Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

1. Integration of a human GOI into the BW25141 E. coli strain

1. Cloning of the GOI
   1. Polymerase chain reaction (PCR) amplify a 500 bp length of a human GOI containing various candidate target sites via standard PCR methods with primers containing NotI and XhoI restriction enzyme sites.
      NOTE: In this experiment, the GOI was the human EMX1 gene.
   2. Digest both the PCR product and the pgrg36¹³ vector with NotI and XhoI restriction enzymes.
   3. Gel purify the desired fragments (500 bp and 12 kb).
   4. Ligate the fragments together using T4 ligase. To do this, mix 50 ng of digested pgrg36 and 6 ng of PCR insert in a reaction volume of 20 μL containing 1x ligase buffer and 0.5 U ligase enzyme. Incubate at room temperature (RT) overnight.
   5. Transform the ligated plasmid into DH5-α competent cells. Grow the resulting transformants on Luria broth (LB) agar plates containing ampicillin (100 μg/mL) at 32 °C and incubate overnight. Pick up a colony and grow it in LB media containing ampicillin overnight. Isolate the plasmid DNA from the E. coli, using a commercially available miniprep Kit. Confirm the insertion of the GOI fragment by Sanger sequencing the plasmid obtained from the mini-plasmid preparation (mini-prep)¹³.
2. Preparation of the BW25141-GOI
   1. Transform the obtained pgrg36-GOI plasmid into the BW25141 E. coli strain. It is essential to use a BW25141 strain in order to minimize the number of false positive colonies.
   2. Grow the transformed cells in the LB buffer at 32 °C overnight. GOI is inserted into the genomic DNA of the BW25141 strain (BW25141-GOI). Remove the pgrg36-GOI plasmid from the BW25141-GOI strain using the standard pgrg36 protocol¹³. Briefly, dilute a colony (approximately 10⁷-fold) and grow it on an LB plate at 42 °C overnight. Streak the colonies on the LB plate and grow them at 42 °C overnight.
   3. Confirm the correct GOI insertion by colony PCR, using the primers suggested in the pgrg36 protocol: 5'-GATGCTGGTGGCGAAGCTGT-3' and 5'-GATGACGGTTTGTCACATGGA-3'. The primer amplifies the genomic DNA insertion site, and the size of the resulting PCR product will be 904 bp plus the size of the insert (500 bp in this case).
   4. Prepare electrocompetent BW25141-GOI cells (a detailed protocol is described in steps 3.2.6–3.2.10).

2. Preparation of the Cas9 variant library

1. Library preparation
   1. Transform the Cas9 vector⁷ into a commercial E. coli mutator strain (Table of Materials) and follow the manufacturer’s instructions to obtain a variant library (the Mutator library).
   2. Perform error-prone PCR on the whole WT Cas9 sequence in the Cas9 vector, using an error-prone PCR kit (Table of Materials).
      NOTE: The low-error rate protocol was adopted in the Sniper-Cas9 case to avoid disrupting the original function of the protein.
   3. Digest the Cas9 vector with appropriate restriction enzymes. Gel purify the PCR product (from step 2.1.2) and the digested backbone.
      NOTE: The size of the SpCas9 gene is about 4.3 kb. XhoI and KpnI were chosen to digest pBLC-SpCas9 vector which was used in the Sniper-Cas9 case.
   4. Assemble the backbone fragment (from step 2.1.3) and the insert amplified using error-prone PCR (from step 2.1.3) via isothermal in vitro recombination.
      NOTE: More than 500 ng of backbone is needed to obtain a high concentration of library (error-prone PCR [EP] library). Two different error-prone PCR kits were used to prepare EP libraries in the Sniper-Cas9 case (EP library I and II).
   5. Purify the products from the assembly (from step 2.1.4) using a DNA purification kit that enables low-volume elution (Table of Materials). Elute with 6 μL of nuclease-free water (NFW) and measure the concentration of DNA.
   6. Transform more than 500 ng of Cas9 library vector (for each of three libraries) into 50 μL of electrocompetent E. coli cells (Table of Materials). See the electroporation protocol in steps 3.2.1-3.2.4. For this library preparation, use 1 mL of SOC medium instead of 250 μL per 50 μL of competent cells.
   7. Make 1:100, 1:1,000, and 1:10,000 dilutions of the mixture containing the recovered cells with SOC medium. Plate the diluted cells on 100 mm LB agar plates supplemented with chloramphenicol (12.5 μg/mL). Plate the remaining cells on a 245 mm² plate. Incubate at 37 °C overnight.
2. Calculation of library complexity
   1. Photograph the dilution plates via a gel documentation system or an ordinary digital camera. Run OpenCFU software¹⁴ and upload the photographs of the dilution plates. Set the counting area inside a plate and remove false colonies.
   2. Manually multiply the number of colonies by the dilution factor to obtain the original number of transformants. Convert these numbers into logarithmic form (base 10). Calculate the average to determine the complexity of the library.
   3. When the desired complexity value is obtained, gather all the colonies on the 245 mm square plate (from step 2.1.7) using a spreader and 20 mL of LB supplemented with chloramphenicol. Do not grow the gathered colonies and purify the plasmid library using a commercial midiprep kit.
      NOTE: The higher the library complexity, the better. When Sniper-Cas9 was identified, a diversity of 3 x 10⁶ was achieved for each library.

3. Positive and negative screening for evolving Cas9

1. Target selection and plasmid construction
   1. Select a target sgRNA spacer sequence in the GOI. Substitute one or two residues in the random nucleotide to produce a mismatched sequence.
      NOTE: Human EMX1 target site 3 (GAGTCCGAGCAGAAGAAGAA with GGG PAM) was used in the Sniper-Cas9 case. Followings are the mismatched sequences used: GAGTCCGAGCAGAAagAGAA, GAacCCGAGCAGAAGAAGAA, GAGTCCGAGCAGAgGAAGAA, and GAGcCCGAGCAGAAGAAGAA.
   2. Insert the mismatched sequence (see step 3.1.1) into the sgRNA plasmid using standard oligonucleotide (oligo) cloning procedures⁷.
   3. Insert the mismatched sequence with a PAM at the 3' end into p11-lacY-wtx1 (Table of Materials) to construct the ccdB plasmid using standard oligo cloning procedures¹⁵.
2. Preparation of Sniper-screening E. coli competent cells
   1. Thaw BW25141-GOI electrocompetent cells on ice.
   2. Add 1 ng each of the ccdB plasmid and the sgRNA plasmid with double mismatches into 50 μL of thawed BW25141-GOI cells. Gently mix the cells by pipetting and move them into a prechilled 0.1 cm electroporation cuvette.
   3. Transform the E. coli with the two plasmids via electroporation. Add 250 μL of SOC medium immediately after electroporation. Gently pipette the solution to mix the cells and the medium. Transfer the mixture to a 1.5 mL microcentrifuge tube.
      NOTE: For maximum efficiency, set the voltage at 1.80 kV, and the runtime should be between 4.8 ms and 5.0 ms.
   4. Recover the transformed cells and incubate them at 32 °C for 1 h with gentle shaking.
   5. Plate 125 μL of the recovered cells on an ampicillin (50 μg/mL)/kanamycin (25 μg/mL) LB agar plate (culture condition). Plate the remaining cells on an ampicillin/kanamycin/arabinose (1.5 mg/mL) LB agar plate (ccdB-expressing condition). Incubate at 32 °C overnight.
   6. Check for the absence of surviving colonies on the ccdB-expressing condition plate. Gather colonies from the culture condition plate using a spreader and culture them in 250 mL of super optimal broth (SOB) medium supplemented with 50 μg/mL of ampicillin and 25 μg/mL of kanamycin at 32 °C, with gentle shaking.
   7. When the optical density at 600 nm (OD₆₀₀) reaches 0.4, chill the flask on ice. Prepare prechilled deionized water and a prechilled 10% glycerol solution (sterilize before use).
   8. Centrifuge (at 4,000 x g for 5 min at 4 °C) the cells and discard the supernatant. Add 200 mL of prechilled deionized water. Resuspend the cells using a 10 mL serological pipette. Repeat this step 3x.
   9. Wash the cells with 50 mL of prechilled 10% glycerol solution. Centrifuge them as before (at 4,000 x g for 5 min at 4 °C).
   10. Discard the supernatant and resuspend the pellet in 300 μL of 10% glycerol solution. Make 50 μL aliquots and freeze them in liquid nitrogen. Store the cells (Sniper-screening cells) at -80 °C.
3. Sniper-screening
   1. Transform the Sniper-screening cells (from step 3.2.10) with 100 ng of the Cas9 variant plasmids from each library (from step 2.2.3.See steps 2.1.1 and 2.1.4). Follow the electroporation steps described in steps 3.2.1–3.2.3.
   2. Transfer 250 μL of the cells to a fresh 1.5 mL microcentrifuge tube. Add 250 pg of ATC to make a final concentration of 10 ng/mL. Recover both ATC-containing and ATC-free cells (see step 3.2.4 for the recovery step).
   3. Plate 25 μL of recovered ATC-free cells on a chloramphenicol/kanamycin LB agar plate (nonselective condition). Add ATC to the recovered ATC-containing cells to make a final concentration of 100 ng/mL on a 245 mm LB plate. Immediately plate the cells on a chloramphenicol/kanamycin/arabinose LB agar plate (selective condition). Incubate overnight at 32 °C.
      NOTE: The size and the number of the LB plate are determined by the size of diversity of the screening covers. In the case of a 100 mm Petri dish with 20 mL of LB, add 2 μg of ATC.
   4. Photograph the plates. Count the number of viable colonies using OpenCFU software¹⁴. (See step 2.2.1) Make sure that the number of colonies on the nonselective plate is at least 10x larger than the diversity of the library to cover all variants.
   5. Calculate the survival frequency as follows.
      Survival frequency = the number of colonies on a selective plate / (the number of colonies on a nonselective plate x 10)
   6. Pool the colonies that survived on the selective plates from all three libraries. Incubate the surviving colonies in 250 mL of LB medium supplemented with 12.5 μg/mL chloramphenicol at 42 °C overnight. Isolate the screened Cas9 library DNA using a midiprep kit.
      NOTE: This step clears the sgRNA plasmid.
   7. Repeat the screening process from steps 3.3.1–3.3.6 until the survival frequency reaches a plateau. Use 10 ng of the selected Cas9 plasmid for transformation and 10 ng/mL of ATC during recovery. Maintain the ATC concentration at 100 ng/mL for the selective condition.
4. Shuffling and the second screening
   1. Shuffle the selected pooled variants using the following DNA-shuffling protocol. PCR amplify the Cas9 insert in the Cas9 plasmid using flanking primers, 150 nucleotides from the insert boundaries. Digest 2 μg of amplified PCR product with DNase I for 1 min at 37 °C.
   2. Purify fragments 70–200 bp in length using 2% agarose gel electrophoresis. PCR amplify the purified fragments. Use the product as a template to PCR amplify the Cas9 insert with appropriate primers flanking Cas9. Use the final PCR product to construct a Cas9 library as described in step 2.1.4.
   3. Prepare new Sniper-screening cells (see section 3.2) with another mismatched sgRNA plasmid (see step 3.1.1). Redo the screening process (sections 3.2–3.3) until the survival rate reaches a plateau. Use 10 ng of the selected Cas9 plasmid for transformation and 10 ng/mL ATC during recovery. Maintain the ATC concentration at 10 ng/mL for the selective condition.
5. Selection of evolved Cas9 mutant plasmids
   1. After the last screening step, randomly pick one hundred colonies from the selective plate and culture them in chloramphenicol-containing LB medium at 42 °C overnight.
   2. Isolate the plasmids using a miniprep procedure and Sanger-sequence the inserts using sequencing primers within Cas9.
   3. Select the top three most frequent variants to test them in human cell lines.

4. Delivery of Cas9 as an RNP with truncated sgRNA

1. Target gene selection using Cas-OFFinder
   1. Pick target sites with Cas-OFFinder (http://www.rgenome.net/cas-offinder/). Select the PAM type appropriate for the specific type of Cas9 and the target genome (human, mouse, zebrafish, etc.). Fill in the Query sequences tab, choose the Mismatch number, and click the Enviar button.
   2. After a few seconds, the on-target (with a mismatch number of ‘0’) and off-target sites will appear. In general, choose the off-target sites with one to three mismatches.
2. Preparation of the sgRNA template
   1. Order crRNA and tracrRNA oligos with the following template sequences, namely crRNA sequence: 5'-TAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAA-3'; tracrRNA sequence: 5'-AGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'. Fill N20 in the crRNA sequence with the target sequence obtained in step 4.1.2. To design truncated sgRNAs, remove bases from the 5' end to obtain N19, N18, or N17 sgRNA sequences.
   2. Prepare the PCR mixture for amplification of the sgRNA-encoding sequence as follows: combine 10 μL of 5x buffer, 0.5 μL of crRNA oligo (100 pmol/μL), 0.5 μL of tracrRNA oligo (100 pmol/μL), 2.5 μL of dNTPs (10 mM each), 0.5 μL of DNA polymerase, and 36 μL of NFW (Table of Materials).
   3. Amplify the template using the following conditions, namely, for the initial denaturation: 1 min at 98 °C; for the denaturation, annealing, and extension: 10 s at 98 °C, 15 s at 54 °C, 20 s at 72 °C, for 25 cycles; for the final extension: 5 min at 72 °C.
   4. Analyze 2 μL of the amplified template DNA (from step 4.2.3) on a 2% agarose gel. Purify the template using a PCR purification kit.
      NOTE: The size of the template DNA is 125 bp.
3. Synthesis of sgRNA
   1. Prepare the reaction mixture for sgRNA synthesis as follows: combine 8.5 μL of template DNA (from step 4.2.3), 1 μL of UTP (25 mM), 1 μL of CTP (25 mM), 1 μL of GTP (25 mM), 1 μL of ATP (25 mM), 4.2 μL of MgCl₂ (100 mM), 4.5 μL of T7 RNA polymerase (50 U/μL), 3 μL of 10x T7 RNA polymerase buffer, 1.2 μL of pyrophosphatase (0.5 U/μL), 0.75 μL of RNase inhibitor (40 U/μL), and 4.2 μL of NFW.
   2. Incubate the reaction mixture at 37 °C overnight (at least for 10 h). Add 0.5 μL of DNase (2 U/μL) to the reaction mixture and incubate at 37 °C for 15–30 min. Purify the sgRNA using an RNA purification kit.
   3. To reduce the toxicity caused by an innate immune response triggered by the 5'-triphosphate on the sgRNA¹⁶, remove the 5'-triphosphate from the guide RNAs with calf intestinal alkaline phosphatase (CIP) as follows: treat 10 µg of in vitro-transcribed RNA with 250 U of CIP for 3 h at 37 °C in the presence of 100 U of RNase inhibitor. Purify the CIP-treated sgRNA using an RNA purification kit.

5. WT- and Sniper-Cas9 protein expression and purification

1. Protein expression in E. coli
   1. Transform pET plasmids¹⁸ encoding His-tagged WT- and Sniper-Cas9 into the BL21 (DE3) E. coli strain.
   2. Inoculate 50 mL of LB medium containing 50 μg/mL kanamycin with a fresh colony harboring the pET-Cas9 expression plasmid and shake it overnight (200 rpm) at 37 °C (preculture).
   3. Transfer 10 mL of overnight culture to 500 mL of fresh LB medium containing 50 μg/mL kanamycin. Incubate the culture with shaking (200 rpm) at 37 °C for 2 h.
   4. Monitor the OD₆₀₀ until the culture reaches the mid-log phase of growth (OD₆₀₀ ≈ 0.6–0.7).
   5. Induce the expression of the WT- or Sniper-Cas9 protein with isopropyl β-D-1-thiogalactopyranoside (IPTG, at a final concentration of 0.25 nM). Incubate the culture at 18 °C overnight.
2. Protein purification
   1. Harvest the cells by centrifugation at 5,000 x g for 10 min at 4 °C.
   2. Resuspend the pellet in the lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 4 mM dithiothreitol [DTT], 5 mM benzamidine, 100 mM phenylmethylsulfonyl fluoride [PMSF], pH 8) at 20 mL per gram wet weight.
   3. Add PMSF, DTT, and lysozyme to a final concentration of 1 mg/mL each and incubate the mixture on ice for 30 min.
   4. Sonicate the cells on ice. Pulse repeatedly for 10 s at 200–300 W with a 10 s cooling period between each pulse, for a total time of 20 min.
   5. Centrifuge the lysate at 6,000 x g for 30 min at 4 °C.
   6. Remove the supernatant to a fresh tube. Add 1 mL of His-Bind agarose resin to 5 mL of cleared lysate and shake gently for 1 h at 4 °C.
   7. Load the lysate/His-Bind Agarose resin mixture onto a column with a capped bottom outlet.
   8. Remove the bottom cap and collect the column flow-through.
   9. Wash the column 2x with wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8); collect the wash fraction for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
   10. Elute the protein 10x with 1 mL of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8), collecting samples for SDS-PAGE.
   11. Concentrate the eluted WT- or Sniper-Cas9 protein using a 100 kDa column filter. Store the samples in a solution of 10 mM Tris-HCl, 150 mM NaCl, and 50% glycerol at -80 °C.

6. RNP delivery

1. Transfection and preparation of cells for RNP delivery
   1. Maintain HEK293T cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C with 5% CO₂.
   2. Mix WT- or Sniper-Cas9 protein (2 μg) with sgRNA (2 μg) and incubate for 10 min at RT to make RNP complexes.
   3. Trypsinize and count the cells. Prepare 2 x 10⁴ cells per one reaction. Wash the cells with phosphate-buffered saline (PBS) and centrifuge. Aspirate the supernatant and resuspend the pellet with electroporation buffer.
   4. Electroporate RNP complexes into the cells using the following settings, namely 1,300 V, 30 ms, and one pulse. Plate the cells on a 48-well plate filled with 500 μL of DMEM supplemented with FBS and antibiotics (as described in step 6.1.1) right after the electroporation. Incubate at 37 °C with 5% CO₂.
   5. Isolate the genomic DNA with a gDNA preparation kit, 48 h after the transfection.

7. Transfection of plasmids encoding Sniper-Cas9 and sgRNA

1. Construction of an sgRNA plasmid
   1. Order forward and reverse oligos with the following template sequences, namely forward: 5'-CACCGNNNNNNNNNNNNNNNNNNNN-3'; reverse: 5'-AAACNNNNNNNNNNNNNNNNNNNNC-3'. Replace N20 with the target sequence obtained in step 4.1.2. Shorten the target sequence to a length of N19, N18, or N17 to synthesize truncated sgRNA.
   2. Anneal both oligos in 1x T4 DNA ligase buffer.
   3. Digest the pRG2 vector with BsaI restriction enzyme.
   4. Gel purify the digested vector (3,300 bp) using a 0.8% agarose gel.
   5. Ligate the annealed oligo and the purified fragment using T4 ligase at 37 °C: mix 50 ng of digested pRG2 and 1 ng of annealed oligo in a reaction volume of 20 μL. Incubate at RT for 15 min.
   6. Transform the ligation mixture into the DH5 alpha strain and grow transformants on LB agar plates containing ampicillin (100 μg/mL) at 37 °C. Confirm the insertion of the oligo in the vector by standard sequencing.
2. Transfection of plasmids encoding Sniper-Cas9 and sgRNA
   1. Maintain HEK293T cells in DMEM supplemented with 10% FBS and 1% antibiotics at 37 °C with 5% CO₂. The day before transfection, trypsinize and count the cells. When working at a 48-well scale, plate 1 x 10⁵ cells per well in 250 μL of complete growth medium. The cells should be 50%–80 % confluent on the day of transfection.
   2. At the 48-well scale, prepare 250 ng of p3s-Cas9 plasmid and 250 ng of sgRNA plasmid for transfection, using a lipid-based transfection reagent. Mix the plasmids in 25 μL of serum free-MEM.
   3. Dilute 1 μL of transfection reagent with 25 μL of serum free-MEM. Incubate the mixture at RT for 5 min. Combine the two mixtures and incubate the resulting solution at RT for 20 min to form plasmid-lipofectamine complexes.
   4. After 20 min of incubation, add 50 μL of the solution containing the plasmid-transfection reagent complexes directly to each well containing cells, and mix gently by rocking the plate back and forth. Incubate the cells at 37 °C in a CO₂ incubator for 48–72 h post transfection before assaying for transgene expression.

8. Calculation of indel frequencies to determine on-target and off-target activities

1. Targeted deep sequencing for the analysis of on-target and potential off-target sites
   1. Isolate genomic DNA from step 6.1.5 or 7.2.4 with a gDNA preparation kit. Generate deep sequencing libraries by PCR amplification of the gDNA with primers targeting on-target and off-target.
   2. Use index primers to label each sample. Subject pooled libraries to paired-end sequencing using a next-generation sequencing machine.
2. Deep sequencing analysis using Cas-Analyzer
   1. Analyze deep sequencing data using the Cas-Analyzer assessment tool¹⁷.
   2. Choose the Fastq files under the Read 1 and Read 2 tabs (Read 1 = XX_SXX_L001_R1_001.fastq, Read 2 = XX_SXX_L001_R2_001.fastq).
   3. Fill out the Basic Information tab and the Analysis Parameters tab. Click the Enviar button.