CRISPR Cas, LAMP, RNase HII, reverse transcriptase, IVD raw material supplier.

Email:  info@magigen.com
News Report
New Arrivals

RNA targeting with CRISPR Cas13a

Source:NatureAuthor:Julia

Magigen is a CRISPR Cas protein magic studio. If you want to buy CRISPR Cas protein, please CLICK HERE: CRISPR Cas protein.

RNA has important and diverse roles in biology. Here, Zhang Feng et al.   demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR–Cas effector Cas13a protein (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding.

Abstract

To achieve robust CRISPR/Cas13a-mediated RNA knockdown, Zhang Feng et al. first evaluated 15 CRIPSR Cas13a orthologues for protospacer flanking site (PFS) preference and activity using a previously described ampicillin-resistance assay (Fig. 1a). This assay monitors CRISPR Cas13a-mediated cleavage of the β-lactamase (ampicillin resistance) transcript, resulting in bacterial death under ampicillin selection, which can be measured by quantifying surviving colonies. Using this approach, we found that the Cas13a orthologue from L. wadei (LwaCas13a) was most active, followed by the previously characterized LshCas13a (from Leptotrichia shahii) (Fig. 1b and Extended Data Fig. 1b). Analysis of the sequenced PFS distributions from the LwaCas13a and LshCas13a screens revealed that most LwaCas13a PFS sequences were depleted (Extended Data Fig. 1c–e). Motif analysis of the depleted PFS sequences at varying thresholds revealed the expected 3′ H motif for LshCas13a, but no significant PFS motif for LwaCas13a (Fig. 1c and Extended Data Fig. 1f, g). This observation is consistent with previous studies, which have shown that CRIPSR LwaCas13a protein is more active than LshCas13a protein as a nucleic-acid sensor. Because of its high activity and lack of PFS in bacteria,We focused on LwaCas13a for further development.

RNA-target-crispr-cas13

Fig. 1

In vitro cleavage reactions with LwaCas13a demonstrated programmable RNA cleavage with a CRISPR RNA (crRNA) encoding a 28-nucleotide (nt) spacer (shorter than the 29–30 nt length found in the native L. wadei CRISPR array. These reactions confirmed the higher cleavage efficiency of LwaCas13a over LshCas13a, and revealed similar biochemical characteristics for the two enzymes, including the ability to cleave the corresponding pre-crRNA transcript. We also explored the crRNA constraints on LwaCas13a cleavage by truncating the spacer, finding that LwaCas13a retained in vitro cleavage activity with spacer lengths as short as 20 nt. Although guide lengths less than 20 nt no longer support catalytic activity, the LwaCas13–crRNA complex may still retain binding activity, providing an opportunity for orthogonal applications with a single enzyme.

We next evaluated the ability of LwaCas13a to cleave transcripts in mammalian cells. We cloned mammalian codon-optimized LwaCas13a into mammalian expression vectors with msfGFP fusions on the C or N terminus and either a dual-flanking nuclear export sequence or nuclear localization sequence (NLS) and evaluated expression and localization (Fig. 1d). We found that msfGFP-fused LwaCas13a constructs expressed well and localized effectively to the cytoplasm or nucleus according to the localization sequence. To evaluate the in vivo cleavage activity of LwaCas13a, we developed a dual-luciferase reporter system that expressed both Gaussia luciferase (Gluc) and Cypridinia luciferase (Cluc) under different promoters on the same vector, allowing one transcript to serve as the LwaCas13a target and the other to serve as a dosing control (Fig. 1e). We then designed guides against Gluc and cloned them into a tRNAVal promoter-driven guide expression vector. We transfected the LwaCas13a expression vector, guide vector, and dual-luciferase construct into HEK293FT cells and measured luciferase activity 48 h after transfection. We found that LwaCas13a–msfGFP–NLS resulted in the highest levels of knockdown (75.7% for guide 1, 72.9% for guide 2), comparable to position-matched short hairpin (sh)RNA controls (78.3% for guide 1, 51.5% for guide 2) (Fig. 1f), which control for accessibility and sequence in the target region; we therefore used this design for all further knockdown experiments. We also found that knockdown is most efficient with a spacer length of 28 nt (73.8%), is dose-responsive both to the input protein and guide vector amounts, and is not sensitive to RNA polymerase III promoter choice.

We next tested knockdown in HEK293FT cells of three endogenous genes: KRAS, CXCR4, and PPIB. We observed varying levels of knockdown; and for KRAS and CXCR4, LwaCas13a knockdown (40.4% for PPIB, 83.9% for CXCR4, 57.5% for KRAS) was similar to RNA interference (RNAi) with position-matched shRNAs (63.0% for PPIB, 73.9% for CXCR4, 44.3% for KRAS) (Fig. 1g). We also found that knockdown of KRAS was possible with either U6 or tRNAVal promoters. Similar results were obtained in the A375 melanoma cell line. In all cases tested, knockdown was abolished by mutating the catalytic domain of LwaCas13a. To test whether LwaCas13a knockdown is efficient in plants, we targeted three rice (Oryza sativa) genes with three guides per transcript and co-transfected LwaCas13a and guide vectors into O. sativa protoplasts (Fig. 1h). After transfection, we observed >50% knockdown with seven out of the nine guides and maximal knockdown of 78.0% (Fig. 1i).

To evaluate the range of efficiency of LwaCas13a knockdown, we tiled guides along the length of four transcripts: Gluc, Cluc, KRAS, and PPIB (Fig. 2a). The Gluc and Cluc tiling screens revealed guides with greater than 60% knockdown (Fig. 2b, c), with the majority of Gluc-targeting guides exhibiting >50% knockdown and up to 83% knockdown. To compare LwaCas13a knockdown with RNAi, we selected the top three performing guides against Gluc and Cluc and compared them to position-matched shRNAs. We found that five out of six top performing guides achieved significantly higher levels of knockdown (P < 0.05) than their matched shRNAs (Extended Data Fig. 3h). For endogenous genes, we found that, while knockdown efficiency was transcript dependent, there was maximal knockdown of 85% and 75% for KRAS and PPIB, respectively (Fig. 2d, e). We selected the top three guides from the KRAS and PPIB tiling screens and observed robust knockdown with LwaCas13a (53.7–88.8%) equivalent to levels attained by shRNA knockdown (61.8–95.2%), with shRNA significantly better for two out of six guides (P < 0.01) and LwaCas13a significantly better for two out of six guides (P < 0.01) (Fig. 2f). LwaCas13a can also mediate significant knockdown of the nuclear transcripts MALAT1 and XIST, whereas position-matched shRNAs showed no detectable knockdown (P > 0.05).

crispr cas13a edit rna

Fig. 2

CRIPSR LshCas13a activity is governed by target accessibility in E. coli, and we therefore used our data from the four tiling screens to investigate whether LwaCas13a activity is higher for guides located in regions of accessibility. We found that the most effective guides were closer together than expected by chance, and predicted target accessibility could explain some of the variation in targeting efficacy (4.4–16% of the variation in knockdown).

Because CRIPSR LwaCas13a can process its own pre-crRNA, it offers the possibility of streamlined multiplexed delivery of LwaCas13a guides13. We designed five different guides against the endogenous PPIB, CXCR4, KRAS, TINCR, and PCAT transcripts and delivered the targeting system as a CRISPR array with 28-nt guides flanked by 36-nt direct repeats (representing an unprocessed direct repeat and a truncated spacer), under expression of the U6 promoter. We found levels of knockdown for each gene that were comparable to single or pooled guide controls (Fig. 2i). To evaluate specificity in this context, we tested multiplexed delivery of three guides against PPIB, CXCR4, and KRAS or three variants where each one of the three guides was replaced with a non-targeting guide. We found that in each case where a guide was absent from the array, only the targeted transcripts were reduced (Fig. 2j).

To further investigate the specificity of LwaCas13a in vivo, we introduced single mismatches into guides targeting either Gluc (Fig. 3a) or endogenous genes, as well as double mismatches, and found that knockdown was sensitive to mismatches in the central seed region of the guide–target duplex, which we additionally confirmed by biochemical profiling. To comprehensively search for off-target effects of LwaCas13a knockdown, we performed transcriptome-wide mRNA sequencing. We targeted the Gluc transcript with LwaCas13a or a position-matched shRNA construct, and found significant knockdown of the target transcript (P < 0.01). Similar results were found for the same comparison when targeting KRAS and PPIB (P < 0.05). Differential expression analysis indicated hundreds of significant off-targets in each of the shRNA conditions but none in LwaCas13a conditions (Fig. 3f), despite comparable levels of knockdown of the target transcripts (30.5%, 43.5%, and 64.7% for shRNA, 62.6%, 27.1%, and 29.2% for LwaCas13a protein, for Gluc, KRAS, and PPIB, respectively) (Fig. 3g). Additional analysis of the Gluc-targeting RNA-seq comparisons suggested the shRNA libraries show higher variability between targeting and non-targeting conditions compared with CRIPSR LwaCas13a because of these off-target effects.

crispr/cas13a protein edit rna

Fig. 3

The collateral activity of CRIPSR LshCas13a has been directly observed biochemically in vitro and indirectly observed through growth suppression in bacteria, but the extent of this activity in mammalian cells is unclear. The multiplexed leave-one-out and RNA-sequencing (RNA-seq) analyses suggested a lack of collateral RNA degradation. We verified this hypothesis by re-analysing the knockdown tiling screens (Fig. 2b–e), finding that expression of the control gene did not correlate with the expression of the targeted gene (Gluc: R = −0.078, P > 0.05; PPIB: R = −0.058, P > 0.05; KRAS: R = −0.51, P < 0.001) . Additionally, in the RNA-seq experiments there were no differentially expressed genes other than the target gene, indicating that LwaCas13a targeting does not lead to an observable cell stress response at the transcriptomic level14, as would be reasonably expected if substantial collateral activity occurred. Furthermore, LwaCas13a-mediated knockdown of targeted transcripts did not affect the growth of mammalian cells expressing similar levels of LwaCas13a (Fig. 3h). Finally, because activation of non-specific RNA nucleases in mammalian cells results in detectable changes in RNA size distribution15, we examined global RNA degradation in cells after LwaCas13a knockdown of Gluc transcripts and found no difference in the RNA integrity between targeting and non-targeting conditions (P > 0.05) .

To expand the utility of CRIPSR LwaCas13a as a tool for studying RNA, we created a catalytically dead variant (dLwaCas13a) by mutating catalytic arginine residues. We quantified RNA binding by dLwaCas13a with RNA immunoprecipitation(Fig 4a) using guides containing the 36-nt direct repeats and 28-nt spacers. We found that pulldown of dLwaCas13a targeted to either luciferase transcripts or ACTB mRNA (Fig. 4b) resulted in significant enrichment of the corresponding target over non-targeting controls (7.8–11.2× enrichment for luciferase and 2.1–3.3× enrichment for ACTB; P < 0.05), validating dLwaCas13a as a reprogrammable RNA binding protein.

crispr cas13a protein target rna

Fig. 4

One application for dLwaCas13a is as a transcript imaging platform. To reduce background noise due to unbound protein, we incorporated a negative-feedback (NF) system based upon zinc finger self-targeting and KRAB domain repression16. Compared with dLwaCas13a, dLwaCas13a–NF effectively translocated from the nucleus to the cytoplasm when targeted to ACTB mRNA. To further characterize translocation of dLwaCas13a–NF, we targeted ACTB transcripts with two guides and found that both guides increased translocation compared with a non-targeting guide (3.1–3.7× cellular/nuclear signal ratio; P < 0.001). To validate dLwaCas13a–NF imaging, we analysed the correlation of dLwaCas13a–NF signal to ACTB mRNA fluorescent in situ hybridization (FISH) signal and found that there was significant correlation and signal overlap for the targeting guides versus the non-targeting guide conditions (R = 0.27 and 0.30 for guide 1 and 2, respectively, and R = 0.00 for the non-targeting guide condition; P < 0.0001).

Using dLwaCas13a–NF, we investigated the accumulation of mRNA into stress granules by combining transcript imaging with visualization of stress granules marker G3BP1. In fixed samples, we found significant correlations between dLwaCas13a–NF fluorescence and G3BP1 levels for ACTB-targeting guides compared with non-targeting controls (R = 0.49 and 0.50 for guide 1 and guide 2, respectively, and R = 0.08 for the non-targeting guide; P < 0.001) (Fig. 4f, g). We next performed stress granule tracking in live cells and found that dLwaCas13a–NF targeted to ACTB localized to significantly more stress granules per cell over time than the corresponding non-targeting control (P < 0.05).

These results show that CRIPSR LwaCas13a can be reprogrammed with guide RNAs to effectively knockdown or bind transcripts in mammalian cells. LwaCas13a knockdown is comparable to RNAi knockdown efficiency, but with substantially reduced off-targets, making it potentially well-suited for therapeutic applications. Furthermore, it can mediate nuclear RNA and multiplexed knockdown. Catalytically inactive dLwaCas13a can be used as a programmable RNA binding protein, which we adapted for live imaging transcript tracking. We anticipate that there will be additional applications for LwaCas13a and dLwaCas13a, such as genome-wide pooled knockdown screening, interrogation of lncRNA and nascent transcript function, pulldown assays to study RNA–protein interactions, translational modulation, and RNA base editing. Importantly, we do not observe any evidence for collateral activity of LwaCas13a in mammalian cells. Our data show LwaCas13a functions in mammalian and plant cells with broad efficacy and high specificity, providing a platform for a range of transcriptome analysis tools and therapeutic approaches.

Methods

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Cloning of orthologues for activity screen and recombinant expression

We synthesized human codon-optimized versions of 15 CRIPSR Cas13a orthologues (Genscript) and cloned them into pACYC184 under a pLac promoter. Adjacent to the Cas13a expression cassette, we cloned the orthologue’s corresponding direct repeats flanking either a β-lactamase-targeting or non-targeting spacer. Spacer array expression was driven by the J23119 promoter.

For purification of LwaCas13a, we cloned the mammalian codon-optimized LwaCas13a sequence into a bacterial expression vector for protein purification (6× His/Twin Strep SUMO, a pET-based expression vector received as a gift from I. Finkelstein, University of Texas-Austin).


Bacterial in vivo testing for Cas13a activity and PFS identity

In brief, CRIPSR Cas13a was programmed to target a 5′ stretch of sequence on the β-lactamase transcript flanked by randomized PFS nucleotides. Cas13a cleavage activity resulted in death of bacteria under ampicillin selection, and PFS depletion was subsequently analysed by next-generation sequencing.

To test for activity of CRIPSR Cas13a orthologues, 90 ng of orthologue expression plasmid with either targeting or non-targeting guide was co-transformed with 25 ng of a previously described β-lactamase target plasmid8 into NovaBlue Singles Competent Cells (Millipore). Post-transformation, cells were diluted, plated on LB-agar supplemented with 100 μg μl−1 ampicillin and 25 μg μl−1 chloramphenicol, and incubated at 37 °C overnight. Transformants were counted the next day.

For determination of LshCas13a and LwaCas13a PFS identity, 40 ng of orthologue expression plasmid with either targeting or non-targeting spacer was co-transformed with 25 ng of β-lactamase target plasmid into two aliquots of NovaBlue GigaSingles (Millipore) per biological replicate. Two biological replicates were performed. Post-transformation, cells were recovered at 37 °C in 500 μl of SOC (ThermoFisher Scientific) per biological replicate for 1 h, plated on bio-assay plates (Corning) with LB-agar (Affymetrix) supplemented with 100 μg μl−1 ampicillin and 25 μg μl−1 chloramphenicol, and incubated at 37 °C for 16 h. Colonies were then harvested by scraping, and plasmid DNA was purified with NuceloBond Xtra EF (Macherey-Nagel) for subsequent sequencing.

Harvested plasmid samples were prepared for next-generation sequencing by PCR with barcoding primers and Illumina flow cell handles using NEBNext High Fidelity 2X Master Mix (New England Biosciences). PCR products were pooled and gel extracted using a Zymoclean gel extraction kit (Zymo Research) and sequenced using a MiSeq next-generation sequencing machine (Illumina).

Computational analysis of PFS

From next-generation sequencing of the LshCas13a and LwaCas13a PFS screening libraries, we aligned the sequences flanking the randomized PFS region and extracted the PFS identities. We collapsed PFS identities to four nucleotides to improve sequence coverage, counted the frequency of each unique PFS, and normalized to total read count for each library with a pseudocount of 1. Enrichment of each distribution was calculated against the pACYC184 control (no protein/guide locus) as −log2(fcondition/fpACYC184), where fcondition is the frequency of PFS identities in the experimental condition and fpACYC184 is the frequency of PFS identities in the pACYC184 control. To analyse a conserved PFS motif, top depleted PFS identities were calculated using each condition’s non-targeting control as follows: −log2(fi,targeting/fi,non-targeting) where fi,targeting is the frequency of PFS identities in condition i with targeting spacer and fi,non-targeting is the frequency of PFS identities in condition i with non-targeting spacer. PFS motifs were analysed for a range of thresholds.

Purification of CRIPSR Cas13a

Purification of LwaCas13a was performed as previously described9. In brief, CRIPSR LwaCas13a bacterial expression vectors were transformed into Rosetta 2(DE3)pLysS singles Competent Cells (Millipore) and 4 l of Terrific Broth 4 growth media were seeded with a starter culture. Cell protein expression was induced with IPTG and after overnight growth, the cell pellet was harvested and stored at −80 °C. Following cell lysis, protein was bound using a StrepTactin Sepharose resin (GE) and protein was eluted by SUMO protease digestion (ThermoFisher). Protein was further purified by cation exchange using a HiTrap SP HP cation exchange column (GE Healthcare Life Sciences) and subsequently by gel filtration using a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences), both steps via FPLC (AKTA PURE, GE Healthcare Life Sciences). Final fractions containing LwaCas13a protein were pooled and concentrated into Storage Buffer (600 mM NaCl, 50 mM Tris-HCl pH 7.5, 5% glycerol, 2 mM DTT) and aliquots were frozen at −80 °C for long-term storage.

Cloning of mammalian and plant expression constructs

The human codon-optimized CRIPSR Cas13a gene was synthesized (Genscript) and cloned into a mammalian expression vector with either a nuclear export sequence or NLS under expression of the EF1-a promoter. Because of the stability conferred by monomeric-super-folded GFP (msfGFP), we fused msfGFP to the C terminus of LwaCas13a. The full-length direct repeat of LwaCas13a was used for cloning the guide backbone plasmid with expression under a U6 promoter. The catalytically inactive LwaCas13a–msfGFP construct (dead LwaCas13a or dLwaCas13a) was generated by introducing R474A and R1046A mutations in the two HEPN domains. A drug-selectable version of LwaCas13a–msfGFP was generated by cloning the protein into a backbone with the Blasticidin selection marker linked to the C terminus via a 2A peptide sequence. The negative-feedback version of the dLwaCas13a–msfGFP construct (dLwaCas13a–NF) was generated by cloning a zinc-finger binding site upstream of the promoter of dLwaCas13a–msfGFP and fusing a zinc finger and KRAB domain to the C terminus.

The reporter luciferase construct was generated by cloning Cypridinia luciferase (Cluc) under expression of the CMV promoter and Gaussia luciferase (Gluc) under expression of the EF1-a promoter, both on a single vector. Expression of both luciferases on a single vector allowed one luciferase to serve as a dosing control for normalization of knockdown of the other luciferase, controlling for variation due to transfection conditions.

For the endogenous knockdown experiments in Fig. 1g, guides and shRNAs were designed using the RNAxs siRNA design algorithm. The prediction tool was used to design shRNAs, and guides were designed in the same location to allow for comparison between shRNA and LwaCas13a knockdown.

For the plant knockdown experiments, the rice actin promoter (pOsActin) was PCR amplified from pANIC6A and LwaCas13a was PCR amplified from human expression LwaCas13a constructs. These fragments were ligated into existing plant expression plasmids such that LwaCas13a was driven by the rice actin promoter and transcription was terminated by the HSP terminator while the LwaCas13a gRNAs were expressed from the rice U6 promoter (pOsU6).


Protoplast preparation

Green rice protoplasts (O. sativa L. ssp. japonica var. Nipponbare) were prepared as previously described with slight modifications. Seedlings were grown for 14 days and protoplasts were resuspended in mMG buffer containing 0.1 M CaCl2. This modified mMG buffer was used to prepare fresh 40% PEG buffer as well as in place of WI buffer. Finally, protoplasts were kept in total darkness for 48 h post-transformation. All other conditions were as previously described.

Nucleic-acid target and crRNA preparation for in vitro reactions and collateral activity assays

To generate nucleic-acid targets, oligonucleotides were PCR amplified with KAPA Hifi Hot Start (Kapa Biosystems). dsDNA amplicons were gel extracted and purified using a MinElute gel extraction kit (Qiagen). The resulting purified dsDNA was transcribed via overnight incubation at 30 °C with a HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs). Transcribed RNA was purified using a MEGAclear Transcription Clean-up kit (Thermo Fisher).

To generate crRNAs, oligonucleotides were ordered as DNA (Integrated DNA Technologies) with an additional 5′ T7 promoter sequence. crRNA template DNA was annealed with a T7 primer (final concentrations 10 μM) and transcribed via overnight incubation at 37 °C with a HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs). The resulting transcribed crRNAs were purified with RNAXP clean beads (Beckman Coulter), using a 2× ratio of beads to reaction volume, supplemented with additional 1.8× ratio of isopropanol (Sigma).

CRIPSR LwaCas13a cleavage and collateral activity detection

For biochemical characterization of CRIPSR LwaCas13a, assays were performed as previously described. In brief, nuclease assays were performed with 160 nM of end-labelled single-stranded (ss)RNA target, 200 nM purified LwaCas13a, and 100 nM crRNA, unless otherwise indicated. All assays were performed in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3). For array processing, 100 ng of in vitro transcribed array was used per nuclease assay. Reactions were allowed to proceed for 1 h at 37 °C (unless otherwise indicated) and were then quenched with proteinase buffer (150 U ml−1 proteinase K, 60 mM EDTA, and 4 M urea) for 15 min at 37 °C. The reactions were then denatured with 4.5 M urea denaturing buffer at 95 °C for 5 min. Samples were analysed by denaturing gel electrophoresis on 10% PAGE TBE-Urea (Invitrogen) run at 45 °C. Gels were imaged using an Odyssey scanner (LI-COR Biosciences).

Collateral activity detection assays were performed as previously described. In brief, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNase Alert v2, Thermo Scientific), 2 μl mouse RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic-acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3). Reactions were allowed to proceed for 1–3 h at 37 °C (unless otherwise indicated) on a fluorescent plate reader (BioTek) with fluorescent kinetics measured every 5 min.

Cloning of tiling guide screens

For tiling guide screens, spacers were designed to target mRNA transcripts at even intervals to fully cover the entire length of the transcript. Spacers (ordered from IDT) were annealed and golden-gate cloned into LwaCas13a guide expression constructs with either a tRNAval promoter (Gluc and Cluc screens) or U6 promoter (all endogenous screens).

Mammalian cell culture and transfection for knockdown with CRIPSR/Cas13a

All mammalian cell experiments were performed in the HEK293FT line (American Type Culture Collection (ATCC)) unless otherwise noted. HEK293FT cells were cultured in Dulbecco’s Modified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (VWR Seradigm) and 1× penicillin–streptomycin (Thermo Fisher Scientific). Cells were passaged to maintain confluency below 70%. For experiments involving A375 (ATCC), cells were cultured in RPMI Medium 1640 (Thermo Fisher Scientific) supplemented with 9% fetal bovine serum (VWR Seradigm) and 1× penicillin–streptomycin (Thermo Fisher Scientific). The HEK293FT cells were not authenticated after receiving them from ATCC and were not checked for mycoplasma contamination.

To test knockdown of endogenous genes, Lipofectamine 2000 (Thermo Fisher Scientific) transfections were performed with 150 ng of LwaCas13a plasmid and 250 ng of guide plasmid per well, unless otherwise noted. Experiments testing knockdown of reporter plasmids were supplemented with 12.5 ng reporter construct per well. Sixteen hours before transfection, cells were plated in 96-well plates at approximately 20,000 cells per well and allowed to grow to 90% confluency overnight. For each well, plasmids were combined with Opti-MEM I Reduced Serum Medium (Thermo Fisher) to a total of 25 μl, and separately 0.5 μl of Lipofectamine 2000 was combined with 24.5 μl of Opti-MEM. Plasmid and Lipofectamine solutions were then combined, incubated for 5 min, and slowly pipetted onto cells to prevent disruption.

Transformation of green rice protoplasts

For the green rice experiments, plasmids expressing each CRIPSR LwaCas13a and the corresponding guide RNA were mixed in equimolar ratios such that a total of 30 μg of DNA was used to transform a total of 200,000 protoplasts per transformation.

Measurement of luciferase activity

Media containing secreted luciferase was harvested 48 h after transfection, unless otherwise noted. Media were diluted 1:5 in PBS and then luciferase activity was measured using BioLux Cypridinia and Biolux Gaussia luciferase assay kits (New England Biolabs) on a Biotek Synergy 4 plate reader with an injection protocol. All replicates were performed as biological replicates.

Harvest of total RNA and quantitative PCR

For gene expression experiments in mammalian cells, cell harvesting and reverse transcription for cDNA generation was performed using a previously described modification of the commercial Cells-to-Ct kit (Thermo Fisher Scientific) 48 h after transfection. Transcript expression was then quantified with qPCR using Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan qPCR probes (Thermo Fisher Scientific) with GAPDH control probes (Thermo Fisher Scientific). All qPCR reactions were performed in 5-μl reactions with four technical replicates in a 384-well format, and read out using a LightCycler 480 Instrument II (Roche). For multiplexed targeting reactions, readout of different targets was performed in separate wells. Expression levels were calculated by subtracting housekeeping control (GAPDH) cycle threshold (Ct) values from target Ct values to normalize for total input, resulting in ΔCt levels. Relative transcript abundance was computed as 2−ΔCt. All replicates were performed as biological replicates.

For gene expression experiments in plant cells, total RNA was isolated after 48 h of incubation using Trizol according to the manufacturer’s protocol. One nanogram of total RNA was used in a SuperScript III Plantinum SYBR Green One-Step qRT–PCR Kit (Invitrogen) according to the manufacturer’s protocol. All samples were run in technical triplicate of three biological replicates in a 384-well format on a LightCycler 480 Instrument (Roche). All PCR primers were verified as being specific on the basis of melting curve analysis and were as follows: OsEPSPS (Os06g04280), 5′-TTGCCATGACCCTTGCCGTTGTTG-3′ and 5′-TGATGATGCAGTAGTCAGGACCTT-3′; OsHCT (Os11g07960), 5′-CAAGTTTGTGTACCCGAGGATTTG-3′ and 5′-AGCTAGTCCCAATAAATATGCGCT-3′; OsEF1a (Os03g08020), 5′-CTGTAGTCGTTGGCTGTGGT-3′ and 5′-CAGCGTTCCCCAAGAAGAGT-3′. Primers for OsEF1a were previously described.

For analysis of RNA quality post-knockdown with CRIPSR LwaCas13a, total RNA was harvested by lysing cells using TRI Reagent and purifying the RNA using the Direct-zol RNA MiniPrep Plus kit (Zymo). Four nanograms of total RNA were analysed using a RNA 6000 Pico Bioanalyzer kit (Agilent).

Computational analysis of target accessibility

To first analyse target accessibility, top guides from the tiling screen were analysed to determine whether they grouped closer together than expected under the assumption that if there were regions of accessibility, multiple guides in that region would be expected to be highly active. Top guides were defined as the top 20% of performing guides for the Gluc tiling screen and top 30% of performing guides for the Cluc, KRAS, and PPIB tiling screens. A null probability distribution was generated for pairwise distances between guides by randomly simulating 10,000 guide positions and then comparing with experimentally determined top guide pairwise distances.

Accessibility was predicted using the RNApl fold algorithm in the Vienna RNA software suite25. The default window size of 70 nt was used and the probability of a target region being unpaired was calculated as the average of the 28 single-nucleotide unpaired probabilities across the target region. These accessibility curves were smoothed and compared with smoothed knockdown curves across each of the four transcripts, and correlations between the two factors and their significance were computed using Pearson’s correlation coefficient with the SciPy Python package (pearsonr function). The probability space of these two factors was also visualized by performing two-dimensional kernel density estimation across the two variables.

RNA sequencing and analysis

For specificity analysis of CRIPSR LwaCas13a knockdown, RNA-seq was performed on mRNA from knockdown experiments involving both LwaCas13a and shRNA constructs. Total RNA was prepared from transfection experiments after 48 h using a Qiagen RNeasy Plus Mini kit. mRNA was then extracted using a NEBNext Poly(A) mRNA Magnetic Isolation Module and RNA-seq libraries were prepared using a NEBNext Ultra Directional RNA Library Prep Kit for Illumina. RNA-seq libraries were sequenced on an Illumina NextSeq instrument with at least 10 million reads per library.

An index was generated using the RefSeq GRCh38 assembly and reads were aligned and quantified using Bowtie and RSEM version 1.2.31 with default parameters26. Transcripts per million (TPM) values were used for expression counts and were transformed to log-space by taking the log2(TPM + 1).

To find differentially expressed genes, Student’s t-test was performed on the three targeting replicates versus the three non-targeting replicates. The statistical analysis was only performed on genes that had a log2(TPM + 1) value greater than 2.5 in at least two of the six replicates. Only genes that had a differential expression greater than 2 or less than 0.75 and a false discovery rate <0.10 were reported to be significantly differentially expressed.

Cross-correlations between replicates and averages of replicates were performed using Kendall’s tau coefficient. The variation of shRNA versus LwaCas13a libraries was analysed by considering the distribution of standard deviations for gene expression across the six replicates (three targeting and three non-targeting replicates) and represented as violin plots.

Cell viability assay

Mammalian cells were transfected with luciferase reporter target, guide plasmid, and either CRIPSR LwaCas13a or drug-selectable LwaCas13a. Twenty-four hours after transfection, cells were split 1:5 into fresh media and drug-selectable LwaCas13a samples were supplemented with 10 μg ml−1 Blasticidin S (Thermo Fisher Scientific). After 48 h of additional growth, cells were assayed for luciferase knockdown, maintenance of LwaCas13a expression via GFP fluorescence measurement on a multimode plate reader (Biotek Neo2), and cell growth by CellTiter-Glo Luminescent Cell Viability Assay (Promega).

Quantifying dLwaCas13a binding with RNA immunoprecipitation

For RNA immunoprecipitation experiments, HEK293FT cells were plated in six-well plates and transfected with 1.3 μg of dLwaCas13a expression plasmid and 1.7 μg of guide plasmid, with an additional 150 ng of reporter plasmid for conditions involving reporter targeting. Forty-eight hours after transfection, cells were washed twice with ice-cold PBS (Sigma) and fixed with 0.2% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min at room temperature. After fixation, the paraformaldehyde was removed, 125 mM glycine in PBS was added to quench crosslinking, and the cells were incubated for 10 min. Cells were washed twice with ice-cold PBS, harvested by scraping, and the cell suspension was centrifuged at 800g for 4 min to pellet the cells. The supernatant was removed and the pellet was washed with PBS before lysis. Cells were lysed with 200 μl of 1× RIPA Buffer (Cell Signaling) supplemented with cOmplete ULTRA Tablets, EDTA-free (Sigma) and ribonuclease inhibitor (Sigma R1158). Cells were allowed to lyse on ice for 10 min and then sonicated for 2 min with a 30 s on/30 s off cycle at low intensity on a Bioruptor sonicator (Diagenode). Insoluble material was pelleted by centrifugation at 16,000g for 10 min at 4 °C, and the supernatant containing cleared lysate was used for pulldown with magnetic beads.

To conjugate antibodies to magnetic beads, 100 μl per sample of Dynabeads Protein A for Immunoprecipitation (Thermo Fisher Scientific) were pelleted by application of a magnet, and the supernatant was removed. Beads were resuspended in 200 μl of wash buffer (PBS supplemented with 0.02% Tween 20 (Sigma)) and 5 μg of rabbit anti-Mouse IgG (Sigma M7023) was added. The sample was incubated for 10 min at room temperature on a rotator to allow antibody to conjugate to the beads. After incubation, beads were pelleted using a magnet, supernatant was removed, and beads were washed twice with wash buffer. The pellet was resuspended in 100 μl wash buffer and split into two 50 μl volumes for conjugation of anti-HA antibody (Thermo Fisher Scientific 26183) or IgG antibody control (Sigma I5381). For each antibody, 2.5 μg of antibody was added with 200 μl wash buffer and incubated for 10 min at room temperature on a rotator. After incubation, beads were pelleted using a magnet and washed twice with wash buffer, and resuspended in 200 μl 1× RIPA with ribonuclease inhibitor (Sigma R1158) and protease inhibitor cocktail (Sigma P8340). One hundred microlitres of sample lysate were added to beads and rotated overnight at 4 °C.

After incubation with sample lysate, beads were pelleted, washed three times with 1× RIPA, 0.02% Tween 20, and then washed with DNase buffer (350 mM Tris-HCl (pH 6.5); 50 mM MgCl2; 5 mM DTT). Beads were resuspended in DNase buffer and TURBO DNase (Life Technologies) was added to a final concentration of 0.08 units per microlitre. DNase was incubated for 30 min at 37 °C on a rotator. Proteins were then digested by addition of Proteinase K (New England Biosciences) to a final concentration of 0.1 units per microlitre and incubated at 37 °C with rotation for an additional 30 min. For denaturation and purification, urea (Sigma) was added to a final concentration of 2.5 M, samples were incubated for 30 min, and RNA was purified using a Direct-Zol RNA miniprep (Zymo Research). Purified RNA was reverse transcribed to cDNA using the qScript Flex cDNA (Quantabio) and pulldown was quantified with qPCR using Fast Advanced Master Mix and TaqMan qPCR probes. All qPCR reactions were performed in 5-μl reactions with 4 technical replicates in 384-well format, and read out using a LightCycler 480 Instrument II. Enrichment was quantified for samples compared with their matched IgG antibody controls.

Translocation measurement of CRIPSR LwaCas13a and LwaCas13a-NF

HEK293FT cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips (Corning) and transfected with 150 ng dLwaCas13a–NF vector and 300 ng guides for imaging ACTB. For translocation experiments, cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100 after 48 h, and mounted using anti-fade mounting medium with DAPI (Vectashield). Confocal microscopy was performed with a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.

Nuclear export of dLwaCas13a-NF with guides targeting ACTB mRNA was analysed by measuring the average cytoplasmic and nuclear msfGFP fluorescence and comparing the ratio across many cells between targeting and non-targeting conditions.

FISH of ACTB transcript

HEK293FT cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips (Corning) and transfected with 75 ng dLwaCas13a–NF vector and 250 ng guides for imaging ACTB. After 48 h, cells were fixed with 4% PFA for 45 min. A QuantiGene viewRNA ISH Cell assay kit (Affymetrix) was used for performing FISH on the cell samples according to the manufacturer’s protocol. After finishing the FISH procedure, coverslips were mounted using anti-fade mounting medium (Vectashield). Confocal microscopy was performed using a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.

Tracking of LwaCas13a to stress granules

HEK293FT cells were plated in 24-well tissue culture plates on poly-d-lysine coverslips (Corning) and transfected with 75 ng dLwaCas13a-NF vector and 250 ng guides for imaging ACTB. For stress granule experiments, 200 μM sodium arsenite was applied for 1 h before fixing and permeabilizing the cells. For immunofluorescence of G3BP1, cells were blocked with 20% goat serum, and incubated overnight at room temperature with anti-G3BP1 primary antibody (Abnova H00010146-B01P). Cells were then incubated for 1 h with secondary antibody labelled with Alexa Fluor 594 and mounted using anti-fade mounting medium with DAPI (Vectashield). Confocal microscopy was performed using a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.

Stress granule co-localization with dLwaCas13a–NF was calculated using the average msfGFP and G3BP1 signal per cell using Pearson’s correlation coefficient. The co-localization analyses were performed in the image analysis software FIJI27 using the Coloc 2 plugin.

For live imaging experiments, HEK293FT cells were plated in 96-well tissue culture plates and transfected with 150 ng dLwaCas13a–NF vector, 300 ng guides for imaging ACTB, and 5 ng of G3BP1–RFP reporter. After 48 h, the cells were subjected to 0 μM or 400 μM sodium arsenite and imaged every 15 min for 2 h on an Opera Phenix High Content Screening System (PerkinElmer) using the spinning disk confocal setting with a 20× water objective. Cells were maintained at 37 °C in a humidified chamber with 50% CO2. Live-cell dLwaCas13a–NF co-localization with G3BP1–RFP in stress granules was measured using Opera Phenix Harmony software (PerkinElmer).

Data availability

CRIPSR LwaCas13a (C2c2) expression plasmids are available from Addgene under UBMTA. Patent applications have been filed relating to work in this manuscript. Support forums and computational tools including relevant code for data analysis are available via the Zhang laboratory website (http://www.genome-engineering.org) and Github (https://github.com/fengzhanglab). High-throughput sequencing data related to this study are available at BioProject under accession number PRJNA383832. Raw data represented in main figures in this study are included in this published article and its Supplementary Information. Additional datasets are available from the corresponding author on reasonable request.


Source file

RNA targeting with CRISPR–Cas13

Omar O. Abudayyeh, Jonathan S. Gootenberg, Patrick Essletzbichler, Shuo Han, Julia Joung, Joseph J. Belanto, Vanessa Verdine, David B. T. Cox, Max J. Kellner, Aviv Regev, Eric S. Lander, Daniel F. Voytas, Alice Y. Ting & Feng Zhang


Related topic

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of COVID19

Clinical validation of a CRISPR Cas13-based detection of SARS-CoV-2 RNA   

A CRISPR Cas13 protocol for detection of COVID-19