Methods

Plasmids and oligonucleotides

Descriptions and Addgene IDs for all plasmids used in this study are available in Sup. Table 1; new plasmids have been deposited with Addgene (https://www.addgene.org/Benjamin_Kleinstiver/). A list of all SpRYgest target sites is provided in Sup. Table 2 that includes spacer sequences, PAMs, and gRNA generation methods. Oligonucleotide sequences and descriptions are available in Sup. Table 3. Target sites for TtAgo are listed in Sup. Table 4. Additional details for plasmids and oligonucleotides (oligos) are provided below in the respective sections. The SpOT-check computed off-target profiles for all gRNAs used in this study are available in Sup. Table 5.

Human cell culture

Human HEK 293T cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FBS (HI-FBS) and 1% penicillin/streptomycin. The supernatant media from cell cultures was analyzed monthly for the presence of mycoplasma using MycoAlert PLUS (Lonza).

Expression of and normalization of SpCas9-containing human cell lysates

Expression plasmids encoding WT SpCas9, SpG, and SpRY each with a -P2A-EGFP signal (RTW3027, RTW4177 and RTW4830, respectively) were used to generate human cell lysates containing SpCas9 proteins. Approximately 20-24 hours prior to transfection, 1.5×105 HEK 293T cells were seeded in 24-well plates. Transfections containing 500 ng of human codon optimized nuclease expression plasmid and 1.5 μL TransIT-X2 were mixed in a total volume of 50 μL of Opti-MEM, incubated at room temperature for 15 minutes, and added to the cells. The lysate was harvested after 48 hours by discarding the media and resuspending the cells in 100 μL of gentle lysis buffer (containing 1X SIGMAFAST Protease Inhibitor Cocktail, EDTA-Free (Sigma), 20 mM Hepes pH 7.5, 100 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM DTT, and 0.1% Triton X-100). The amount of SpCas9 protein was approximated from the whole-cell lysate based on EGFP fluorescence. SpCas9 lysates were normalized to 180 nM fluorescien (Sigma) based on a standard curve. Fluorescence was measured in 384-well plates on a DTX 880 Multimode Plate Reader (Beckman Coulter) with λex = 485 nm and λem= 535 nm.

Production of gRNAs

The DNA substrates required to transcribe gRNAs were generated via two methods. First, plasmids for IVT of SpCas9 gRNAs were generated by annealing and ligating duplexed oligos (see Sup. Table 3) corresponding to spacer sequences into BsaI-digested MSP3485 for T7 promoter-driven transcription of gRNAs. The derivative pT7-spacer-gRNA plasmids were digested with HindIII (NEB) to permit run-off transcription near the 3’ end of the SpCas9 gRNA. Secondly, oligo-derived DNA templates for IVT were generated by combining a target specific oligo (encoding a T7 promoter, spacer sequence, and partial sequence of the SpCas9 crRNA) and a common SpCas9 gRNA scaffold oligo (oKAC682), and then incubating with either Klenow Fragment (3’→5’ exo-) (New England Biolabs (NEB), M0212L) in 1x NEBuffer 2 at 37 °C for 30 minutes, or Q5 polymerase (NEB) using the following program: 2 minutes 98 °C; 5 cycles of (10 seconds 98 °C, 10 seconds 65 °C, 30 seconds 72 °C); 5 minutes 72 °C. Plasmid or oligo-derived transcription templates were cleaned up using a MinElute PCR Purification Kit (Qiagen). SpCas9 gRNAs were transcribed at 37 °C for 16 hours using the T7 RiboMAX Express Large Scale RNA Production Kit (Promega). For gRNAs utilized in in vitro cleavage reactions containing SpRY from human cell lysates, the 37 °C incubation was followed by the addition of 1 μL RQ1 DNase at 37 °C for 15 minutes to degrade the DNA template. The DNase treatment step was omitted when preparing most gRNAs utilized for scaled-up SpRYgest reactions with purified SpRY protein. Following transcription and optional DNase treatment, gRNAs were purified using paramagnetic beads (prepared as previously described28; GE Healthcare Sera-Mag SpeedBeads from Fisher Scientific, washed in 0.1X TE and suspended in 20% PEG-8000 (w/v), 1.5 M NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA pH 8 and 0.05% Tween20) and refolded by heating to 90 °C for 5 minutes and then cooling to room temperature at 1 °C every 2 seconds. Synthetic gRNAs were purchased from Synthego.

For one-pot gRNA IVT reactions, we utilized two general methods. First, gRNAs were generated using the EnGen sgRNA Synthesis Kit (NEB, E3322S) according to the manufacturer recommended protocol, or the EnGen sgRNA Synthesis Kit with increased oligo concentrations (final concentrations of 0.75 μM target-specific oligo and 0.75 μM common SpCas9 gRNA scaffold oligo (oKAC682)). The DNase step was omitted. Second, we also optimized a separate one-pot gRNA synthesis method using other commercial reagents. In this second method, 20 μL one-pot reactions were assembled containing final amounts or concentrations of 2.5 U Klenow Fragment (3’→5’ exo-), target-specific oligo at 0.5 or 1.5 μM (for standard or scaled-up reactions, respectively), common SpCas9 gRNA scaffold oligonucleotide (oKAC682) at 0.25 or 0.75 μM (for standard or scaled-up reactions, respectively), 125 μM dNTPs, 1x RiboMAX Express T7 Buffer (Promega, P1320), and 2 μL T7 Express Enzyme Mix (Promega, P1320) and incubated at 37 °C for 4 hours unless otherwise indicated. Appropriately scaled 5 μL reactions were assembled for smaller-scale one-pot reactions. For one-pot gRNAs used in SpRYgest reactions, the Promega recommended RQ1 DNase treatment was omitted and no clean-up of the gRNA was performed. To quantify gRNA yield, separate IVT reactions were performed that included the RQ1 DNase step and were purified using paramagnetic beads. Note that gRNA yield will vary based on incubation time.

Expression and purification of SpRY and SpRY-HF1 proteins

E. coli codon optimized SpRY and SpRY-HF1 coding sequences including an N-terminal MKIEE tag and C-terminal SV40 NLS and 6x histidine tag were synthesized (GenScript, NJ, USA) and cloned into pET28 expression vectors. The SpRY and SpRY-HF1 expression constructs and were used to express and purify the proteins as described previously29. Briefly, E. coli strain NiCo21(DE3) (C2529H from NEB) harbouring the recombinant construct was grown in 1-2 L of LB medium with 40 μg/mL Kanamycin at 30°C until mid-log phase. Overexpression of the target protein was induced by adding IPTG to a final concentration of 0.4mM with shaking overnight at 18°C. Cells were harvested and target protein expression was assessed by SDS PAGE prior to purification. Cells were disrupted by sonication in breakage buffer (50mM Tris-HCl (pH8.0), 300mM NaCl, 1mM EDTA, 1mM DTT, 2% (v/v) glycerol) supplemented with PMSF. The supernatant was passed through HiTrap DEAE Sepharose (Cytiva, MA, USA) in column buffer (20mM Tris(pH7.5) and 250mM NaCl) followed by subsequent purification on a HisTrap HP column (Cytiva). After 16x column volume wash in buffer containing 20mM T ris pH7.5, 250mM NaCl, 40mM imidazole, target proteins were eluted using a 40mM to 750mM imidazole gradient in the same buffer. Pooled fractions containing the proteins were further purified by loading onto HiTrap heparin HP columns (Cytiva), washed with 6 column volumes of a buffer containing 20mM Tris (pH8.0), 1mM EDTA, and 1mM DTT, and eluted using a 0.25 to 2M NaCl gradient in the same buffer. Pooled fractions were dialyzed in SEC column buffer (20mM HEPES (pH8.0), 250mM KCl, and 1mM DTT) and concentrated using an Amicon® Ultra-15 Centrifugal Filter Unit with 100 kDa molecular weight cut-off. Concentrated fractions were loaded on to a HiLoad 16/600 Superdex 200 pg column (Cytiva) using a 1 mL sample loop. Size exclusion chromatography was performed in SEC column buffer with a flow rate of 0.5mL/min. Eluted fractions were assessed by SDS-PAGE, pooled, dialyzed in storage buffer (20mM Tris (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT and 50% (v/v) glycerol), and stored at −20°C. Protein concentration was determined by Bradford assay using BSA for standards.

In vitro cleavage reactions using SpCas9 from lysates

Plasmid KAC833 linearized with HindIII (NEB) was used as the DNA substrate for most in vitro cleavage reactions unless otherwise stated. SpCas9 ribonucleoprotein (RNP) complexes were formed by mixing 9 μL of SpCas9-containing normalized whole-cell lysate (normalized to 180 nM Fluorescein) with 11.25 pmol of transcribed or synthetic gRNA, and incubating for 5 minutes at 37 °C. Cleavage reactions were initiated by the addition of 34.82 fmol of linearized plasmid (digested with HindIII (NEB)) and buffer to bring the total reaction volume to 22.5 μL with a final composition of 10 mM HEPES pH 7.5, 150 mM NaCl, and 5 mM MgCl2. Reactions were performed at 37 °C and aliquots were terminated at timepoints of 1,6, 36 and 216 minutes by removing 5 μL aliquots, mixing with 5 μL of stop buffer (50 mM EDTA and 2 mg/ml Proteinase K (NEB)), and incubating at room temperature for 10-minutes. Cleavage fragments were purified using paramagnetic beads and quantified via QIAxcel capillary electrophoresis (Qiagen). The relative abundances of substrate and products were analyzed using QIAxcel ScreenGel Software (v1.5.0.16, Qiagen) and plotted using GraphPad Prism 9 (v9.2.0).

In vitro cleavage reactions using purified SpRY

Small-scale in vitro cleavage reactions were performed as described above, except using 0.6-1 μM purified SpRY per reaction pool instead of 9 μL of SpCas9-containing normalized whole-cell lysate (0.6 μM in Sup. Figs. 9a and 9b and 1 μM in 9c). For scaled-up digests, 4 μg of supercoiled plasmid DNA was incubated at 37 °C for 3 hours with purified SpRY protein at a final concentration of 1 μM and IVT gRNA (prepared without DNase treatment) at a final concentration of 2 μM in Buffer 3.1 (NEB). Reactions were stopped by the addition of 1 μL of Proteinase K (NEB) and incubated at room temperature for 15 minutes. Cleavage fragments were resolved by 0.8% agarose gel electrophoresis with 1 μL of 1 kb Plus DNA Ladder (NEB) and visualized by ethidium bromide staining. Digestion products were purified using a QIAquick Gel Extraction Kit (Qiagen).

Molecular cloning reactions using purified SpRY

The C-terminal P2A-EGFP sequence was added to SaABE8e or pCMV-PE2 (Addgene IDs 138500 and 132775, respectively), and the N-terminal BPNLS was added to an SpCas9 plasmid similar to pCMV-T7-SpCas9 (Addgene plasmid ID 139987) via isothermal assembly. Reactions contained approximately 5 μL of isothermal assembly mix (prepared similar to as previously described12) or NEBuilder HiFi (NEB), 0.01 pmol of plasmid linearized via SpRYgest, and 0.03 pmol of PCR product insert in a final volume of 10 μL, and incubated at 50°C for 60 minutes. Cloning reactions were transformed into chemically competent XL1-Blue E. coli cells and grown at 37 °C for approximately 16 hours. Individual colonies were grown overnight at 37 °C, miniprepped (Qiagen), and fidelity of cloning was verified via Sanger sequencing. Saturation mutagenesis plasmid libraries for were constructed by incubating 0.02 pmol of BPK848 (linearized via SpRYgest) with 0.1 pmol of ~60bp ssDNA oligo (either oBK9102 or oBK9104) with 10 μL NEBuilder HiFi DNA Assembly Master Mix (NEB) in a final volume of 20 μL, and incubated at 50 °C for 15 minutes. NEBuilder reactions were cleaned up via MinElute (Qiagen) and eluted in 10 μL water, transformed into 100 μL of electrocompetent XL1-Blue E. coli, and recovered in 3 mL of SOC for 1 h at 37 °C. Next, 2 μL of the transformation recovery media was plated on LB + chloramphenicol, where the number of colonies following overnight at 37 °C were used to estimate library complexity. The remaining recovery was grown overnight in 150 mL LB + chloramphenicol and plasmid DNA was isolated by MaxiPrep (Qiagen). The complexity of the SpCas9 catalytic and PAM domain libraries were estimated to be 77,400 and 292,600 respectively. Plasmid libraries were sequenced via Sanger sequencing and NGS. For NGS, PCR amplicons were generated from the plasmids using primer pairs oKAC1589/oKAC1590 (for the catalytic domain) or oKAC1591/oKAC1592 (for the PI domain) and sequenced on a MiSeq (Illumina) to a depth of 12,378 and 409,221 reads for the catalytic and PI domain libraries, respectively. The resulting data was analyzed using CRISPResso230 to generate allele tables.

In vitro cleavage reactions using TtAgo

For TtAgo reactions, 5’-phosphorylated DNA guides were either purchased from Integrated DNA technologies or generated by incubating an unmodified oligonucleotide with T4 Polynucleotide Kinase (NEB) at 37 °C for 30 minutes, followed by heat-activation at 65°C for 20 min. Complexes of TtAgo programmed with ssDNA guides were prepared by combining final concentrations of 1 pmol TtAgo (NEB) and 2 pmol 5’-phosphorylated ssDNA guides and incubating at 70 °C for 20 minutes. Cleavage reactions were performed by combining TtAgo complexes with either 79.85 fmol of linearized KAC833 plasmid substrate (digested with PvuI, NEB) or 79.85 fmol supercoiled plasmid DNA (KAC1151 or MNW95) in ThermoPol buffer (NEB) with a final concentration of 10mM MgSO4. Reactions were performed at 80 °C for 60 minutes and terminated by the addition of 1 μL of Proteinase K (NEB). Cleavage fragments from pre-linearized substrates were purified using paramagnetic beads and quantified and analyzed as described above. Cleavage fragments from scaled-up plasmid DNA digests were resolved by 0.8% agarose gel electrophoresis and visualized by ethidium bromide staining.

Bacterial-based positive selection assay

Target plasmids for the selection assays were generated by cloning duplexed oligonucleotides into XbaI and SphI-digested p11-lacY-wtx1 (Addgene ID 69056)16 as previously described7, which contains an arabinose-inducible ccdB toxin gene. The derivative toxin-expressing plasmids contain target sites harboring either NGG or NGA PAMs (BPK740 and BPK754, respectively). To perform the selections, electrocompetent E. coli BW25141(λDE3)17 containing a toxin-expressing plasmid were transformed with BPK848-derived plasmids that express the SpCas9 variant libraries (encoding randomized codons in specified positions) in addition to a gRNA, both from separate T7 promoters. Following a 60-minute recovery in SOC media, transformations were spread on LB plates containing either chloramphenicol and 10 mM dextrose (non-selective) or chloramphenicol + 10 mM arabinose (selective). Transformation efficiency was assessed based on colony count from non-selective plates. The catalytic library selection resulted in approximately 9e4 colonies (from sampling approximately 87x library coverage). The PI domain library selections for NGG PAMs and NGA PAMs resulted in approximately 6e5 and 6.4e4 colonies (from sampling approximately 18x and 2x coverage of the libraries, respectively). Surviving colonies from selective plates were picked as single colonies for miniprep (Qiagen) followed by Sanger sequencing to verify the identities of the mutated amino acids.

Abstract

While restriction enzymes (REs) remain the gold-standard for manipulating DNA in vitro, they have notable drawbacks including a dependence on short binding motifs that constrain their ability to cleave DNA substrates. Here we overcome limitations of REs by developing an optimized molecular workflow that leverages the PAMless nature of a CRISPR-Cas enzyme named SpRY to cleave DNA at practically any sequence. Using SpRY for DNA digests (SpRYgests), we establish a method that permits the efficient cleavage of DNA substrates at any base pair. We demonstrate the effectiveness of SpRYgests using more than 130 gRNAs, illustrating the versatility of this approach to improve the precision of and simplify several cloning workflows, including those not possible with REs. We also optimize a rapid and simple one-pot gRNA synthesis protocol, which reduces cost and makes the overall SpRYgest workflow comparable to that of RE digests. Together, SpRYgests are straightforward to implement and can be utilized to improve a variety of DNA engineering applications.

Competing Interest Statement

K.A.C. and B.P.K are inventors on patents and/or patent applications filed by Mass General Brigham that describe genome engineering technologies. B.P.K. is a consultant for Avectas Inc., EcoR1 capital, and ElevateBio, and is an advisor to Acrigen Biosciences and Life Edit Therapeutics.


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