STAR Methods

A target expression threshold dictates invader defense and autoimmunity by CRISPR-Cas13

Strains and plasmids

The strains and plasmids used in this study are listed in Table S2. The main plasmids used are pZ003 (LshCas13a), pFT50 (crRNA backbone), pFT50-repeat (crRNA backbone with repeat) and pFT62 (target backbone). Spacers were inserted into the backbone by digestion with BsmBI and ligation with Instant sticky-end ligase master mix (NEB, M0370S). Q5 mutagenesis or Gibson assembly were used to modify the target plasmid or the nuclease backbone. All of the oligos used in this research can be found in Table S3.

Transformation-based targeting assays

The transformation assays were conducted in two ways: targeting a genomically-encoded transcript and a plasmid-encoded transcript. For genomically-encoded transcripts, biological replicates containing the nuclease plasmid were inoculated overnight in LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L of dH2O) with chloramphenicol (Cm, 34 μg/mL). After 16 h, the ABS600 was measured and the samples were normalized, back-diluted 1:50 in fresh LB with Cm, and grown until an ABS600 of 0.6 - 0.8. Cultures were placed on ice and made electrocompetent by washing the pellet twice with 10% glycerol. Then, 50 ng of the crRNA plasmids were transformed into 40 μL of competent cells using the E. coli 1 program on the MicroPulser Electroporator (Bio-rad). After 1 h of recovery in 500 μL of SOC medium (SOB medium: 20 g Tryptone, 5 g Yeast Extract, 0.5 g NaCl, 800 mL dH2O and 10 mL 250 mM KCl adjusted to pH 7. To SOB medium added 5ml 2M MgCl2, 20 ml of 1 M Glucose), 10-fold dilutions of the cultures in 1X PBS (10X PBS: 80 g NaCL, 2 g KCl, 17,7 g Na2HPO4*2 H2O, 2.72 g KH2PO4, fill up to 1 L with mqH2O, set pH to 7.4 and autoclave) were prepared and 5 μL spot dilutions were plated on Cm and ampicillin (Amp, 100 μg/mL) LB plates and incubated at 37°C for 16-18 h.

For the plasmid-encoded transcripts, cultures containing the nuclease plasmid and the target plasmid were transformed with the crRNA plasmid. The rest of the procedure matched that followed for the genome targeting assay. The synthetic sequence expressed on a plasmid (pFT62) contains part of the mRFP1 gene sequence, which does not match the E. coli genome. Fragments from mRNAs (target plus 20 bp upstream and downstream) were cloned in pFT62 in place of the synthetic target. Finally, to see if cloning an entire gene on a plasmid would change the targeting outcome, full gene sequences with RBS and stop codon have been cloned in pFT62 through Gibson assembly.

Flow cytometry analysis

Plasmids expressing GFP under different Anderson promoters were cloned by Q5 mutagenesis to introduce the different promoters in the pUA66-PJ23119 GFP plasmid. Cells containing the nuclease plasmid and the GFP plasmid were inoculated overnight in LB medium with kanamycin (Kan, 50 μg/mL) and Cm, then normalized, back-diluted to ABS600 = 0.02 and cultured until ABS600 ≈ 0.8. Cells were then pelleted and resuspended in 1X PBS before being applied to an Accuri C6 Plus analytical flow cytometer (BD Biosciences). 30,000 events were obtained by gating on living cells, and the mean of the FL1-H values were quantified as GFP fluorescence. Final fluorescence values were obtained by subtracting the autofluorescence of cells not expressing GFP.

L-arabinose induction of Cas13a-mediated targeting in E. coli

The plasmid with the synthetic target under control of the arabinose-inducible promoter was cloned by introducing the PBAD promoter in pFT62 through Gibson assembly. To validate this system, E. coli MG1655 ΔaraBAD Pcon-araFGH cells were transformed with the nuclease, the gRNA and the arabinose-inducible target plasmids. Three biological replicates were inoculated overnight in LB supplemented with Amp, Cm, and Kan as well as 0.2% glucose to reduce background expression from the PBAD promoter. The samples were then pelleted, resuspended in LB with antibiotics and then back-diluted to ABS600 = 0.01 with or without 0.2% L-arabinose. Culture turbidity was recorded over time on a Synergy Neo2 or H1 fluorescence microplate reader (BioTek) for 16 h by measuring ABS600 every 3 min.

Assessment of collateral RNA cleavage

Using the validated arabinose-inducible setup, collateral RNA cleavage was visualized on an agarose gel. Cells with the nuclease, gRNA, and target plasmids were grown overnight in Cm, Amp, Kan LB with 0.2% glucose, washed in LB with antibiotics to remove glucose, and back-diluted to ABS600 = 0.01. The cells were cultured until ABS600 ≈ 0.4, after which each sample was split in equal volumes with or without the inducer. After 1 h from induction, the ABS600 of each sample was measured, and the same number of cells (2250 ABS600×mL) was snap frozen on dry ice. The next day, total RNA was extracted using the Directzol RNA mini-prep plus kit (Zymo research). Then 1 μg of each RNA was mixed 2:3 with an RNA loading dye (2x) (for 50 mL: 625 µL Bromophenol blue 2%, 625 µL 2% Xylene Cyanol, 1800 µL of 0.5 M EDTA pH = 8.0, 46,821 mL Formamide), heated at 70°C for 10 min, placed on ice, and resolved on a 1% TBE gel at 120 V for 40 min. RiboRuler High Range RNA ladder (Thermo Scientific, SM1821) was used as a size marker.

Library design and validation

The reference genome and annotation of E. coli K12 MG1655 (NC_000913.3) was used for gRNA library design. First, all potential 32-nt guides were designed for protein-coding genes (limited to the CDS) and rRNAs with non-GU PFS and GC content between 40% and 60%, resulting in an average of 484 guides per gene. To reduce the size of the library, and considering the unknown effect of the targeting location within a gene on guide efficiency, each gene was divided into a maximum of 10 sections with equal length. Within each section, guides were filtered based on the strength of local secondary structure, defined as ΔG, in both repeat-guide sequence and the mRNA targeting region (including a region of 2 times length of gRNA before and after the target). ΔG was calculated as the energy difference between the unconstrained minimum free energy (MFE) structure and the constrained MFE structure with no base pairs, estimated using RNAfold from the Vienna RNA Package (Lorenz et al., 2011) version 2.4.12. Sequences (either guide or flanking primer sequences) containing BsmBI restriction sites or homopolymer stretches of more than four consecutive nucleotides were excluded to facilitate synthesis and cloning. The guide with the lowest secondary structure strength in each section was selected, resulting in a library of 25,997 guides, including 25,470 guides targeting protein-coding genes, 127 guides targeting rRNAs, and 400 randomized non-targeting guides as negative controls. A sequence containing a universal primer binding site and the BsmBI restriction site was added to the guides to amplify the oligo library and digest it before ligating it into the backbone (see Table S4). The library was synthesized by Twist Bioscience.

The base backbone pFT50 was slightly modified to insert the direct repeat before the GFP dropout site to be able to limit the insertion size to the spacer itself. A BsmBI restriction site present in pFT50 was also eliminated through Q5 mutagenesis.

Guide library cloning and verification

The library was amplified with Kapa Hifi polymerase (20 ng DNA) for 10 cycles following the manufacturer’s instructions (Ta = 64°C; 30 s denaturation, 20 s annealing, 15 s extension) using primers SPCpr 349/350. 5 µL library (150 nM) and 5 µL of backbone (50 nM) were mixed in 25 µL of total reaction volume. The mixture was subjected to 50 cycles of BsmBI digestion (3 min at 42°C) and ligation (T4 ligase - 5 min at 16°C) with a final digestion at 55°C for 60 min to ensure complete removal of the backbone, followed by a 10 minute heat inactivation at 80°C. The sample was then ethanol precipitated, and 5 µg were transformed into fresh electrocompetent Top10 cells (90 µL). The transformation was conducted with two separate batches of electrocompetent cells to ensure enough transformants were obtained. After recovering the two cultures in 500µL of SOC medium shaking at 37°C for 1 h, the recovered cultures were back-diluted into 150 mL LB with Amp and cultured with shaking at 37°C for 12 h. The next day, plasmid DNA from the culture was isolated using the ZymoPURE II Maxiprep Kit (Zymo Research, D4203) and further purified by ethanol precipitation.

Guide library screen

Two replicates of E. coli MG1655 cells with or without the nuclease plasmid were inoculated overnight, then the next day the ABS600 was normalized and the cells back-diluted to ABS600 ≈ 0.1 in fresh LB with or without Cm. Once each culture reached ABS600 ≈ 0.8, cells were made electrocompetent by washing twice with 10% glycerol and finally resuspended in 480 μL 10% glycerol. For each sample, six separate transformations were conducted each with 1 μg of library DNA (40 μL/transformation). Transformed cells were then recovered in 500 μL SOC medium for 1 h with shaking at 37°C. The six reactions were combined to yield 3 mL of culture per condition. Serial dilutions of this culture were made, and 100 μL of 1:10,000 dilutions were plated for the targeting and no-Cas13a samples with the appropriate antibiotics (Cm and Amp, or Amp only), yielding a theoretical library coverage of ∼9,500. The remaining culture was diluted 1:100 in LB with Amp and Cm to ABS600 = 0.06 and cultured for 12 h with shaking at 37°C. Finally, the library was isolated with the ZymoPure II Plasmid Midiprep Kit (Zymo Research, D4200).

Next-generation sequencing of the guide libraries

The guides sequences from the purified library DNA were amplified with Kapa Hifi polymerase using primers oEV-315/316 (NT1), oEV-317/318 (NT2), oEV-319/320 (T1), oEV-321/322 (T2). 10 ng of DNA were included in a 50-μL PCR reaction for 15 amplification cycles (15 s at 98°C, 30 s at 64°C, 30 s extension). The amplification products were purified using Ampure beads and further amplified with primers oEV-323/324 (NT1), oEV-325/326 (NT2), oEV-327/328 (T1), oEV-329/330 (T2) to add the appropriate indices and Illumina adaptors. For this reaction, the same settings were used with the only difference being the amount of input DNA (25 ng) and the number of cycles (10). The resulting amplification products were purified with Ampure beads and resolved on a gel to verify the presence of the correct amplicon. The samples were submitted for Sanger sequencing and Bioanalyzer analysis as a quality check. Finally, samples were submitted for next-generation sequencing at the NextSeq 500 sequencer (Illumina) with a 150 bp paired-ends kit (130 million reads) to obtain 1000-fold coverage. To increase the library diversity, 20% of phiX phage was spiked-in.

To correlate transcript expression levels with guide depletion, we measured transcript levels in E. coli MG1655 under conditions paralleling the library screen. Briefly, two replicates of cells containing the plasmid cBAD33 (empty backbone for nuclease plasmid) were cultured overnight and then normalized to ABS600 = 0.06 in LB with Cm and cultured to ABS600 ≈ 0.5 or ABS600 ≈ 0.8. At those growth points, cells were pelleted and snap-frozen for RNA extraction with the Directzol RNA mini-prep plus kit (Zymo Research, R2071). The samples were also DNase-treated with TURBO DNase (Thermo Fisher Scientific, AM2239) and quality verified using a Bioanalyzer 2100 (Agilent). Finally, rRNA was removed with the Rybo-off rRNA depletion kit (Vazyme Biotech, N407-01) and the samples were sequenced on a NovaSeq 6000 (Illumina) with 50-bp paired-end reads.

The resulting NGS data were deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE179913 for the genome-wide screen ( and GSE179914 for transcriptomic analysis (

Screen analysis and machine learning model

For analysis of the genome-wide screen, after merging using BBMerge (version 38.69) with parameters “qtrim2=t, ecco, trimq=20, -Xmx1g, mix=f”, paired-end sequence reads with a perfect match were assigned to gRNA sequences. After filtering guides for at least 1 count per million reads in at least 2 samples, the library sizes were normalized using the read counts for non-targeting guides with the trimmed mean of M-values (TMM) method in edgeR (Robinson and Oshlack, 2010; Robinson et al., 2009) (version 3.28.0). Differential abundance of gRNAs between targeting samples and control samples lacking the Cas13a nuclease was assessed using the edgeR quasi-likelihood F test after fitting a generalized linear model. The translation initiation rate of each gene was predicted using RBS calculator (version 1.0) (Salis, 2011).

For RNA seq analysis, sequencing reads were aligned to the E. coli K12 MG1655 genome (NC_000913.3) using STAR (Dobin et al., 2013) (version 2.7.4a) with parameters “--alignIntronMax 1 --genomeSAindexNbases 10 --outSAMtype BAM SortedByCoordinate” and the count of reads mapping to each gene was obtained using HTSeq (Anders et al., 2015) (version 0.9.1) with parameters “-i locus_tag -r pos --stranded reverse --nonunique none -t gene”, followed by calculating transcripts per million (TPM).

The machine learning regression model was developed with 144 features as predictors and the log2FC values of gRNAs from the genome-wide screen as targets using auto-sklearn version 0.10.0 (Feurer et al., 2019) with all possible estimators and preprocessors included and parameters ’ensemble_size’: 1, ’resampling_strategy’: ’cv’, ’resampling_strategy_arguments’: {’folds’: 5}, ’per_run_time_limit’: 360, and ’time_left_for_this_task’: 3600. Features included gene expression level (log2 transformed TPM at OD 0.4), gene essentiality, gene id, gene length, (percent) targeting position in gene, delta G of repeat-gRNA and mRNA targeting region, and the one-hot-encoded PFS sequence and gRNA sequence. Gene essentiality information in LB Lennox medium was obtained from EcoCyc (Keseler et al., 2017) ( The optimal histogram-based gradient boosting model was evaluated using 10-fold cross-validation and interpreted using TreeSHAP version 0.36.0 (Lundberg et al., 2020).

Targeting assay with lower nuclease expression

In order to look at targeting with varied nuclease expression, we used a plasmid expressing the nuclease under the constitutive promoter PJ23108 (Pw). At first we assessed the relative promoter strength of Pw and the native promoter (Pnative) by cloning the gfp gene in the nuclease backbone in place of the nuclease itself through Gibson assembly. Each gfp plasmid was transformed with the target plasmid into E. coli MG1655 cells, and the ‘Flow cytometry analysis’ protocol was followed to measure fluorescence of the two constructs. We then performed the transformation-based targeting assay with the low expressed nuclease, the crRNA (T1) and different Anderson promoters (P1 to P8) cloned in front of the synthetic target sequence.

Cell-free transcription-translation assays

Plasmids encoding the nuclease, a crRNA, a target sequence, and deGFP were used to assess the targeting activity in a cell-free transcription-translation assay. The nuclease was either under the control of Pnative or Pw and the crRNA encoded either a targeting (T) spacer or non-targeting (NT) control. To assess differences in targeting activity when the nuclease is under the control of different promoters, the two nuclease constructs were added separately with either the targeting or the non-targeting crRNA and the targeted plasmid to myTXTL Sigma 70 Master Mix (Arbor Biosciences, 507005), with a final concentration of 2 nM, 1 nM and 0.5 nM, respectively. The samples were incubated for 2 h at 29°C, before a plasmid encoding deGFP was added to a final concentration of 0.5 nM. The samples were then incubated at 29°C for 16 h in a plate reader (BioTek Synergy Neo2) and fluorescence was measured every three minutes (excitation, emission: 485 nm, 528 nm). All shown data was produced using the Echo 525 Liquid Handler (Beckman Coulter). The assays were therefore scaled down to 3 µl reactions per replicate, with four replicates each. As part of the analysis, the background fluorescence from myTXTL mix and water samples was subtracted from all samples. Grubb’s test was performed using the values after 16 h to identify outliers between replicates (α = 0.1). If no outliers were identified, the first of the four replicates was discarded. The graph shows the average deGFP fluorescence over time together with the standard deviation.

Infection experiments with MS2 phage

MS2 phage concentration (PFU/mL) has been calculated by performing a plaque assay. Transcription-based targeting assays have been performed by transforming E. coli CGSC 4401 cells (F+) expressing the nuclease and differentially abundant target rep or cp gene transcripts with the correspondent targeting guides and by calculating the reduction in colony numbers compared to a non-targeting guide. E. coli cells containing the active or dead nuclease and the guide encoding plasmids have been grown overnight in selective LB medium, back-diluted to ABS600 = 0.05 and let grow until ABS600 ≈ 0.3. The samples have then been normalized to ABS600 = 0.3 and aliquoted in a 96 well plate (Thermo Scientific, 167008) together with different amounts of phages (MOI 0.1 and 5) or the respective volume of LB for the no-infection control. Cell growth has been recorded over 16 h by measuring ABS600 every three minutes in a plate reader at 37°C.

Tolerance to targeted kanR gene

E. coli colonies containing the nuclease and crRNA (K1, K2) plasmids were transformed with the targeted plasmid, which has a cloDF13 ori, different promoters in front of the kanR gene (P2, P5, P8), and a non-targeted hygromycin (Hyg, 100 μg/mL) resistance cassette. The plasmid was cloned in two steps by Gibson assembly with pUA66 as the backbone. K1 and K2 were targeting two different regions within the kanR transcript. The procedure is analogous to the transformation assay with a plasmid-encoded transcript, with the targeted plasmid selected on Hyg. The next day, colonies from the spot dilutions were counted, and colonies from the samples showing a negligible reduction in transformation compared to the non-targeting control were inoculated overnight in LB with Cm, Amp and Hyg. Then the cultures were washed to remove Hyg and back-diluted to ABS600 = 0.01 in either Hyg or Kan. The growth curves for the different conditions were measured over time on the microplate reader for 14 h at 37°C by measuring ABS600 every 3 min. For generating the heatmap, 12 h time points were selected to compare the different conditions and the resulting graph is the average of 9 biological replicates.

Data analysis and image visualization

Microsoft Excel was used to analyze the data, and GraphPad Prism was used to generate the bar plots and heatmaps. The graphs were then modified in Adobe Illustrator to construct the final figures. Transformation fold-reduction in the transformation assays was calculated as the ratio between non-targeting and targeting colonies.

Statistical analyses

All statistical analyses were performed using a Welch’s t test assuming unequal variances. P-values above 0.05 or average values lower than the reference average were considered non-significant. Statistical comparisons for the transformation assays relied on log values, which assumes the samples are normally distributed on a log scale.

Article TitleA target expression threshold dictates invader defense and autoimmunity by CRISPR-Cas13


Immune systems must recognize and clear foreign invaders without eliciting autoimmunity. CRISPR-Cas immune systems in prokaryotes manage this task by following two criteria: extensive guide:target complementarity and a defined target-flanking motif. Here we report an additional requirement for RNA-targeting CRISPR-Cas13 systems: expression of the target transcript exceeding a threshold. This finding is based on targeting endogenous non-essential transcripts, which rarely elicited dormancy through collateral RNA degradation. Instead, eliciting dormancy required over-expressing targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as the principal determinant of cytotoxic autoimmunity and revealed that the threshold shifts with the guide:target pair. This expression threshold ensured defense against a lytic bacteriophage yet allowed tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as a third criterion for immune activation by RNA-targeting CRISPR-Cas systems, buffering against autoimmunity and distinguishing pathogenic and benign invaders.


  • Cas13-induced dormancy requires RNA target levels to exceed an expression threshold
  • The expression threshold can prevent cytotoxic self-targeting for endogenous transcripts
  • The threshold shifts depending on the CRISPR RNA guide:target pair
  • The threshold allows cells to distinguish pathogenic and benign infections

Competing Interest Statement

C.L.B. is a co-founder and member of the Scientific Advisory Board for Locus Biosciences as well as a member of the Scientific Advisory Board for Benson Hill. The other authors have no conflicts of interest to declare.

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