Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Engineering LwaCas13a to enhance RNA knockdown efficiency (A) Schematic diagram of the mammalian dual-fluorescence reporter system. (B) Sequence alignment of LwaCas13a and its homologous proteins. Candidate mutagenic regions are marked in the red box. (C) Comparison of the cleavage effect of LwaCas13a mutant and WT in HEK293T cells. The fluorescence intensity level of EGFP represents the targeted cleavage effect of Cas13a (left); the fluorescence intensity level of mCherry represents the collateral cleavage effect of Cas13a (right). (D) Structure-guided mutation and corresponding activity assessment. Mutation candidates in the HEPN1-II domain are marked in red lines. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 4). ∗ p < 0.05; ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Knockdown, Fluorescence, Sequencing, Comparison, Mutagenesis, Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Activity profiling of Cas13a mutations (A) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a double mutants. (B) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a triple mutants. (C) The targeted (EGFP) and collateral (mCherry) cleavage activities of all the Cas13a beneficial mutants. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 4). ∗∗∗ p < 0.001.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Activity Assay, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Screening and performance of crRNA mutants (A) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with 3' end C 3 N extensions. (B) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with 3' end U 3 V (A/C/G) or U 4 V (A/C/G) extensions. (C) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with 5' end N 4 extensions. (D) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with crRNA mutants with both 5' and 3' end beneficial extensions. (E) The targeted (EGFP) and collateral (mCherry) cleavage activities of Cas13a combined with all the beneficial crRNA mutants. Data were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test, comparing each group to the non-targeting (NT) crRNA control. Data are presented as median ± IQR ( n = 4). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Cytotoxicity evaluation of CRISPR-Cas13a system with engineered crRNAs (A) Cell viability was determined using CCK-8 reagent at 24, 48, 72, and 96 h post-transfection. The line chart is the overall graph. WT, wild-type LwaCas13a; NT-crRNA, nontarget crRNA; T-crRNA, target crRNA. (B) Bar charts show specific data for each time point. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: CRISPR, CCK-8 Assay, Transfection, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Engineered CRISPR-Cas13a system exhibits superior cleavage activity in mammalian cells (A) Cleavage activity was determined by dual fluorescence system in HEK293T cells. NT, nontarget crRNA; T, target crRNA ( n = 4). (B) Knockdown efficiency of six endogenous transcripts was determined by qPCR ( n = 3). (C and D) Antiviral assays targeting MuV HN (left) and P (right) genes. Viral titers in Vero-E6 cell supernatants were determined by qPCR ( n = 3). Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: CRISPR, Activity Assay, Fluorescence, Knockdown, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Engineered Cas13a mutant and crRNA mutants contribute to enhancing Cas-crRNA interactions (A) BLI kinetic analysis of the interaction between wild-type/mutant Cas13a and wild-type/mutant crRNAs. Kinetic parameters were from a 1:1 binding model. (B) Comparison of the KD values in graph A. Data were compared using a two-tailed unpaired Mann-Whitney U test. Data are presented as median ± IQR ( n = 4). ∗ p < 0.05; ns, not significant.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: Mutagenesis, Binding Assay, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications

doi: 10.1016/j.omtn.2025.102811

Figure Lengend Snippet: Structural modeling of wild-type and engineered CRISPR-Cas13a system (A) Structural diagram of the binary complex between wild-type/mutant Cas13a and wild-type crRNA, along with a local magnification. (B) Structural diagram of the binary complex between mutant Cas13a and M3crRNA, along with a local magnification. Mutant amino acid residues are labeled in rose; amino acid residues in contact with the 34th to 42nd bases of crRNA are labeled in pink.

Article Snippet: Human codon-optimized Cas13a expression plasmid was obtained from Addgene (#90097).

Techniques: CRISPR, Mutagenesis, Labeling

Journal: Biosensors & bioelectronics

Article Title: Reining in Cas13a activity with N-terminal removable tags expands Cas13a based molecular sensing and enables precise gene interference.

doi: 10.1016/j.bios.2023.115138

Figure Lengend Snippet: Fig. 1. N-terminus composite SUMO tag (His-Twinstrep-Smt3-) reins in Cas13a activation by obstructing crRNA docking. (A) A tandem purification procedure (immobilized nickel, in buffer digestion, and ion exchange chromatography) resulted in SUMO-Cas13a and Cas13a prep of high yield and purity. (B) Standard collateral cleavage assay revealed full suppression of target RNA dependent activation of Cas13a by SUMO tag. (C) The suppression was evident on the graphic bar plot of enzyme activity (V0) derived from standard collateral cleavage assay. (D) Spiking of 0.15 U SUMO protease Ulp1 in ongoing collateral cleavage assay released RNase activity of SUMO-Cas13a. (E) EMSA gel shit assay comprised of 200 nM crRNA and 100, 200 and 400 nM SUMO-Cas13a or Cas13a in collateral cleavage buffer was conducted. Cas13a and Ulp1 digested SUMO-Cas13a caused significant up-shift at low protein concentration while SUMO-Cas13a only did at highest concentration.

Article Snippet: We established a lenti-viral construct for simultaneous expression of SUMO-Cas13a effective enzyme and crRNA cloning cassette (Fig. 6B) based on the widely adapted LentiCRISPRv2GFP (Addgene #82416) system.

Techniques: Activation Assay, Purification, Ion Exchange Chromatography, Cleavage Assay, Activity Assay, Derivative Assay, Protein Concentration, Concentration Assay

Journal: Molecular cell

Article Title: CRISPR-Cas13 inhibitors block RNA-editing in bacteria and mammalian cells

doi: 10.1016/j.molcel.2020.03.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides crRNAs This study Table S4 Recombinant DNA Plasmids This study Table S5 Antibodies mouse monoclonal GFP antibody Biolegend Cat# 668206 anti-his tag anti-body Thermo Fisher Scientific Cat# TA150087 Bacterial and Virus Strains E. coli c-3000 ATCC Cat# 15597 NEB 5-alpha Competent E. coli New England BioLabs Cat# C2987H BL21 Competent E. coli New England BioLabs Cat# C2530H E. coli bacteriophage MS2 ATCC Cat# 15597-B1 Leptotrichia buccalis DSM 1135 ATCC Cat# 14201 E. coli c3000-derived strains This study Table S6 Chemicals, Peptides, and Recombinant Proteins LB Broth Base Thermo Fisher Scientific Cat# 12780029 LB Agar Fisher Scientific Cat# BP1425-500 SOC medium New England BioLabs Cat# B9020S ampicillin Fisher Scientific Cat#11593027 chloramphenicol Sigma Cat# C0378 kanamycin Fisher Scientific Cat# 25389-94-0 gentamicin ACROS Cat# 1405-41-0 IPTG Invitrogen Cat# 15529019 penicillin-streptomycin Thermo Fisher Scientific Cat# 10378016 fetal bovine serum VWR Seradigm Cat# 97068-085 LipofectAmine 3000 Thermo Fisher Scientific Cat# L3000015 Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 RMPI 1640 Medium Thermo Fisher Scientific Cat# 11875119 protease inhibitor cockt Thermo Fisher Scientific Cat# 78425 Critical Commercial Assays QIAGEN Plasmid Mini Kit QIAGEN Cat# 12125 myTXTL Sigma 70 Master Mix Kit Arbor Bioscience Cat# 507024 AMPure XP beads Beckman Coulter Cat# A63882 Dynabeads™ Sheep anti-Mouse IgG Invitrogen Cat# 11201D Pierce™ His Protein Interaction Pull-Down Kit Thermo Fisher Scientific Cat# 21277 Pierce™ Gaussia-Firefly Luciferase Dual Assay Kit Thermo Fisher Scientific Cat# 16181 High Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 Maxima SYBR Green qPCR Master Mix Thermo Fisher Scientific Cat# K0252 Direct-zol™ RNA MiniPrep kit Zymo Reseach Cat# R2052 the CellTiter 96 AQueous One Solution Cell Proliferation assay Promega Cat# G3580 Pierce LDH Cytotoxicity Assay Kit Thermo Fisher Scientific Cat# {"type":"entrez-nucleotide","attrs":{"text":"C20300","term_id":"1632571","term_text":"C20300"}} C20300 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216 Open in a separate window KEY RESOURCES TABLE An integrated approach is designed to screen anti-CRISPRs (AcrVIA) candidates A series of prophages are discovered in regions encoding AcrVIAs AcrVIAs inhibit Cas13a RNA targeting in bacteria and human cells AcrVIAs impede programmable RNA base editing by blocking dCas13a-based toolkit

Techniques: Recombinant, Virus, Protease Inhibitor, Plasmid Preparation, Luciferase, Reverse Transcription, SYBR Green Assay, Proliferation Assay, LDH Cytotoxicity Assay

Journal: Molecular cell

Article Title: CRISPR-Cas13 inhibitors block RNA-editing in bacteria and mammalian cells

doi: 10.1016/j.molcel.2020.03.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides crRNAs This study Table S4 Recombinant DNA Plasmids This study Table S5 Antibodies mouse monoclonal GFP antibody Biolegend Cat# 668206 anti-his tag anti-body Thermo Fisher Scientific Cat# TA150087 Bacterial and Virus Strains E. coli c-3000 ATCC Cat# 15597 NEB 5-alpha Competent E. coli New England BioLabs Cat# C2987H BL21 Competent E. coli New England BioLabs Cat# C2530H E. coli bacteriophage MS2 ATCC Cat# 15597-B1 Leptotrichia buccalis DSM 1135 ATCC Cat# 14201 E. coli c3000-derived strains This study Table S6 Chemicals, Peptides, and Recombinant Proteins LB Broth Base Thermo Fisher Scientific Cat# 12780029 LB Agar Fisher Scientific Cat# BP1425-500 SOC medium New England BioLabs Cat# B9020S ampicillin Fisher Scientific Cat#11593027 chloramphenicol Sigma Cat# C0378 kanamycin Fisher Scientific Cat# 25389-94-0 gentamicin ACROS Cat# 1405-41-0 IPTG Invitrogen Cat# 15529019 penicillin-streptomycin Thermo Fisher Scientific Cat# 10378016 fetal bovine serum VWR Seradigm Cat# 97068-085 LipofectAmine 3000 Thermo Fisher Scientific Cat# L3000015 Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 RMPI 1640 Medium Thermo Fisher Scientific Cat# 11875119 protease inhibitor cockt Thermo Fisher Scientific Cat# 78425 Critical Commercial Assays QIAGEN Plasmid Mini Kit QIAGEN Cat# 12125 myTXTL Sigma 70 Master Mix Kit Arbor Bioscience Cat# 507024 AMPure XP beads Beckman Coulter Cat# A63882 Dynabeads™ Sheep anti-Mouse IgG Invitrogen Cat# 11201D Pierce™ His Protein Interaction Pull-Down Kit Thermo Fisher Scientific Cat# 21277 Pierce™ Gaussia-Firefly Luciferase Dual Assay Kit Thermo Fisher Scientific Cat# 16181 High Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 Maxima SYBR Green qPCR Master Mix Thermo Fisher Scientific Cat# K0252 Direct-zol™ RNA MiniPrep kit Zymo Reseach Cat# R2052 the CellTiter 96 AQueous One Solution Cell Proliferation assay Promega Cat# G3580 Pierce LDH Cytotoxicity Assay Kit Thermo Fisher Scientific Cat# {"type":"entrez-nucleotide","attrs":{"text":"C20300","term_id":"1632571","term_text":"C20300"}} C20300 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216 Open in a separate window KEY RESOURCES TABLE An integrated approach is designed to screen anti-CRISPRs (AcrVIA) candidates A series of prophages are discovered in regions encoding AcrVIAs AcrVIAs inhibit Cas13a RNA targeting in bacteria and human cells AcrVIAs impede programmable RNA base editing by blocking dCas13a-based toolkit

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Luciferase, SYBR Green Assay, Proliferation Assay, LDH Cytotoxicity Assay

Journal: Molecular cell

Article Title: CRISPR-Cas13 inhibitors block RNA-editing in bacteria and mammalian cells

doi: 10.1016/j.molcel.2020.03.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides crRNAs This study Table S4 Recombinant DNA Plasmids This study Table S5 Antibodies mouse monoclonal GFP antibody Biolegend Cat# 668206 anti-his tag anti-body Thermo Fisher Scientific Cat# TA150087 Bacterial and Virus Strains E. coli c-3000 ATCC Cat# 15597 NEB 5-alpha Competent E. coli New England BioLabs Cat# C2987H BL21 Competent E. coli New England BioLabs Cat# C2530H E. coli bacteriophage MS2 ATCC Cat# 15597-B1 Leptotrichia buccalis DSM 1135 ATCC Cat# 14201 E. coli c3000-derived strains This study Table S6 Chemicals, Peptides, and Recombinant Proteins LB Broth Base Thermo Fisher Scientific Cat# 12780029 LB Agar Fisher Scientific Cat# BP1425-500 SOC medium New England BioLabs Cat# B9020S ampicillin Fisher Scientific Cat#11593027 chloramphenicol Sigma Cat# C0378 kanamycin Fisher Scientific Cat# 25389-94-0 gentamicin ACROS Cat# 1405-41-0 IPTG Invitrogen Cat# 15529019 penicillin-streptomycin Thermo Fisher Scientific Cat# 10378016 fetal bovine serum VWR Seradigm Cat# 97068-085 LipofectAmine 3000 Thermo Fisher Scientific Cat# L3000015 Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 RMPI 1640 Medium Thermo Fisher Scientific Cat# 11875119 protease inhibitor cockt Thermo Fisher Scientific Cat# 78425 Critical Commercial Assays QIAGEN Plasmid Mini Kit QIAGEN Cat# 12125 myTXTL Sigma 70 Master Mix Kit Arbor Bioscience Cat# 507024 AMPure XP beads Beckman Coulter Cat# A63882 Dynabeads™ Sheep anti-Mouse IgG Invitrogen Cat# 11201D Pierce™ His Protein Interaction Pull-Down Kit Thermo Fisher Scientific Cat# 21277 Pierce™ Gaussia-Firefly Luciferase Dual Assay Kit Thermo Fisher Scientific Cat# 16181 High Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 Maxima SYBR Green qPCR Master Mix Thermo Fisher Scientific Cat# K0252 Direct-zol™ RNA MiniPrep kit Zymo Reseach Cat# R2052 the CellTiter 96 AQueous One Solution Cell Proliferation assay Promega Cat# G3580 Pierce LDH Cytotoxicity Assay Kit Thermo Fisher Scientific Cat# {"type":"entrez-nucleotide","attrs":{"text":"C20300","term_id":"1632571","term_text":"C20300"}} C20300 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216 Open in a separate window KEY RESOURCES TABLE An integrated approach is designed to screen anti-CRISPRs (AcrVIA) candidates A series of prophages are discovered in regions encoding AcrVIAs AcrVIAs inhibit Cas13a RNA targeting in bacteria and human cells AcrVIAs impede programmable RNA base editing by blocking dCas13a-based toolkit

Techniques: Recombinant, Virus, Protease Inhibitor, Plasmid Preparation, Luciferase, Reverse Transcription, SYBR Green Assay, Proliferation Assay, LDH Cytotoxicity Assay

Journal: Molecular cell

Article Title: CRISPR-Cas13 inhibitors block RNA-editing in bacteria and mammalian cells

doi: 10.1016/j.molcel.2020.03.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides crRNAs This study Table S4 Recombinant DNA Plasmids This study Table S5 Antibodies mouse monoclonal GFP antibody Biolegend Cat# 668206 anti-his tag anti-body Thermo Fisher Scientific Cat# TA150087 Bacterial and Virus Strains E. coli c-3000 ATCC Cat# 15597 NEB 5-alpha Competent E. coli New England BioLabs Cat# C2987H BL21 Competent E. coli New England BioLabs Cat# C2530H E. coli bacteriophage MS2 ATCC Cat# 15597-B1 Leptotrichia buccalis DSM 1135 ATCC Cat# 14201 E. coli c3000-derived strains This study Table S6 Chemicals, Peptides, and Recombinant Proteins LB Broth Base Thermo Fisher Scientific Cat# 12780029 LB Agar Fisher Scientific Cat# BP1425-500 SOC medium New England BioLabs Cat# B9020S ampicillin Fisher Scientific Cat#11593027 chloramphenicol Sigma Cat# C0378 kanamycin Fisher Scientific Cat# 25389-94-0 gentamicin ACROS Cat# 1405-41-0 IPTG Invitrogen Cat# 15529019 penicillin-streptomycin Thermo Fisher Scientific Cat# 10378016 fetal bovine serum VWR Seradigm Cat# 97068-085 LipofectAmine 3000 Thermo Fisher Scientific Cat# L3000015 Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 RMPI 1640 Medium Thermo Fisher Scientific Cat# 11875119 protease inhibitor cockt Thermo Fisher Scientific Cat# 78425 Critical Commercial Assays QIAGEN Plasmid Mini Kit QIAGEN Cat# 12125 myTXTL Sigma 70 Master Mix Kit Arbor Bioscience Cat# 507024 AMPure XP beads Beckman Coulter Cat# A63882 Dynabeads™ Sheep anti-Mouse IgG Invitrogen Cat# 11201D Pierce™ His Protein Interaction Pull-Down Kit Thermo Fisher Scientific Cat# 21277 Pierce™ Gaussia-Firefly Luciferase Dual Assay Kit Thermo Fisher Scientific Cat# 16181 High Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 Maxima SYBR Green qPCR Master Mix Thermo Fisher Scientific Cat# K0252 Direct-zol™ RNA MiniPrep kit Zymo Reseach Cat# R2052 the CellTiter 96 AQueous One Solution Cell Proliferation assay Promega Cat# G3580 Pierce LDH Cytotoxicity Assay Kit Thermo Fisher Scientific Cat# {"type":"entrez-nucleotide","attrs":{"text":"C20300","term_id":"1632571","term_text":"C20300"}} C20300 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216 Open in a separate window KEY RESOURCES TABLE An integrated approach is designed to screen anti-CRISPRs (AcrVIA) candidates A series of prophages are discovered in regions encoding AcrVIAs AcrVIAs inhibit Cas13a RNA targeting in bacteria and human cells AcrVIAs impede programmable RNA base editing by blocking dCas13a-based toolkit

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Luciferase, SYBR Green Assay, Proliferation Assay, LDH Cytotoxicity Assay

Journal: Molecular cell

Article Title: CRISPR-Cas13 inhibitors block RNA-editing in bacteria and mammalian cells

doi: 10.1016/j.molcel.2020.03.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides crRNAs This study Table S4 Recombinant DNA Plasmids This study Table S5 Antibodies mouse monoclonal GFP antibody Biolegend Cat# 668206 anti-his tag anti-body Thermo Fisher Scientific Cat# TA150087 Bacterial and Virus Strains E. coli c-3000 ATCC Cat# 15597 NEB 5-alpha Competent E. coli New England BioLabs Cat# C2987H BL21 Competent E. coli New England BioLabs Cat# C2530H E. coli bacteriophage MS2 ATCC Cat# 15597-B1 Leptotrichia buccalis DSM 1135 ATCC Cat# 14201 E. coli c3000-derived strains This study Table S6 Chemicals, Peptides, and Recombinant Proteins LB Broth Base Thermo Fisher Scientific Cat# 12780029 LB Agar Fisher Scientific Cat# BP1425-500 SOC medium New England BioLabs Cat# B9020S ampicillin Fisher Scientific Cat#11593027 chloramphenicol Sigma Cat# C0378 kanamycin Fisher Scientific Cat# 25389-94-0 gentamicin ACROS Cat# 1405-41-0 IPTG Invitrogen Cat# 15529019 penicillin-streptomycin Thermo Fisher Scientific Cat# 10378016 fetal bovine serum VWR Seradigm Cat# 97068-085 LipofectAmine 3000 Thermo Fisher Scientific Cat# L3000015 Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 RMPI 1640 Medium Thermo Fisher Scientific Cat# 11875119 protease inhibitor cockt Thermo Fisher Scientific Cat# 78425 Critical Commercial Assays QIAGEN Plasmid Mini Kit QIAGEN Cat# 12125 myTXTL Sigma 70 Master Mix Kit Arbor Bioscience Cat# 507024 AMPure XP beads Beckman Coulter Cat# A63882 Dynabeads™ Sheep anti-Mouse IgG Invitrogen Cat# 11201D Pierce™ His Protein Interaction Pull-Down Kit Thermo Fisher Scientific Cat# 21277 Pierce™ Gaussia-Firefly Luciferase Dual Assay Kit Thermo Fisher Scientific Cat# 16181 High Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific Cat# 4368814 Maxima SYBR Green qPCR Master Mix Thermo Fisher Scientific Cat# K0252 Direct-zol™ RNA MiniPrep kit Zymo Reseach Cat# R2052 the CellTiter 96 AQueous One Solution Cell Proliferation assay Promega Cat# G3580 Pierce LDH Cytotoxicity Assay Kit Thermo Fisher Scientific Cat# {"type":"entrez-nucleotide","attrs":{"text":"C20300","term_id":"1632571","term_text":"C20300"}} C20300 Experimental Models: Cell Lines HEK293T ATCC Cat# CRL-3216 Open in a separate window KEY RESOURCES TABLE An integrated approach is designed to screen anti-CRISPRs (AcrVIA) candidates A series of prophages are discovered in regions encoding AcrVIAs AcrVIAs inhibit Cas13a RNA targeting in bacteria and human cells AcrVIAs impede programmable RNA base editing by blocking dCas13a-based toolkit

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Luciferase, SYBR Green Assay, Proliferation Assay, LDH Cytotoxicity Assay