, 8.7e–3, 6e–6. (D) Empirical cumulative distribution function of safely depleted B. zoohelcum spacers over all genes from 5′ UTR into gene and from 3′ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum .

    Article Snippet: The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques:

    Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. ( B) .

    Journal: Molecular cell

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1016/j.molcel.2016.12.023

    Figure Lengend Snippet: Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. ( B) .

    Article Snippet: The mammalian codon-optimized gene for Cas13b ( B. zoohelcum ) was synthesized (GenScript) and inserted into a bacterial expression vector (6x His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: CRISPR

    Cas13b is a programmable single-stranded RNase with collateral activity. (A) Schematics showing the RNA secondary structure of the cleavage target alone (left top), cleavage target in complex with a targeting 30nt spacer (left middle), and crRNAs with either the short or long direct repeat (left bottom). For the cleavage target alone, the dark bases show the spacer-targeted region. Numbers along the targets represent nucleotide distance from the 5’ labeled end. Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (right). Reactions were incubated for 10 minutes. The short and long direct repeat crRNAs cleave ssRNA with similar efficiency. The ssRNA target is 5’ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5’ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5’ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5’ to the 3’ end of the target. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the 5’ PFS and 3’ PFS sequences are indicated by the gold bars. The gold letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the preference for the NAN or NNA 3’ PFS after 60 minutes of incubation (bottom). The 5’ PFS is tested as A, and the 3’ PFS is tested as ANN. The gold 3’ PFS letters represent the RNA bases at the second and third 3’ PFS position within each target ssRNA. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 7 ) is substituted with seven monomers of the variable loop base in the gel (top). Denaturing gel showing cleavage bands by variable loop base (bottom). The targets were incubated for 30 minutes. (E) Denaturing gel showing BzCas13b collateral cleavage activity after 30 minutes of incubation, with schematic of cleavage experiment to the right. Two crRNAs (A and B) target substrate 1 (1A and 1B) or substrate 2 (2A and 2B).

    Journal: bioRxiv

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1101/092577

    Figure Lengend Snippet: Cas13b is a programmable single-stranded RNase with collateral activity. (A) Schematics showing the RNA secondary structure of the cleavage target alone (left top), cleavage target in complex with a targeting 30nt spacer (left middle), and crRNAs with either the short or long direct repeat (left bottom). For the cleavage target alone, the dark bases show the spacer-targeted region. Numbers along the targets represent nucleotide distance from the 5’ labeled end. Denaturing gel demonstrating short direct repeat and long direct repeat crRNA-mediated ssRNA cleavage (right). Reactions were incubated for 10 minutes. The short and long direct repeat crRNAs cleave ssRNA with similar efficiency. The ssRNA target is 5’ labeled with IRDye 800. Three cleavage sites are observed. (B) Schematic showing three numbered protospacers for each colored 5’ PFS on a body-labeled ssRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the requirement for a D 5’ PFS (not C) (bottom). Reactions were incubated for 60 minutes. crRNAs correspond to protospacer numbered from the 5’ to the 3’ end of the target. (C) Schematic of a body-labeled ssRNA substrate being targeted by a crRNA (top). The protospacer region is highlighted in blue, and the 5’ PFS and 3’ PFS sequences are indicated by the gold bars. The gold letters represent the altered sequences in the experiment. Denaturing gel showing crRNA-guided ssRNA cleavage activity demonstrating the preference for the NAN or NNA 3’ PFS after 60 minutes of incubation (bottom). The 5’ PFS is tested as A, and the 3’ PFS is tested as ANN. The gold 3’ PFS letters represent the RNA bases at the second and third 3’ PFS position within each target ssRNA. (D) Schematic showing the secondary structure of the body labeled ssRNA targets used in the denaturing gel. The variable loop of the schematic (represented as N 7 ) is substituted with seven monomers of the variable loop base in the gel (top). Denaturing gel showing cleavage bands by variable loop base (bottom). The targets were incubated for 30 minutes. (E) Denaturing gel showing BzCas13b collateral cleavage activity after 30 minutes of incubation, with schematic of cleavage experiment to the right. Two crRNAs (A and B) target substrate 1 (1A and 1B) or substrate 2 (2A and 2B).

    Article Snippet: BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: Activity Assay, Labeling, Incubation

    HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27. (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic and results. A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Denaturing gel showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5’ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum wildtype and R116A/H121A/R1177A/H1182A Cas13b.

    Journal: bioRxiv

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1101/092577

    Figure Lengend Snippet: HEPN domains mediate RNA cleavage by Cas13b, whose activity is repressed by Csx27. (A) Protospacer design for MS2 phage plaque drop assay to test RNA interference (left). Plaque drop assay for full B. zoohelcum VI-B1 locus (center) and bzcas13b (right). (B) DNA interference assay schematic and results. A target sequence is placed in frame at the start of the transcribed bla gene that confers ampicillin resistance or in a non-transcribed region of the same target plasmid. Target plasmids were co-transformed with bzcas13b plasmid or empty vectors conferring chloramphenicol resistance and plated on double selection antibiotic plates. (C) Denaturing gel showing ssRNA cleavage activity of WT and HEPN mutant BzCas13b. The protein and targeting crRNA complexes were incubated for 10 minutes. (D) Electrophoretic Mobility Shift Assay (EMSA) graph showing the affinity of BzCas13b proteins and targeting crRNA complex to a 5’ end labeled ssRNA. The EMSA assay was performed with supplemental EDTA to reduce any cleavage activity. (E) Quantification of MS2 phage plaque drop assay with B. zoohelcum wildtype and R116A/H121A/R1177A/H1182A Cas13b.

    Article Snippet: BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: Activity Assay, Sequencing, Plasmid Preparation, Transformation Assay, Selection, Mutagenesis, Incubation, Electrophoretic Mobility Shift Assay, Labeling

    Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants. (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR-or long-DR-containing CRISPR arrays from the B. zoohelcum genome by either wildtype or HEPN mutant BzCas13b (D1, R116A/H121A; D2, R1177A/H1182A; Q, R116A/H121A/R1177A/ H1182A). The schematic shows fragment lengths of a cleaved CRISPR array.

    Journal: bioRxiv

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1101/092577

    Figure Lengend Snippet: Cas13b from the VI-B1 locus processes a CRISPR array with two direct repeat variants. (A) RNA-Sequencing of the native VI-B1 locus from Bergeyella zoohelcum ATCC 43767. (B) Denaturing gel showing cleavage products of in vitro synthesized short-DR-or long-DR-containing CRISPR arrays from the B. zoohelcum genome by either wildtype or HEPN mutant BzCas13b (D1, R116A/H121A; D2, R1177A/H1182A; Q, R116A/H121A/R1177A/ H1182A). The schematic shows fragment lengths of a cleaved CRISPR array.

    Article Snippet: BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: CRISPR, RNA Sequencing Assay, In Vitro, Synthesized, Mutagenesis

    Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS. (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5’ PFS (D) and 3’ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli. (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum spacers, with abundances colored by type of spacer.

    Journal: bioRxiv

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1101/092577

    Figure Lengend Snippet: Heterologous expression of Cas13b mediates knockdown of E. coli essential genes by a double-sided PFS. (A) Design of E. coli essential gene screen to determine targeting rules of nucleic acid interference. (B) Manhattan plots of mean spacer depletions mapped over 45 genes and aggregated across normalized gene distance for either the full B. zoohelcum VI-B1 locus (left) or cas13b alone (right), with non-targeting spacers in gray, safely depleted spacers ( > 5σ above mean depletion of non-targeting spacers) above blue line, and strongly depleted spacers (top 1% depleted) above red line. For the full locus, 36,142 targeting spacers and 630 non-targeting spacers passed QC filter. Of the targeting, 367 are strongly depleted and 1672 are safely depleted. For cas13b alone, 35,272 targeting spacers and 633 non-targeting spacers passed QC filter. Of the targeting, 359 are strongly depleted and 6374 are safely depleted. (C) Weblogo of sequence motifs of strongly depleted B. zoohelcum spacers. (D) Normalized PFS score matrix, where each score is the ratio of number of safely depleted B. zoohelcum spacers to total number of spacers for a given PFS, scaled so that maximum PFS score is 1. (E) Spacers targeting kanamycin to validate PFS targeting rules of 5’ PFS (D) and 3’ PFS (NAN or NNA). (F) Schematic of kanamycin validation screen for B. zoohelcum cas13b in E. coli. (G) Results from kanamycin validation screen; spacer abundances versus control for individual B. zoohelcum spacers, with abundances colored by type of spacer.

    Article Snippet: BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: Expressing, Sequencing

    Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b. (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. (B) A schematic phylogenetic tree of the subtype VI-B loci. Loci with csx27 (brown) comprise variant VI-B1; loci with csx28 (gold) comprise variant VI-B2.

    Journal: bioRxiv

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1101/092577

    Figure Lengend Snippet: Discovery of two Class 2 CRISPR-Cas systems, subtype VI-B1 and VI-B2, containing Cas13b. (A) Bioinformatic pipeline to discover putative Class 2 CRISPR loci lacking Cas1 and Cas2. (B) A schematic phylogenetic tree of the subtype VI-B loci. Loci with csx27 (brown) comprise variant VI-B1; loci with csx28 (gold) comprise variant VI-B2.

    Article Snippet: BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: CRISPR, Variant Assay

    Efficient RNA targeting by Cas13b is correlated with local RNA accessibility. (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b) against 10 6 Monte Carlo simulations: empirical P-values from left to right of 3e-6, 1e- 6, 8.7e-3, 6e-6. (D) Empirical cumulative distribution of safely depleted B. zoohelcum spacers over all genes from 5’ UTR into gene and from 3’ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum spacers.

    Journal: bioRxiv

    Article Title: Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

    doi: 10.1101/092577

    Figure Lengend Snippet: Efficient RNA targeting by Cas13b is correlated with local RNA accessibility. (A) Methodology of secondary structure-mediated spacer efficiency analysis of E. coli essential gene screen data with Vienna RNAplfold. (B) Optimization of top 1 accuracy (computationally predicted most accessible spacer matches the top experimentally depleted spacer) and top 3 accuracy (computationally predicted top spacer falls in top 3 experimentally depleted spacers) on randomly selected B. zoohelcum training dataset using RNAplfold, first with u start and u end , and then with W and L . (C) Performance of optimized RNAplfold model on randomly selected B. zoohelcum testing dataset (48 cohorts for full B. zoohelcum VI-B1 locus, 56 cohorts for bzcas13b) against 10 6 Monte Carlo simulations: empirical P-values from left to right of 3e-6, 1e- 6, 8.7e-3, 6e-6. (D) Empirical cumulative distribution of safely depleted B. zoohelcum spacers over all genes from 5’ UTR into gene and from 3’ UTR into gene. Yellow line separates UTR and gene, red line is theoretical cumulative distribution function of uniformly distributed spacers, and blue line is empirical cumulative distribution of safely depleted B. zoohelcum spacers.

    Article Snippet: BzCas13b Protein Purification The mammalian codon-optimized gene for Cas13b (B. zoohelcum) was synthesized (GenScript) and inserted into a bacterial expression vector (6× His/Twin Strep SUMO, a pET based vector received as a gift from Ilya Finkelstein) after cleaving the plasmid with the BamHI and NotI restriction enzymes and cloning in the gene using Gibson Assembly® Master Mix (New England Biolabs).

    Techniques: