Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

Abstract

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.

Keywords: Cas13a; RNA-targeting; Type VI CRISPR-Cas; anti-CRISPR; bacteriophage; bioinformatics; prophage; reproducibility.

Figure 1:

Gene organization of reported Acrs. Misnamed aca genes and adjacent acr genes as depicted in Lin et al. 2020 (left) and with ORFs redrawn to reflect their true scale and spacing (right).

Figure 2:

Misidentification of Acr homologs. (A) Reported Acr homologs with percent identity for each match indicated in Lin et al. 2020. BLASTp alignments demonstrating no significant homologs are present in the target genomes for (B) AcrIC1 or (C) AcrIIC4.

Figure 3:

Plaque assays with MS2 phage (10-fold dilutions from left to right) on lawns expressing LwaCas13a or LbuCas13a and a cognate crRNA targeting MS2. With the exception of the “non-targeting” strain, the others possess an MS2 targeting crRNA and either an empty pET16b plasmid, or one encoding acrVIA1–7, as indicated. Due to the inconsistent appearance of the clearings, an independently sourced C3000 wild-type strain was also procured, and plaque assays conducted to confirm the titer and robustness of the phage lysate.

Figure 4:. Target plasmid transformation assay to measure Acr function.

(A) Assay schematic. E. coli strains carried plasmids with Cas13a from L. wadei, L. buccalis, or L. seeligeri and a crRNA targeting the kanamycin resistance marker of a second plasmid (or non-targeting crRNA control), which also candidate Acr proteins. Cas13a targeting of the kanR marker restricts transformation of the target plasmid, unless the Acr inhibits Cas13a. (B) Results of transformation assay. The indicated recipient strains (columns) were transformed with the indicated plasmids (rows), serially diluted and spotted onto media selecting for both plasmids. nt, non-targeting crRNA.

Figure 5:. Assessment of AcrVIA-mediated inhibition of LwaCas13a.

(A, B) Knockdown of endogenous PPIB (panel A) or KRAS (panel B) transcripts by LwaCas13a in the presence of various amounts of Acr expression plasmids was determined via RT-qPCR analysis (n = 3 biological replicates; dots represent the mean of three technical triplicate qPCR values with SD shown). PPIB or KRAS RNA levels were normalized to ACTB and knockdown was determined by comparison to a non-targeting LwaCas13a control. Increasing amounts of plasmids encoding the anti-CRISPR proteins AcrVIA5, AcrVIA4, AcrIIA4, and AcrIIA5 were added to a consistent amount of LwaCas13a nuclease and gRNA plasmids. The molar ratios of the Acr to LwaCas13a expression plasmids were approximately 0.12, 0.72, and 4.32 for the 2 ng, 12 ng, and 72 ng treatments, respectively.