Bacterial strains and culture conditions
Plasmid culture and extraction, as well as plasmid transformation assays were performed with New England Biolabs Turbo Competent E. coli. Bacteria were grown in Lysogeny Broth (LB) supplemented with antibiotics: kanamycin (50 µg/mL), chloramphenicol (25 µg/ml), and anhydrotetracyline (100 ng/mL). 0.2% arabinose was added to induce expression of Cas13 from Para promoters.
Cas13 plasmids for transformation assay were cloned into the vector pAM38 containing p15A origin of replication, chloramphenicol resistance marker, and an arabinose-inducible Para promoter. Coding sequences for LwaCas13a, LbuCas13a, or LseCas13a were inserted downstream of the Para promoter, and crRNA constructs were inserted downstream of the cas13 coding sequence driven by a synthetic promoter (J23119). These plasmids were constructed by three piece Gibson assembly as described below and validated by Sanger sequencing. Lists of plasmids, oligonucleotide primers, and crRNAs can be found in Supplementary Tables.
LwaCas13a plasmids: Three piece Gibson assembly using (1) SalI/HindIII-digested pAM38, (2) LwaCas13a coding sequence and Shine-Dalgarno sequence amplified from Addgene #91865 using primers oAM1496 and oAM1497, and (3) J23119-driven crRNA gBlock fragments synthesized by IDT was used to generate pAM491 (LwaCas13a kan crRNA) and pAM494 (LwaCas13a non-targeting crRNA).
LbuCas13a plasmids: Three piece Gibson assembly using (1) SalI/HindIII-digested pAM38, (2) LbuCas13a coding sequence codon-optimized for E. coli (synthesized by Genewiz) and Shine-Dalgarno sequence amplified using primers oAM211 and oAM1477, and (3) J23119-driven crRNA gBlock fragments synthesized by IDT was used to generate pAM492 (LbuCas13a kan crRNA) and pAM475 (LbuCas13a non-targeting crRNA).
LseCas13a plasmids: Three piece Gibson assembly using (1) SalI/HindIII-digested pAM38, (2) LseCas13a coding sequence codon-optimized for E. coli (synthesized by Genewiz) and Shine-Dalgarno sequence amplified using primers oAM207 and oAM994, and (3) J23119-driven crRNA gBlock fragments synthesized by IDT was used to generate pAM526 (LseCas13a kan crRNA) and pAM319 (LseCas13a non-targeting crRNA).
Acr plasmids: Acrs were expressed from pAM326 (carrying kanamycin resistance marker and pWV01 origin of replication) and driven by an anhydrotetracycline-inducible Ptet promoter. Acr coding sequences were synthesized by Genewiz, amplified using primers oAM1529 and oAM1530, and inserted with Ptet fragment into HindIII/EagI-digested pAM326 via three piece Gibson assembly to make pAM495 (AcrVIA1), pAM496 (AcrVIA2), pAM497 (AcrVIA3), pAM498 (AcrVIA4), pAM499 (AcrVIA5), pAM500 (AcrVIA6), pAM501 (AcrVIA7). The AcrVIA1Lse plasmid pAM383 was generated previously (Meeske et al. 2020).
Plasmid transformation assay
NEB Turbo competent E. coli were transformed with either empty vector or plasmids harboring LwaCas13a, LbuCas13a, or LseCas13a and expressing non-targeting or targeting crRNAs, and selected on LB agar containing chloramphenicol and 0.2% glucose to repress Cas13a expression. Each transformed strain was made chemically competent by resuspension of exponentially growing cells in ⅕ culture volume of ice cold TFB I (10 mM CaCl2,30 mM potassium acetate, pH 5.8, 100 mM RbCl, 50mM MnCl2, 15% glycerol), incubation on ice for 15 minutes, then pelleting and resuspension in 1/25 culture volume of ice cold TFB II (10 mM MOPS pH 6.5, 10 mM RbCl, 75 mM CaCl2, 15% glycerol). Chemically competent cells were transformed by heat shock at 42°C with 100 ng Acr plasmids or empty vector control, then recovered for 1 hour in LB. Tenfold serial dilutions of recovered transformants were made and 5 µL of each dilution was spotted onto LB agar supplemented with kanamycin, chloramphenicol, arabinose, and anhydrotetracycline. Plates were photographed after 1 day of incubation at 37°C.
Human cell culture and transfections
Human HEK 293T cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FBS (HI-FBS) and 1% penicillin/streptomycin. Supernatants from cell cultures were tested monthly for mycoplasma using MycoAlert PLUS (Lonza). For transfections, approximately 2 × 104 cells per well were seeded in 96-well plates. Approximately 18-22 hours after seeding, transfections were performed using TransIT-X2 (Mirus) transfection reagent with 60 ng of LwaCas13a plasmid (Addgene ID 91924), 20 ng of gRNA plasmid (generated by cloning oligos into an LwaCas13a crRNA entry plasmid, LTH151, Addgene ID 171129), and with 0, 2, 12, or 72 ng of Acr plasmid. The human codon optimized Acr constructs for AcrVIA4 and AcrVIA5 were synthesized by Twist Biosciences and cloned into a pCMV backbone (plasmid IDs LTH956 and LTH957 respectively; see Supplementary Sequences), similar to how we previously generated the human cell expression constructs for AcrIIA4 and AcrIIA5 (Addgene IDs 133801 and 133802, respectively) (see Supplementary Sequences). The total amount of DNA in each transfection remained constant (152 ng), with a pCMV-null plasmid (Addgene ID 171128) used to balance DNA amounts when titrating the Acr plasmid. The plasmid mixtures were combined with 0.54 ul of TransIT-X2 and volume of Opti-MEM (ThermoFisher) to a final volume of 15 ul, incubated at room temperature for 15 minutes, and then applied to the HEK 293T cells. All experiments were performed with 3 independent biological replicates.
RNA extraction and RT-qPCR
At 48 hours post-transfection, total RNA was extracted from transfected cells using an RNeasy Plus Mini Kit (Qiagen; cat. no. 74136). Purified RNA was reverse transcribed using a High-Capacity RNA-to-cDNA kit (ThermoFisher; cat. no. 4388950) using up to 250 ng of RNA as input. Prior to qPCR, the cDNA library for each sample was diluted 1:20. Samples for qPCR were prepared in technical triplicate with 5 ul of Fast SYBR Green Master Mix (ThermoFisher; cat. no. 4385610), 3 ul diluted cDNA, and 2 ul qPCR primer pairs specific to the target (IDT; Supplementary Table 1). Control reactions amplifying ACTB were set up in parallel for each cDNA library. Reaction cycling was performed using a Roche LightCycler480. To determine the fold change of each sample, the expression levels were normalized to a negative control transfection containing LwaCas13a plasmid and an empty pUC19 backbone plasmid (Addgene ID 133961). Each qPCR reaction was analyzed for purity by melting curve analysis to confirm a single PCR product.
CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements, like bacteriophages and plasmids. To overcome CRISPR immunity, phages have evolved diverse families of anti-CRISPR proteins (Acrs), each of which inhibits the nucleic acid binding or cleavage activities of specific Cas protein families. Recently, Lin et al. (2020) described the discovery and characterization of 7 different Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems, which use the nuclease Cas13a to perform RNA-guided RNA cleavage. In this Matters Arising article, we detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses, as well as reported experiments involving bacterial strains that are impossible to construct. The authors were unable to provide their published strains with which we might reproduce their experiments. We independently tested the Acr sequences described in Lin et al. (2020) in two different Cas13 inhibition assays, but could not detect anti-CRISPR activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are bona fide type VI anti-CRISPR proteins.