Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems

Abstract

The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9’s RNA-targeting potential.

1. Introduction

CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated cas genes provide bacteria and archaea with adaptive defense against horizontal gene transfer ^1–3. CRISPR-Cas systems are very widespread, present in almost half of the sequenced prokaryotic genomes. CRISPR immunity is accomplished through three steps: 1). The integration of a short segment of the invader’s genome into the CRISPR array as a new “spacer”, forming an immunological memory that primes the microbe for future defense ¹. 2). The biogenesis of CRISPR RNAs (crRNAs) from newly acquired spacers, and the assembly of Cas protein effector complexes guided by crRNAs ². 3). Recognition and interference of nucleic acid targets. Directed by crRNAs, the Cas effector enzymes locate and destroy target sequences complementary to the CRISPR guide ^4–6. CRISPR systems are remarkably diverse and are categorized into two major classes, six types and numerous subtypes based on the cas operon composition; Class I CRISPR systems rely on multi-protein complexes for target destruction whereas Class II systems utilize single effector enzymes^7,8.

How the Cas9 and Cas12 (Class 2, type II and V effectors) families of crRNA-guided DNA endonucleases operate have been elucidated in vitro using biochemical approaches ^5,6,9, setting the stage for their adoption as powerful eukaryotic genome editing tools that have revolutionized biomedical research and gene therapy ^9–13. Programmed by a crRNA and the tracrRNA co-factor, Cas9 identifies the dsDNA target through recognition of a short protospacer adjacent motif (PAM) and crRNA-target complementarity; the HNH and RuvC nuclease domains of Cas9 carry out DNA scissions on the target and non-target strands (TS and NTS), respectively ^5,6. Catalytically dead Cas9s (dCas9s), which retains their target binding capability but can no longer generate DNA double strand breaks ⁵, have also proven to be useful for the delivery of various effector domains to intended genomic sites, to achieve locus-specific gene regulation, epigenome modification, base editing, chromosomal loci imaging, etc. [for comprehensive CRISPR technology reviews, see^13,14]. Several families of Cas9-specific Acrs have been discovered^15–21, and they inhibit Cas9-mediated DNA cleavage or genome editing via distinct mechanisms (for comprehensive anti-CRISPR reviews, see²²).

While the vast majority of Class II CRISPR systems target DNA, there are a few capable of targeting RNA. For example, type VI effector Cas13 is a promiscuous RNase that requires activation by RNA-guided RNA recognition ^23,24, and has been repurposed for in vivo knockdown of mammalian transcripts ²⁵. Furthermore, Cas9 orthologs from Streptococcus pyogenes (Spy), Staphylococcus aureus (Sau), Neisseria meningitidis (Nme), and Campylobacter jejuni (Cje) have all been shown to possess crRNA-guided ribonuclease activities ^26–29. These discoveries fundamentally expanded Cas9’s targeting capacities, and potentiated novel CRISPR-Cas9 based strategies to manipulate specific RNA transcripts in vivo, including SpyCas9-enabled visualization and elimination of specific mRNAs in mammalian cells ^30,31, and SauCas9-mediated programmable defense against RNA phage MS2 for bacterial cells ²⁶. Most of these RNA-targeting Cas9s recognize sequence-specific RNA targets in a PAM-independent manner ^26–28; in stark contrast to their targeting of dsDNA substrates, which is strictly PAM-dependent.

As more and more Cas9 orthologs are identified and utilized as RNA-guided DNA endonucleases and gene-editors, it is of importance to fully characterize their CRISPR-programmable RNA-targeting potential. This kind of investigation can potentially expand the toolkit for transcriptome engineering, and help decipher the biological roles for RNA-targeting CRISPRs. Furthermore, insights can also be gained about how transcripts produced from the Cas9-targeted genomic sites would be affected during genome engineering. Here we describe detailed methods to recombinantly purify Cas9 proteins, to prepare guide RNAs and ssRNA substrates, to assay Cas9-mediated in vitro binding to and cleavage of RNA targets, and to assess how anti-CRISPRs affect Cas9’s ribonuclease activities.

2. Methods

To test if any particular CRISPR-Cas9 system has an RNA-guided ribonuclease activity using in vitro approaches, the Cas9 protein of interest needs to be purified recombinantly and the corresponding crRNA and tracrRNA species be created by in vitro transcription. Potential ssRNA targets should be synthesized or purchased in radiolabeled or fluorescently-labeled form. These individual components are then mixed together under various conditions to systematically assess Cas9-catalyzed binding to and cleavage of the RNA targets (Figure 1). All protein dilution and cleavage/binding reaction assembly steps are performed on ice unless stated otherwise.

Figure 1. Workflow for testing Cas9’s RNA-targeting property in vitro.

This chart describes the major steps involved in the characterization of Cas9’s CRISPR-guided, ribonuclease activity.

2.1. Cloning of Cas9 and Anti-CRISPR (Acr) expression constructs

Cas9 and Acrs are recombinantly expressed in Escherichia coli (E. coli) and purified using a series of affinity and size exclusion columns (Figure 2). To express the Cas9 proteins and Acrs of interests, their coding sequences are first cloned into bacterial protein expression vectors such as the popular T7 protein expression system. The coding sequences can be PCR amplified directly from the genomic DNA of the native bacterial strain(s), or synthesized chemically if no genomic DNA material is available. Commonly used affinity tags such as 6XHis-or MBP- tags can be appended to the N- or C- terminus to aid Cas9 purification. In our experience, 6XHis tag mediated purification gives lower yield for certain Cas9 (e.g. NmeCas9) mainly due to low tag binding efficiency. As a workaround, we recommend using heparin column, which consistently gives high yield and good separation between Cas9 proteins and other native E. coli proteins. To determine if codon optimization is needed for optimal Cas9 or Acr expression in E. coli, the online tool “GenScript Rare Codon Analysis” can be used.

Figure 2. Workflow for the purification of recombinant Cas9 and anti-CRISPR proteins.

Untagged Cas9 is purified with a heparin column followed by a size exclusion column. 6xHis tagged-Acrs are expressed and purified by an initial Ni-NTA affinity step. The tag is then removed with TEV protease, and the untagged proteins are purified away from the 6xHis tag through either a 2^nd nickel column or a size exclusion column.

To clone a FLAG-tagged NmeCas9, we assembled the wild type NmeCas9 ORF into pET-28b vector (Novagen) digested with NcoI and NotI using Gibson assembly (New England Biolabs [NEB]). A FLAG tag was then inserted to the C-terminus of Nmecas9 using Q5 site-directed mutagenesis (NEB). The FLAG-tag is not essential for purification, but incorporated to help the tracking of NmeCas9 by western blot in downstream applications. Constructs for expressing RuvC, or HNH, or double active site mutants of cas9 can be further generated using Q5 site-directed mutagenesis.

The anti-CRISPR genes for type II systems (e.g. AcrIIC1_Nme¹⁷ and AcrIIA4¹⁶) were either directly synthesized as gBlocks (Integrated DNA Technologies [IDT]) or PCR amplified from existing plasmids, and ligated into pET-28b via NdeI and HindIII sites. To avoid appending any tag onto the small Acr proteins, a Tobacco Etch Virus (TEV) protease cleavage site was incorporated between the Acr gene and the His tag on pET-28b through the PCR primers, to allow the removal of the 6XHis tag during purification.

Reagents:

DNA oligonucleotides and gBlocks (IDT)

pET-28b (+) vector DNA (Novagen, 69865)

Q5 site-directed mutagenesis kit (NEB, E0554S)

T4 DNA ligase (NEB, M0202S)

Competent E. coli JM109 cells for cloning (Promega, L2005)

2.2. Expression and purification of Cas9 and Acr Proteins

The expression constructs for Cas9s and the Acrs are transformed into BL21 (DE3) (Novagen) competent cells. To induce protein expression, a 3 mL overnight culture from a single transformant is used to inoculate 1 L of LB broth with appropriate antibiotics. The large culture is grown to OD₆₀₀ of 0.4–0.6, cooled on ice, induced with 0.5 mM IPTG, then grown at 18°C for 16 hrs. On the following day, the induced culture is pelleted and stored at −20°C until use.

The following protocol is used for the purification of NmeCas9 without using any affinity tag, and should be broadly applicable to other Cas9 proteins with minor modifications. See Figure 3 for representative SDS-PAGE images for NmeCas9 purification.

Figure 3. Representative SDS-PAGE of NmeCas9 purification.

Coomassie stained 10% SDS-PAGE gels of the NmeCas9 purification procedure. (A) Analysis of cell lysis and fractions from the heparin column. Note the significant reduction of NmeCas9 band intensity in the heparin flow through, relative to the soluble fraction. The 850 mM NaCl elution fractions were pooled and loaded onto S200. (B) Elution fractions from the S200 size exclusion column. Fractions marked by the star were pooled, concentrated, and saved.

1. Resuspend the E. coli pellet in Cas9 lysis buffer. Lyse the cells by sonication.

2. Pellet insoluble material by centrifugation at 30,000 g for 15 min at 4°C.

3. Load supernatant onto a 5 mL heparin column at a flow rate of 2 mL/min using FPLC.

4. Wash the column with 30 mL of Cas9 heparin column wash buffer at a flow rate of 3 mL/min.

5. Elute the bound proteins using a step gradient of pH 7.5 PBS buffer, with 600 mM, 850 mM, and 2 M NaCl, 10 mL per gradient and collect 5 mL fractions.

6. Analyse 5 μL of each fraction on SDS-PAGE, with Coomassie staining.

7. Pool all Cas9 containing fractions together and concentrate it down to 1 mL using an Amicon Ultra-15 10,000 MWCO spin filter.

8. Load the concentrated sample onto a HiPrep 16/60 Sephacryl S-200 HR size exclusion column. Elute with an isocratic elution over 1.5 column volumes at 0.5 mL/min with Cas9 Sephacryl S-200 buffer, and collect 2.5 mL fractions.

9. Analyze 5 μL of each fraction on SDS-PAGE, with Coomassie staining.

10. Pool all Cas9 containing fractions and concentrate down to 0.5 mL using an Amicon Ultra-15 10,000 MWCO spin filter.

11. Measure protein concentration with a UV spectrometer at 280 nm. See Table 1 for conversion factors.

12. Make 10 μL aliquots. Flash freeze in liquid nitrogen and store at −80°C.

Table 1.

Properties of NmeCas9 and Acrs

Protein	Molecular Weight	Molar Extinction coefficient	1 A280 unit equals to	1 mg/mL equals to
NmeCas9-FLAG	124.3 kDa	106075	1.17 mg/mL	8.0 μM
AcrIIC1	9.8 kDa	25940	0.38 mg/mL	101.7 μM
AcrIIC2	14.3 kDa	21095	0.68 mg/mL	69.6 μM
FLAG-AcrIIC3	14.6 kDa	11585	1.26 mg/mL	68.4 μM

The following protocol is for purifying Acrs that contain a TEV cleavable 6xHis tag (except for AcrIIC_Nme3).

1. Resuspend the E. coli pellet in Acr lysis buffer. Lyse cells by sonication.

2. Pellet insoluble material by centrifugation at 30,000 g for 15 min at 4°C.

3. Wash 1 mL Ni-NTA slurry with Acr Ni column wash buffer.

4. Mix cleared E. coli lysate with washed Ni-NTA resin. Incubate at 4°C for 1 hr.

5. Pour the mixture into a disposable chromatography column. Wash the resin with 20 mL of Acr Ni column wash buffer.

6. Elute the bound proteins with 10 mL Acr Ni column elution buffer.

7. Add 100 μg TEV protease to the eluted protein and dialyze against Acr dialysis buffer overnight at 4°C.

8. Wash 1 ml Ni-NTA slurry with Acr dialysis buffer.

9. Incubate dialyzed protein with washed Ni-NTA beads at 4°C for 1 hour.

10. Pour the mixture into a disposable column. Collect flow-through, which contains Acrs with the 6xHis tag cleaved off.

11. Concentrate the flow-through using an Amicon Ultra-15 3,000 MWCO spin concentrator.

12. Add glycerol to final concentration of 10%. Measure protein concentration with a UV spectrometer at 280 nm. See Table 1 for conversion factors.

13. Make 10 μL aliquots. Flash freeze in liquid nitrogen and store at −80°C.

    Note: FLAG-AcrIIC3 tends to bind to Ni resin, even without a 6X-His tag. Therefore, for purifying FLAG-AcrIIC3, follow steps n-r instead of h-m, after step g.

14. Concentrate the dialyzed protein down to 1 mL using an Amicon Ultra-15 3,000 MWCO spin concentrator.

15. Load the concentrated sample onto a HiPrep 16/60 Sephacryl S-200 HR column. Elute with an isocratic elution over 1.5 column volumes at 0.5 mL/min with Acr dialysis buffer, and collect 2.5 mL fractions.

16. Analyze 5 μL of each fraction on SDS-PAGE. Pool all AcrIIC3 containing fractions and concentrate down to 0.5 mL using an Amicon Ultra-15 10,000 MWCO spin filter. Add glycerol to final concentration of 10%.

17. Measure protein concentration with a UV spectrometer at 280 nm. See Table 1 for conversion factors.

18. Make 10 μL aliquots. Flash freeze in liquid nitrogen and store at −80°C.

Equipment:

ÄKTA Start Protein Purification System FPLC (GE Healthcare, 29-0220-94)

Frac30 Fraction Collector (GE Healthcare, 29-0230-51)

HiTrap™ Heparin HP (GE Healthcare, 17-0407-03)

HiPrep Sephacryl S-200 HR (GE Healthcare, 17-1166-01)

Buffers and reagents:

Competent BL21 (DE3) E. coli cells (Novagen, 69450)

Amicon Ultra-15, 10 KDa MWCO, Centrifugal Filter Unit (Sigma-Aldrich, UFC901008)

10x FastBreak Cell Lysis Reagent (Promega, V8571)

Econo-Pac Chromatography Columns (Biorad, 7321010)

Cas9 lysis buffer: 1xPBS pH 7.5, 350 mM NaCl, 0.5 mM TCEP, 1x FastBreak cell lysis reagent

Cas9 heparin column wash buffer: 1xPBS pH 7.5, 350 mM NaCl, 0.5 mM DTT

Cas9 Sephacryl S-200 buffer: 1xPBS pH7.5, 350 mM NaCl, 0.5 mM DTT

Acr Lysis buffer: 20 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.5 mM TCEP and 1x FastBreak cell lysis reagent

Ni-NTA Agarose (Qiagen, 30210)

Acr Ni column wash buffer: 20 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.5 mM DTT

Acr Ni column elution buffer: 20 mM HEPES pH 7.5, 300 mM NaCl, 500 mM imidazole, 0.5 mM DTT

Acr dialysis buffer: 20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM DTT

TEV protease (Sigma-Aldrich, T4455)

Bio-Safe Coomassie Stain (Biorad, 1610786)

Dithiothreitol [DTT] (Fisher Scientific, BP17225)

HEPES (Sigma-Aldrich, H4034)

Bond-Breaker TCEP Solution, Neutral pH (Thermo Fisher Scientific, 77720)

Imidazole (Sigma-Aldrich, I202)

2.3. In vitro transcription (IVT) and gel purification of crRNA and tracrRNA

The crRNAs and tracrRNA used for in vitro cleavage/binding assays are generated by in vitro transcription, followed by 15% denaturing PAGE purification. Transcription templates could be gel-purified PCR products, linearized plasmids, or annealed DNA oligo pairs. We chose to create our transcription templates by annealing pairs of DNA oligos, because this approach is faster and the IVT yield is reliably great. The forward DNA oligo carries a T7 promoter followed by sequences corresponding to a mature crRNA or tracrRNA naturally exist in microbes. If the 1^st nt of the small RNA is not a guansosine, it should be changed to guanosine to ensure robust T7 transcription. Or alternatively, an extra guanosine can be added before this 1^st nt. For representative oligo designs and the Nme crRNA/tracrRNA sequences, see Rousseau et al.²⁷. To anneal the oligos for IVT, 100 pmol of the forward template oligo is mixed with 100 pmol of the reverse complement oligo in 25 μL oligo annealing buffer. This mixture is then incubated at 95°C for 5 min in a heating block, then slow cooled in the heating block to room temperature. For each small RNA species, we usually set up one 20 μL IVT reaction using the AmpliScribe T7-Flash Transcription Kit (Epicentre) using 8 pmol annealed oligos as template. After overnight incubation at 37°C, the entire IVT reaction is treated with RNase-free DNase, mixed with an equal volume of 2X Gel Loading Buffer II (Invitrogen) and resolved on a large 15% denaturing PAGE using a V16-2 vertical gel apparatus (Apogee), and a 10-well comb with 1.5 mm thick spacers. A typical 20 μL IVT reaction is loaded into 1–2 wells. If purifying multiple RNAs on the same gel, leave one empty well in between different samples to prevent cross contamination.

To purify the in vitro transcribed RNAs, this 15% denaturing polyacrylamide gel is removed from the glass plates, sandwiched between two layers of clear plastic wrap and placed on a Fluor-coated TLC plate (Sigma-Aldrich). We then shine UV light on the gel using a hand-held UV source to locate the IVT products using the UV shadowing technique. The location of the RNA band to be excised are marked on the plastic wrap, cut out using a clean razor blade, and placed into a 1.7 mL eppendorf tube with four holes punctured at its bottom by a hot 23-gauge needle. This tube (with gel slice) is then placed onto a 2.0 mL tube and spun in a microcentrifuge for 1 min at 14,000 g. This step forces the gel through the tiny holes and shreds it into tiny pieces. Alternatively, the gel slice can be crushed inside an intact 1.7 mL tube using a plastic pestle. The shredded gel pieces are soaked overnight in 1.5 mL RNA elution buffer with agitation. On the next day, we transfer the elution supernatant (some gel chunk carryover is ok) onto a Costar Spin-X centrifuge tube filter column (Corning), and spin it at 1,500 g for 1 min. The flow-through is split into two 1.5 mL eppendorf tubes, mixed with 10 μg glycogen and equal volumes of isopropanol, and precipitated at −20°C for half an hour. We then centrifuge the precipitation mixture at maximum speed 20,000 g for 30 min at 4°C, and wash the pellet twice with 0.5 mL 70% ice-cold ethanol. Liquid is removed from the pellet as much as possible. The final pellet is air dried for 3 min and resuspended in 10 μL RNase-free water. The RNA concentration can be determined using a Nanodrop at wavelength 260 nm before long-term storage at −80°C. We also quality check the RNA by analyzing a small aliquot on a Biorad Criterion sized 15% denaturing PAGE (see next section).

Equipment:

NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, 840274100)

V16-2 vertical gel apparatus (Apogee, 31071010)

Hand-held UV lamp (UVP 95000705, shortwave 254 nm)

Reagents and buffers:

Caution: RNase-free reagents should be used throughout all RNA experiments.

UltraPure DNase/RNase-Free water (Invitrogen, 10977023)

AmpliScribe T7-Flash Transcription Kit (Epicentre, ASF3507)

DNA oligos and ultramers (IDT)

1X oligo annealing buffer: 100 mM NaCl, 10 mM Tris, pH 8.5, RNase-free.

2X Gel Loading Buffer II (ThermoFisher, AM8547. 95% formamide, 18 mM EDTA and 0.025% each of SDS, Xylene Cyanol, and Bromophenol Blue)

Thin-layer chromatography (TLC) plate for UV shadowing (Sigma-Aldrich, Z185329)

Single-edge razor blade (VWR, 55411-050)

RNase-free plastic pestle (Fisher, 12-141-368)

RNA elution buffer: 0.3 M NaCl-TE (300 mM NaCl, 10 mM Tris pH7.5, 1 mM EDTA)

PrecisionGlide needle 23G (BD, 305145)

Costar Spin-X Centrifuge Tube Filter (Corning, 8163)

Glycogen, 20 mg/mL (Roche, 10901393001)

Tris base (Fisher Scientific, BP152)

Ethylenediaminetetraacetic acid [EDTA] (Sigma-Aldrich, EDS)

2.4. Cas9-catalyzed in vitro RNA cleavage

The crRNAs and ssRNA substrates used in our experiments are all designed based on native spacers from CRISPR-containing bacteria strains and their natural DNA target sequences. In theory, crRNA can be reprogrammed to contain any spacer (i.e. guide) sequence of interest. Accordingly, the ~35–50 nt ssRNA substrate should include a segment complementary to the crRNA spacer. Furthermore, it is better to have RNA cleavage products longer than 15 nt, so that their migration on the denaturing PAGE will not be impeded by the salt front. Fluorescently labeled ssRNA targets, with 5’-Cy5 and 3’−6-FAM, are purchased from Integrated DNA Technologies. We usually clean up the purchased ssRNA by gel purification, to remove any smaller RNA species that might have resulted from modest degradation. Gel purification is carried out the same way as described previously for in vitro transcription products, except that a thinner gel is used and the RNA is visualized directly (they appear yellow-green due to 6-FAM) without UV shadowing.

We usually prepare a master stock (e.g. 250 mL) of 15% urea-acrylamide solution, which can be stored at 4°C for up to two months. Before setting up in vitro cleavage reactions, we prepare a fresh 15% denaturing urea-polyacrylamide gel by transferring 15 mL of this gel stock solution into a conical tube, and adding 150 μL of 10% APS solution and 15 μL TEMED. This entire mixture is then immediately poured into an empty Criterion gel cassette (Biorad, 1.0 mm thickness), with the 18-well comb inserted back on. The gel normally polymerizes within 20–30 min and can be stored temporarily at 4°C. All Cas9 cleavage reactions are carried out in 1X RNA cleavage buffer at 37°C for 30 min. For NmeCas9, we typically use 250–500 nM NmeCas9 protein and equal moles of dual RNAs (crRNA and tracrRNA) in a cleavage reaction with 25–50 nM fluorescently labeled RNA substrate added at the last step. For any new Cas9 ortholog never characterized before, experiments with varying concentrations of Cas9 RNP, ssRNA substrate in combination with different reaction time are highly recommended. Furthermore, co-factor requirements (e.g. tracrRNA, PAM, metal ion, nuclease motifs, salt concentration, crRNA spacer length, etc.) and mismatch tolerance for Cas9-catalyzed RNA cleavage can be defined as described earlier ^26–29. To quality check the Cas9 protein prep, a PAM-containing dsDNA target should also be analyzed in a control experiment; and robust dsDNA cleavage indicates that the Cas9 RNP is functional. The dsDNA target can be a circular plasmid or an annealed DNA oligo pair.

Below is a detailed protocol for an in vitro RNA cleavage assay.

1. Dilute a small aliquot of Cas9 protein stock to 5 μM using fresh Cas9 dilution buffer.

2. Assemble in vitro RNA cleavage reactions in 0.65 mL or PCR tubes:

   200 mM HEPES pH 7.5, 1 mM EDTA, 5 mM fresh DTT	1 μL
   100 mM MgCl2	1 μL
   1 M KCl	1.5 μL
   RNase-free water	2.5 μL
   5 μM Cas9 stock	1 μL
   5 μM crRNA	1 μL
   5 μM tracrRNA	1 μL
   0.5 μM fluorescently labeled RNA substrate	1 μL
   Total volume	10 μL

3. Mix well; incubate at 37°C for half an hour.

4. To quench finished reactions, add 1 μL of 10 mg/mL Proteinase K to each 10 μL reaction, mix well, incubate at 37°C for 15 min.

5. Pre-run the 15% urea-polyacrylamide gel in 1X TBE buffer for 15 min at 200 V.

6. Add two volumes of freshly prepared 1.5X Formaldehyde-Formamide Loading dye to quenched cleavage reactions. Heat the samples at 95°C for 3 min, snap chill on ice.

7. Thoroughly flush the wells with a pipette or syringe. Load 10 μL into one well for each reaction. Save the rest of sample at −80°C.

8. Run the gel in 1X TBE buffer for 45 min at 200 V.

9. Open the cassette and transfer the gel to the Biorad Blot/UV/Stain-Free Sample Tray. FAM- or Cy5- labeled RNA can be visualized and imaged with appropriate filters on a Biorad Chemidoc MP imaging system.

We found that the 2X Gel Loading Buffer II (ThermoFisher) works well to separate cleaved DNA oligos or IVT products on denaturing urea-polyacrylamide gels. But unfortunately, this loading dye cannot fully denature the crRNA-ssRNA target duplex (Figure 4). What works best is our homemade Formaldehyde-Formamide loading dye, which fully denatures crRNA-RNA target duplexes, offers great resolution and doesn’t interfere with Cy5 detection. Due to the ability of formaldehyde to crosslink protein to nucleotides, it is important to digest the Cas9 protein using proteinase K before adding the loading dye.

Figure 4. Comparison of different denaturing RNA gel loading buffers.

NmeCas9-catalyzed in vitro RNA cleavage reactions are mixed with either Gel loading buffer II or our homemade Formaldehyde-Formamide loading buffer, heated at 95°C, snap-chilled on ice and ran on a 15% urea-PAGE. Gel loading buffer II cannot completely resolve the crRNA-target RNA duplex, whereas the Formaldehyde-Formamide loading buffer effectively denatures all RNA species.

Equipment:

ChemiDoc MP Imaging System (Biorad, 12003154)

Blot/UV/Stain-Free Sample Tray for ChemiDoc (Biorad, 12003028)

Criterion vertical midi-format electrophoresis Cell (Biorad, 1656001)

Criterion empty cassettes, 18 wells (Biorad, 3459902, 1.0 mm thickness)

Reagents and buffers:

5’-Cy5, 3’-FAM labeled ssRNA oligonucleotide (Integrated DNA Technologies)

40% Acrylamide/Bis Solution, 19:1 (Biorad, 1610145)

Urea (Sigma, U5378)

TEMED (Tetramethylethylenediamine, Sigma, T9281)

Formaldehyde solution, 37% (Sigma, F1635)

Formamide deionized (Sigma, F9037)

Proteinase K 20 mg/mL (Thermo Scientific, EO0491)

Boric acid (Sigma-Aldrich, B6768)

10X TBE buffer: Mix 108 g Tris base, 55 g boric acid, 40 mL of 0.5 M EDTA pH 8.0, add H2O to 1L, pass through 0.22 µm filter before storage.

15% urea-acrylamide stock solution: Mix 105 g urea, 93 mL 40% acrylamide/Bis 19:1, 25 mL 10X TBE, add H₂O to a final volume of 250 mL. Store at 4°C for up to two months.

10% APS: dissolve 1 g ammonium persulfate (Sigma, A3678) into 10 mL sterile water. Store at 4°C for up to a month.

Cas9 dilution buffer: 20 mM HEPES pH7.5, 1mM DTT.

1X RNA cleavage buffer condition: 20 mM HEPES pH 7.5, 150 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, and 10 mM MgCl₂.

1.5X Formaldehyde-Formamide Loading dye: 1.5X TBE, 2.3 M formaldehyde, 53% formamide, 20 mM EDTA pH 8.0, 2.5 mg/mL Orange G. Prepare fresh in a chemical hood.

Orange G (Sigma-Aldrich, O3756)

2.5. Testing the effects of anti-CRISPR (Acr) proteins on RNA cleavage by Cas9

The ability of known Acrs to inhibit Cas9’s ribonuclease activity can be analyzed in vitro using the RNA cleavage assay. Increasing amounts of purified Type II Acr proteins can be pre-mixed with the Cas9 protein of interest in 1X RNA cleavage buffer for 10 min at room temperature. The tracrRNA and crRNA are then added for RNP assembly to occur at room temperature for another 10 min. Next, we add fluorescently labeled RNA substrate and carry out the cleavage reaction as described above. Typically, Acrs proteins are tested at 0.5- to 10- fold molar excess over Cas9. A Type I CRISPR-specific inhibitor (e.g. AcrE2³²) can serve as a negative control that would not block Cas9’s activity. It is also possible to gain insights about whether an Acr interferes with crRNA loading onto Cas9. To this end, Cas9-catalyzed RNA (or dsDNA) cleavage efficiencies under two different scenarios should be compared. One is to allow Cas9 to complex with its small RNA partners before the addition of Acr; the other is to incubate Cas9 with Acr before the addition of crRNA and tracrRNA. For any Acr that specifically block the crRNA loading step, we expect to see a strong blockage of in vitro substrate cleavage when Acr is added before crRNA and tracrRNA, but minimal inhibitory effect when Acr is added after Cas9 RNP complex formation³³.

Below is a step-by-step protocol for testing the impact of Acr on in vitro RNA cleavage.

1. Dilute a small aliquot of Cas9 protein stock to 5 μM using fresh Cas9 dilution buffer.

2. Dilute an aliquot of Acr protein to 2.5, 5, 15, and 30 μM stocks, using 1X Acr storage buffer.

3. Assemble in vitro RNA cleavage reactions in 0.65 mL or PCR tubes:

   Scenario 1 –

   Add Acr after Cas9 RNP assembly

   200 mM HEPES pH 7.5, 1 mM EDTA, 5 mM fresh DTT	1 μL
   100 mM MgCl2	1 μL
   1 M KCl	1.5 μL
   RNase-free water	1.5 μL
   5 μM Cas9 stock	1 μL
   5 μM crRNA	1 μL
   5 μM tracrRNA	1 μL
   Mix well; incubate at RT for 10 min to assemble RNP.
   Add 0, 2.5, 5, 15, or 30 μM Acr stock	1 μL
   Mix well; incubate at RT for 10min.
   Add 0.5 μM fluorescently labeled RNA substrate	1 μL
   Total volume	10 μL

   Scenario 2 –

   Add crRNA and tracrRNA after Cas9-Acr assembly.

   200 mM HEPES pH 7.5, 1 mM EDTA, 5 mM fresh DTT	1 μL
   100 mM MgCl2	1 μL
   1 M KCl	1.5 μL
   RNase-free water	1.5 μL
   5 μM Cas9 stock	1 μL
   Add 0, 2.5, 5, 15, or 30 μM Acr stock	1 μL
   Mix well; incubate at RT for 10 min.
   Add 5 μM crRNA	1 μL
   Add 5 μM tracrRNA	1 μL
   Mix well; incubate at RT for 10min.
   Add 0.5 μM fluorescently labeled RNA substrate	1 μL
   Total volume	10 μL

4. Mix well; incubate at 37°C for half an hour.

5. Analyze the cleavage reactions by denaturing PAGE.

Reagents and buffers:

1X Acr storage buffer: 20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM DTT

2.6. RNA Cleavage site mapping

Cas9’s exact cleavage sites on the ssRNA substrate can be determined by running the cleavage reaction on RNA sequencing gel, in comparison to RNA ladders generated by treating the same substrate with limited RNases or chemicals. At least two ladders generated with different methods are needed to accurately determine precise cleavage position. Commonly used ladders include alkaline hydrolysis, RNase T1 and RNase A ladders, etc. Alkaline hydrolysis generally has no base preference and creates a ladder with single base resolution. RNase T1 specifically cleaves after unpaired G residues in single stranded RNAs³⁴. RNase A cleaves after unpaired C or U residues³⁵. The selection of ladders would depend on the sequence composition of the specific RNA target and predicted cleavage sites.

Alkaline hydrolysis or various RNase digestions produce RNA products with different forms of 3’ termini, including 3’-OH, 3’-phosphate or 2’,3’-cyclic phosphate^36,37. These modifications would affect the mobility of the RNA products during gel electrophoresis (Figure 5), which may complicate accurate mapping of RNA cleavage sites. Therefore, it is important to treat RNA cleavage products and all the RNA ladders before gel electrophoresis, using the T4 polynucleotide kinase [PNK] (NEB) that converts these types of 3’ modifications into 3’- hydroxyl groups. Then, product migration on RNA sequencing gels would faithfully indicate the precise cleavage sites. Furthermore, by comparing the band migration patterns before and after T4 PNK treatment, what kind of 3’ ends are formed by Cas9-catalyzed RNA cleavage can also be inferred (Figure 5 [3’-OH for NmeCas9]).

Figure 5. Migration patterns for Cas9 cleavage products and various RNA ladders, before and after T4 PNK treatment.

The same 5’-FAM labeled ssRNA target is treated with RNase T1 (A), alkaline hydrolysis (B), and NmeCas9 (C). The reactions are resolved on a RNA sequencing gel, with or without de-phosphorylation by T4 PNK. The uncleaved full-length RNA substrate is indicated by *. Note the changes in migration pattern for (A) and (B), but not (C). A representative band in each panel is marked by arrowheads, highlighting the effects of de-phosphorylation of the RNA 3’ termini by T4 PNK.

The following protocols are for mapping the RNA cleavage site by NmeCas9 using alkaline hydrolysis and RNase T1 ladders.

2.6.1. Generation of RNase T1 ladder (pre- PNK treatment)

1. To a 1.5 mL tube, add 5 μL 10x RNase T1 cleavage buffer, 25 pmol of fluorescently labeled ssRNA target, 150 μg yeast tRNA, add water to 46 μL.

2. Add 4 μL of 1 U/μL RNase T1. Mix well, and incubate at 37°C for 5 min.

3. Add 50 μL phenol: chloroform. Vortex for 15 sec. Centrifuge at 18,000 g for 1 min. Carefully pipet the aqueous phase into a clean 1.5 mL tube without disturbing the organic phase.

4. Add 5 μL 3 M sodium acetate (pH 5.2) and 1 μL of 20 mg/mL glycogen to the extracted aqueous phase. Mix well. Then mix in 150 μL 100% ethanol, and incubate at −20°C for 1–2 hrs.

5. Centrifuge at 18,000 g for 15–30 min at 4°C to pellet the RNA.

6. Discard the supernatant. Wash the pellet twice with 500 μL ice cold 70% ethanol.

7. Air-dry the pellet for 3 min, and then dissolve the pellet in 10 μL RNase-free H₂O.

2.6.2. Generation of alkaline hydrolysis ladder (pre-PNK treatment)

1. In a 1.5ml tube, mix 45 μL alkaline hydrolysis buffer and 5 μL of 5 μM fluorescently labeled ssRNA target. Incubate at 95°C for 20 min.

2. Add 50 μL phenol: chloroform, and vortex for 15 sec. Centrifuge at 18,000 g for 1 min. Carefully pipet the aqueous phase into a clean 1.5 ml tube without disturbing the organic phase.

3. Add 5 μL 3 M sodium acetate (pH 5.2) and 1 μL of 20 mg/mL glycogen to the extracted aqueous phase. Mix well. Then add 150 μL 100% ethanol. Mix well and incubate at −20°C for 1–2 hrs.

4. Centrifuge at 18,000 g for 15 min at 4°C to pellet the RNA.

5. Discard the supernatant. Wash the pellet twice with 500 μL ice cold 70% ethanol.

6. Air-dry the pellet, and dissolve the pellet in 10 μL RNase-free H₂O.

2.6.3. PNK treatment and clean up for the ladders

1. To a 1.5 ml tube, add 5 μl of 10x T4 PNK buffer, 10 μl purified RNA from above (hydroxyl or RNase T1 ladders), 34 μl water and 1 μl of 10 U/μL T4 PNK. Mix well. Incubate at 37°C for 30min.

2. Add 50 μl phenol: chloroform. Vortex for 15 sec. Centrifuge at 18,000 g for 1min. Carefully pipet the aqueous phase into a clean 1.5 ml tube without disturbing the organic phase.

3. Add 5 μL 3 M sodium acetate (pH 5.2) and 1 μL of 20 mg/mL glycogen to the extracted aqueous phase. Mix well. Then add 150 μL 100% ethanol. Mix well and incubate at −20°C for 1–2 hrs.

4. Centrifuge at 18,000 g for 15–30 min at 4°C to pellet the RNA.

5. Discard the supernatant. Wash the pellet twice with 500 μL ice-cold 70% ethanol.

6. Air-dry the pellet, and dissolve it in 10 μL RNase-free water. Store the RNA at −80°C.

2.6.4. Clean-up of RNA cleavage by NmeCas9 (with vs. without PNK treatment)

1. Assemble the in vitro cleavage reaction as described in section 2.4 a-b.

2. Add 1 μL T4 PNK (or 1 μL of RNase-free water as control) directly to the RNA cleavage reaction, mix well and incubate at 37°C for 1 hr.

3. Add equal volume of phenol: chloroform. Vortex for 15 sec. Centrifuge at 18,000 g for 1 min. Carefully pipet the aqueous phase into a clean 1.5 mL tube without disturbing the organic phase.

4. Add 5 μL 3 M sodium acetate (pH 5.2) and 1 μL of 20 mg/mL glycogen to the extracted aqueous phase. Mix well. Then add 150 μL 100% ethanol. Mix well and incubate at −20°C for 1–2 hrs.

5. Centrifuge at 18,000 g for 15 min at 4°C to pellet the RNA.

6. Discard the supernatant. Wash the pellet twice with 500 μL ice cold 70% ethanol.

7. Air-dry the pellet, and dissolve it in 10 μL RNase-free water. Store at −80°C.

2.6.5. Mapping cleavage site with denaturing urea-PAGE

1. Prepare a 15% urea-polyacrylamide gel as describe in section 2.4, but using the V16-2 vertical system (Apogee) with a thin 0.4 mm comb and spacer set.

2. Pre-run the gel in 1X TBE buffer with a constant wattage for 20–30 min to warm the gel up to 50–60°C.

3. While the gel is in pre-running, mix 3 μL of Cas9 cleavage products, RNase T1 ladder and alkaline hydrolysis ladder (from sections 2.6.1 through 2.6.4) with 6 L freshly prepared 1.5X Formaldehyde-Formamide loading dye, respectively. Heat the samples at 95°C for 3 min, snap chill on ice.

4. Thoroughly flush the wells with a pipette or syringe to get rid of the urea.

5. Load the samples, run the gel in 1X TBE buffer with constant wattage. Note: keep the gel above 55°C to efficiently denature all RNA species. The temperature of the gel could be monitored with a digital laser temperature gun.

6. Transfer the gel to the Biorad Blot/UV/Stain-Free Sample Tray. FAM- or Cy5-labeled RNA can be visualized and imaged with appropriate filters on a Biorad Chemidoc MP imaging system.

Equipment, reagents and buffers:

Digital laser temperature gun (Etekcity, Lasergrip 1080)

RNase T1 (Thermo Fisher Scientific, EN0541)

T4 PNK (NEB, M0201S)

Phenol:chloroform:iso-amyl alcohol (25:24:1), low pH (VWR, 97064-710)

Yeast tRNA 10 mg/mL (Thermo Fisher Scientific, AM7119)

Glycogen, 20 mg/mL (Roche, 10901393001)

10X RNase T1 cleavage buffer: 0.5 M Tris pH 7.5, 20mM EDTA

Alkaline hydrolysis buffer: 50 mM NaHCO₃, pH 9.2, 1 mM EDTA and 6.75 ng/μL yeast tRNA

2.7. Characterize crRNA-guided binding of Cas9 to RNA targets

How the Cas9 RNP engages its ssRNA target can be investigated using the RNA electrophoretic mobility shift assay (EMSA) or RNA gel shift assay. We assemble in vitro binding reactions in a similar way as for cleavage reactions, except that MgCl₂ is omitted to render Cas9 catalytically inactive and 30 μg/mL of heparin is included in the buffer to reduce non-specific RNA binding. Before setting up the binding reactions, we prepare a fresh 6% native polyacrylamide-0.5X TBE gel by mixing 2.25 mL of 40% acrylamide/Bis 19:1, 0.75 mL 10X TBE buffer, 12 mL ddH₂O, 150 μL 10% APS, and 15 μL TEMED. This mixture is immediately poured into an empty Biorad criterion gel cassette, with the 18-well comb inserted back on. Let the gel polymerize for 20–30 min and store temporarily at 4°C.

The following is a step-by-step protocol for RNA EMSA.

1. Dilute a small aliquot of Cas9 protein stock to 5 μM using fresh Cas9 dilution buffer.

2. Assemble in vitro binding reactions in 0.65 mL eppendorf tubes or PCR tubes:

   200 mM HEPES pH 7.5, 1 mM EDTA, 5 mM fresh DTT	1 μL
   Heparin 300 μg/mL	1 μL
   0.5 M KCl	1 μL
   RNase-free water	3 μL
   5 μM Cas9 stock	1 μL
   5 μM crRNA	1 μL
   5 μM tracrRNA	1 μL
   Mix well; incubate at RT for 10 min to assemble RNP.
   Add 0.5 μM fluorescently labeled ssRNA target	1 μL
   Total volume	10 μL

3. Incubate at 37˚C for half an hour.

4. Pre-run the 6% native-polyacrylamide gel at 4°C in 0.5X TBE buffer, for 15 min at 200 V.

5. Add 2 μL 60% glycerol to each finished 10 μL binding reaction, and mix well.

6. Flush the gel wells with a pipette or syringe. Load half of the entire mixture (6 μL, containing 10% glycerol) into one well for each reaction. To help monitor the progress of gel electrophoresis, run a buffer only control containing xylene cyanol and bromophenol blue in the last lane. Run the gel at 200 V until the bromophenol blue dye reaches the bottom of the gel.

7. Open the cassette and transfer the gel to the Biorad Blot/UV/Stain-Free Sample Tray. FAM- or Cy5- labeled RNA can be visualized and imaged with appropriate filters on a Biorad ChemiDoc MP imaging system.

Additional reagents and buffers:

Heparin sodium salt (Sigma, H4784)

1X RNA binding condition: 20 mM HEPES pH 7.5, 30 μg/mL heparin, 50 mM KCl, 0.1 mM EDTA, and 0.5 mM DTT.

In a binding reaction that contains all key components (e.g. Cas9, crRNA, tracrRNA, etc), we usually observe several higher molecular weight shifts forming on the fluorescently labeled RNA target. It is important to set up a series of controls where individual components are left out from the reaction: gel shifts that depend on both Cas9 and a matching crRNA are likely formed by sequence-specific Cas9 binding events; whereas gel shifts that form in all crRNA-containing reactions, regardless of Cas9’s presence, probably reflect crRNA-target binding only. A titration experiment with varying concentrations of the Cas9 RNP in the binding assay will help determine the K_d. Moreover, co-factor requirements (e.g. nuclease motifs, PAM, salt concentration, etc.) and mismatch tolerance for crRNA-guided binding by Cas9 can also be investigated using RNA EMSA^26,27,29.

2.8. Are RNA cleavage products released from the Cas9 RNP?

SpyCas9 is reported as a single turnover enzyme that holds on to all four ends of cleavage products after cutting the dsDNA target ^38,39. To investigate if NmeCas9 RNP releases its RNA cleavage products, we carry out the RNA cleavage-shift experiment by setting up a regular in vitro RNA cleavage assay and resolving the reaction on denaturing- and native- polyacrylamide gels in parallel. The 15% urea-PAGE helps track the fraction of RNA probe that is cleaved into smaller products; while the 6% native-PAGE reveals if Cas9 RNP releases the products after RNA cleavage occurs. It is highly recommended to use an ssRNA target labeled with two distinct fluorophores at two ends, so that both the 5’ and 3’ cleavage products can be visualized from one experiment. We found that under the conditions tested, NmeCas9 cleaves the crRNA-complementary ssRNA target into two fragments by a ~40-50% efficiency²⁷. The 5’ products mostly run as a faster migrating species on the native-PAGE, indicating that they are released from the Cas9 RNP. On the contrary, the 3’ cleavage products that bear ~20 nts of complementary to the crRNA are not released, as they all exist in higher molecular weight shifts ²⁷. Of note, this cleavage-shift experiment is conceptually different from RNA EMSA described in the earlier section. In RNA EMSA, Cas9 is catalytically inactive due to either the lack of Mg²⁺ or an active site mutation in HNH nuclease domain, and this offers an opportunity to test Cas9’s binding to intact ssRNA targets, without any RNA scission events. The cleavage-shift experiment allows potential RNA cleavage to happen first, and then analyze the association of cleavage products with crRNA-loaded Cas9 by native-PAGE.

The following is a step-by-step protocol for an RNA cleavage-shift experiment.

1. Dilute a small aliquot of Cas9 protein stock to 5 μM using fresh Cas9 dilution buffer.

2. Assemble a regular in vitro cleavage reaction in 0.65 mL or PCR tubes:

   200 mM HEPES pH 7.5, 1 mM EDTA, 5 mM fresh DTT	1 μL
   100 mM MgCl2	1 μL
   1 M KCl	1.5 μL
   RNase-free water	2.5 μL
   5 μM Cas9 stock	1 μL
   5 μM crRNA	1 μL
   5 μM tracrRNA	1 μL
   0.5 μM fluorescently labeled RNA substrate	1 μL
   Total volume	10 μL

   Note*: Do not include heparin in any regular 37°C cleavage reaction, because co-presence of Mg²⁺ and heparin will trigger certain RNA probes to appear as aggregates on native-PAGE.

3. Mix well, and incubate at 37°C for 30 min.

4. Pre-run a 15% urea-polyacrylamide gel in 1X TBE buffer, at 200 V for 15 min. Pre-run a 6% native-polyacrylamide gel at 4°C in 0.5X TBE buffer, at 200 V for 15 min.

5. Split each finished cleavage reaction into two 5-μL halves.

6. Mix one half reaction with 1 μL 60% glycerol, and run on a 6% native polyacrylamide gel in 0.5X TBE buffer at 4°C, for 35 min at 200 V.

7. For the other half of the reaction, mix with 1 μL 5 mg/ml Proteinase K, incubate at 37°C for 15 min. Then add 12 μL freshly prepared 1.5X Formaldehyde-Formamide loading dye, heat at 95°C for 3 min, snap chill on ice.

8. After flushing the wells with a pipette or syringe, load 10 μL of these mixtures onto a 15% urea-polyacrylamide gel. Run the gel in 1X TBE buffer for 45 min at 200 V.

9. For both types of gels, open the cassettes and visualize the FAM- or Cy5- labeled RNAs as described in earlier sections.

3. Results and discussions

Using the protein purification protocol described in section 2.2, we routinely obtain > 5 mg purified Cas9 protein from 2 L induced E. coli culture. The use of a heparin column as the first purification step offers two obvious benefits. First, > 85% purity from cleared crude E. coli lysate can be achieved after this step, eliminating the need for any affinity chromatography. Second, the heparin column helps efficiently get rid of nucleic acids associated with Cas9, which tends to bind non-specifically to DNA or RNA. This is because heparin mimics the high negative charge of nucleic acids, and can act as their competitor. If heparin columns are not used, care should be taken to eliminate nucleic acids during Cas9 purification before using the Cas9 preps for biochemical assays.

The N. meningitidis CRISPR-Cas9 system is a type II-C prototype previously shown to restrict DNA natural transformation in the native bacterial host⁴⁰, and can also serve as high-fidelity, eukaryotic genome editing^41–44 or manipulation^45–47 platforms. Using protocols described in the Method section, we discovered an intrinsic crRNA-guided ribonuclease activity for NmeCas9 (Figure 4)²⁷. NmeCas9 catalyzes robust in vitro cleavage of ssRNA target in a crRNA-guided, tracrRNA-dependent, and PAM-independent fashion²⁷. We also found that RNA cleavage by NmeCas9 likely generates products with 3’ hydroxyl ends, because the migration pattern for the 5’ cleavage products looks the same before and after T4 PNK treatment (Figure 5C). On the contrary, certain bands in the RNase T1 or alkaline hydrolysis RNA ladders migrate differently with or without PNK treatment (Figure 5A–B), consistent with the 2’, 3’- cyclic phosphate nature of RNase T1 formed termini^36,37. This also highlights the need to treat all sequencing ladders and Cas9 cleavage products with T4 PNK before gel analysis, in order to accurately map out the RNA cleavage sites.

We use fluorescently labeled RNA oligonucleotides as substrates for Cas9-catalyzed cleavage, circumventing the need for using radioactive RNA probes. Fluorescent probes have much longer shelf lives if stored properly, greatly reducing the effort and cost associated with repeated labeling and purification of radioactive probes. Furthermore, distinct fluorophores (e.g. 6-FAM and Cy5) can be used simultaneously to track behaviors of different probes in the same reaction, or of different products resulted from the same probe. One common concern for using fluorescent nucleic acid probes is the signal detection limit. With the Biorad ChemiDoc MP imaging system, we can easily detect an RNA band of only 4 fmole of 6-FAM-labeled oligonucleotide on a polyacrylamide gel.

The discovery of anti-CRISPRs against Class II CRISPR systems added a new twist to the arms race between phage and bacteria, and also has profound evolutionary implications. How Cas9 inhibitors exert their function, and their technological utilities in gene editing are under intensive investigation²². The Acr purification procedure outlined in section 2.2 works well for most of Type II Acrs. For AcrIIC_Nme3 that tends to have mild aggregation issue, two procedures can enhance solubility: adding an N-terminal FLAG tag to the Acr¹⁷, or expressing a SUMO-Acr fusion before removing the SUMO tags³³. As more diverse anti-CRISPR genes are identified across bacterial phyla, exiting purification protocols likely need to be tweaked for novel Acrs. The amount of each Acr needed to elicit sufficient inhibition may also vary, and therefore should be determined empirically for any given Acr-Cas9 pair. The order in which the Cas9-sgRNA-Acr reaction is assembled may or may not affect the inhibitory outcome, depending on the exact mechanism how that Acr blocks Cas9’s function³³.

4. Conclusion

Here we present a method for purifying Cas9 proteins and characterizing their CRISPR-guided, RNA-targeting potential using in vitro approaches. This method uses fluorescently labeled RNA probes, which eliminate the use of hazardous radioactive material while still providing high detection sensitivity. Although originally developed for N. meningitidis Cas9 and its anti-CRISPRs, this method is broadly applicable to diverse Cas9 orthologs. With minor modifications, it can also assist the investigation of other Class II CRISPR effector enzymes.

Highlights.

• We detail a method of assessing the RNA targeting potential for diverse CRISPR-Cas9 systems

• Cas9’s ribonuclease activity is characterized with purified protein, fluorescent RNA substrate, and synthesized guide RNA.

• Binding of Cas9 RNP to RNA target and product release after cleavage are also assayed in vitro

• This method is developed for the Neisseria meningitidis Cas9 and its anti-CRISPRs, but is broadly applicable to other Cas9 orthologs.