Journal: Cell host & microbe

Article Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.

doi: 10.1016/j.chom.2019.05.004

Figure Lengend Snippet: Figure 1. Structure of LbCas12a-crRNA-AcrVA1 Ternary Complex (A) Domain organization of LbCas12a. (B) Cryo-EM map of LbCas12a-crRNA-AcrVA1 complex color-coded as in (A). AcrVA1 is in cyan. (C) Cartoon presentation of the overall structure of LbCas12a-crRNA-AcrVA1. The blue and red boxes indicate the interaction interfaces between AcrVA1 and LbCas12a. (D) Close-up view of crRNA and AcrVA1. Cryo-EM density is shown in mesh. Shown below is a schematic of the crRNA used in this study, with disordered segment shown in a gray background. (E) Fitting of AcrVA1 to the corresponding cryo-EM densities. See also Figures S1–S3; Tables S1 and S2; Video S1.

Article Snippet: The inactive LbCas12a (D832A) plasmid was obtained from Addgene (Chen et al., 2018).

Techniques: Cryo-EM Sample Prep

Journal: Cell host & microbe

Article Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.

doi: 10.1016/j.chom.2019.05.004

Figure Lengend Snippet: Figure 2. Interactions between LbCas12a and AcrVA1 (A) Close-up view of LbCas12a-AcrVA1 in- teractions involved in the NUC lobe. Observed interactions are shown as red dashed lines. (B) Close-up view of LbCas12a-AcrVA1 in- teractions involved in the REC1 domain in the REC lobe. (C) In vitro DNA cleavage assay of LbCas12a in the presence of wild-type, truncated, and mutated AcrVA1. (D) Superimposition of the structures of AcrVA1- bound LbCas12a-crRNA (this study, LbCas12a shown in transparent red) and the dsDNA-bound LbCas12a-crRNA (PDB: 5XUS, LbCas12a shown in transparent gray). Target (TS) and non-target strands (NTSs) of the PAM duplex are colored in magenta and black, respectively. (E and F) Interactions between LbCas12a and PAM duplex from a structure of LbCas12a-crRNA- dsDNA complex (PDB: 5XUS). See also Figure S3 and Video S1.

Article Snippet: The inactive LbCas12a (D832A) plasmid was obtained from Addgene (Chen et al., 2018).

Techniques: In Vitro, DNA Cleavage Assay

Journal: Cell host & microbe

Article Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.

doi: 10.1016/j.chom.2019.05.004

Figure Lengend Snippet: Figure 3. AcrVA1 Cleaves crRNA in a Cas12a-Dependent Manner (A) A TBE-urea denaturing gel showing crRNA cleavage by the AcrVA1-bound LbCas12a-crRNA complex. dLbCas12a: dead mutant LbCas12a. (B) D95A/S96A mutant reduced the RNase activity of AcrVA1. (C) Electrostatic potential surface of AcrVA1. The positively charged helices a1-2 function as a clamp that tethers the seed segment of crRNA (green). (D) Representative denaturing gels showing the crRNA cleavage by either the WT AcrVA1 or the mutants of the helices a1-2. (E) AcrVA1 mutants with reduced RNase activity (R41A, H42A, and H45A) failed to inhibit LbCas12a. (F) Key residues for RNase activity (R41, H42, and H45) are located in helix a2 and are close to the +5 position of crRNA. See also Figure S3.

Article Snippet: The inactive LbCas12a (D832A) plasmid was obtained from Addgene (Chen et al., 2018).

Techniques: Mutagenesis, Activity Assay

Journal: Cell host & microbe

Article Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.

doi: 10.1016/j.chom.2019.05.004

Figure Lengend Snippet: Figure 4. Structure of LbCas12a-crRNA-AcrVA4 Complex (A and B) Cryo-EM maps of AcrVA4 dimer in complex with one copy (A) or two copies (B) of LbCas12a-crRNA. Color codes are as in Figure 1A. Two AcrVA4 molecules are shown in cyan and teal, respectively. (C and D) Cartoon presentation of LbCas12a-crRNA-AcrVA4 structures corresponding to (A) and (B). (E) Close-up view of crRNA and AcrVA4. Cryo-EM density is shown in mesh. (F) Fitting of AcrVA4 dimer to the corresponding cryo-EM densities. (G) Cartoon presentation of AcrVA4. See also Figures S4 and S5; Tables S1 and S2; Video S2.

Article Snippet: The inactive LbCas12a (D832A) plasmid was obtained from Addgene (Chen et al., 2018).

Techniques: Cryo-EM Sample Prep

Journal: Cell host & microbe

Article Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.

doi: 10.1016/j.chom.2019.05.004

Figure Lengend Snippet: Figure 5. Interactions between LbCas12a and AcrVA4 (A) AcrVA4 bound to the pocket created by crRNA, the WED and REC2 domains, and the BH motif. (B) Detailed interactions between AcrVA4 and WED domain. Interactions are shown as red dashed lines. (C) Detailed interactions between AcrVA4 and the BH motif. (D) Detailed interactions between AcrVA4 and LbCas12a-crRNA involving REC2 domain and crRNA. (E) In vitro DNA cleavage assay of LbCas12a in the presence of the wild-type and mutated AcrVA4. See also Video S1.

Article Snippet: The inactive LbCas12a (D832A) plasmid was obtained from Addgene (Chen et al., 2018).

Techniques: In Vitro, DNA Cleavage Assay

Journal: Cell host & microbe

Article Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.

doi: 10.1016/j.chom.2019.05.004

Figure Lengend Snippet: Figure 6. Inhibition Mechanism of AcrVA4 (A) Superimposition of structures of AcrVA4-bound LbCas12a-crRNA (this study) and dsDNA-bound LbCas12a-crRNA (PDB: 5XUS). (B) Structure alignment shows that dsDNA binding induced substantial movements of the residues involved in AcrVA4 binding. Shifts in the residues are indicated by black arrows. The conformation of Glu894 in AcrVA4-bound structure is incompatible with crRNA-DNA heteroduplex in DNA-bound structure, and vice versa. (C) AcrVA4 directly interacts with the BH motif and thus would prevent its movement. The lid region is in a closed conformation. (D) The BH motif is involved in conformational transition of the lid region upon dsDNA binding. The lid region is in an open conformation. (E) The BH motif contributes to stabilization of the crRNA-DNA hybridization upon dsDNA binding. The interaction between the BH motif and the heteroduplex is indicated by red dashed lines. (F) Structural comparisons of AcrVA4-bound LbCas12a-crRNA (colored, this study) with LbCas12a-crRNA (gray, PDB: 5ID6), PAM-bound FnCas12a-crRNA (gray, PDB: 6GTC), and R-loop-bound FnCas12a before cleavage (gray, PDB: 5NFV) and after cleavage (gray, PDB: 5MGA). Shown below is a close-up view and the lid region in each state. See also Figure S5.

Article Snippet: The inactive LbCas12a (D832A) plasmid was obtained from Addgene (Chen et al., 2018).

Techniques: Inhibition, Binding Assay, DNA Hybridization

Journal: Cell host & microbe

Article Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.

doi: 10.1016/j.chom.2019.05.004

Figure Lengend Snippet: Figure 7. Structure of Cas12a-crRNA- AcrVA4 in Complex with dsDNA (A) Cryo-EM map of LbCas12a-crRNA-AcrVA4- dsDNA complex color-coded as in Figure 1A. Target strand (TS) and non-target strand (NTS) are colored in magenta and black, respectively. (B) Close-up view of the partially formed crRNA- DNA heteroduplex in the context of cryo-EM density map (upper panel) compared to the crystal structure of fully formed crRNA-DNA heteroduplex (bottom panel, PDB: 5XUS). See also Figure S6.

Article Snippet: The inactive LbCas12a (D832A) plasmid was obtained from Addgene (Chen et al., 2018).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Programmable RNA detection with CRISPR-Cas12a

doi: 10.1101/2023.01.29.525716

Figure Lengend Snippet: a. Schematic representation of a crRNA-Cas12a complex performing trans- cleavage of ssDNA reporters following the recognition of two split-activators. b-d. Fold change at t=60 minutes of in vitro trans- cleavage assay with Cas12a orthologs (red = LbCas12a, green = AsCas12a, orange = ErCas12a) activated by individual truncated ssDNA activators of length 6–20 nt e-g. Heat maps representing fold change at t=60 minutes of an in vitro trans- cleavage assay activated by combinations of truncated ssDNA activators of different lengths ranging from 6–14 nt in the Pp and Pd regions. The reactions contained 25 nM truncated ssDNA GFP-activators, 60 nM Cas12a, and 120 nM crGFP and were incubated for 60 min at 37°C. Error bars represent SD (n=3). Statistical analysis was performed using a two-tailed t-test where ns = not significant with p > 0.05, and the asterisks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001) denote significant differences.

Article Snippet: Briefly, plasmids expressing LbCas12a and AsCas12a were obtained from Addgene (a gift from Zhang lab and Doudna lab) and directly used for protein expression.

Techniques: In Vitro, Cleavage Assay, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: Programmable RNA detection with CRISPR-Cas12a

doi: 10.1101/2023.01.29.525716

Figure Lengend Snippet: a. Schematic of WT vs SAHARA CRISPR-Cas systems for the detection of a target nucleic acid. b. ssDNA activators were designed with point mutations across the length of the activator. GFP-activator mutants were designed for a WT CRISPR activator (24-nt) and a SAHARA split activator system (12-nt +12-nt). The mutation location is identified by ‘M’ following the nucleotide number where the base has been changed to guanine (3’ to 5’ direction). c-e. Comparison of fold changes for the in vitro trans- cleavage assay between WT and SAHARA activator mutants normalized to the WT activator for Cas12a orthologs (c: LbCas12a, d: AsCas12a, and e: ErCas12a). Comparison of RFU values at t=60 min for the in vitro trans- cleavage assay between WT and SAHARA. Statistical analysis was performed using a two-tailed t-test where ns = not significant with P > 0.05, and the asterisks (*P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001) denote significant differences.

Article Snippet: Briefly, plasmids expressing LbCas12a and AsCas12a were obtained from Addgene (a gift from Zhang lab and Doudna lab) and directly used for protein expression.

Techniques: CRISPR, Mutagenesis, Comparison, In Vitro, Cleavage Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Programmable RNA detection with CRISPR-Cas12a

doi: 10.1101/2023.01.29.525716

Figure Lengend Snippet: a-d. PAM sequence tolerance of Cas12a orthologs (red = LbCas12a, green = AsCas12a, orange = ErCas12a) coupled with SAHARA. Comparison of trans- cleavage activity among S12 dsDNA activators containing different PAM sequences (n=3). The PAM sequences TTTA, AAAT, and VVVN were assessed. e-g. Cas12a orthologs tolerate a wide range of GC contents in the crRNA and S12 dsDNA for RNA detection (n=3). h-j. The trans- cleavage activity of Cas12a with varying concentrations of S12 after incubation for 60 min at 37°C. Error bars represent SD (n=3).

Article Snippet: Briefly, plasmids expressing LbCas12a and AsCas12a were obtained from Addgene (a gift from Zhang lab and Doudna lab) and directly used for protein expression.

Techniques: Sequencing, Comparison, Activity Assay, RNA Detection, Incubation