Modification of CRISPR-Cas12f1 systems for gRNA pull-down
Plasmid-borne SpCas12f1 and AsCas12f1 CRISPR systems described earlier1 were engineered to also encode a 10×His:MBP tag fused to N-terminus of the cas12f1 gene. Additionally, a sequence encoding the tobacco etch virus (TEV) protease recognition sequence (ENLYFQS) was also included. The SpCas12f1 plasmid was digested with EcoNI and NcoI restriction enzymes (NEB) and the backbone was isolated by agarose gel purification (Qiagen). Next, a synthesized DNA fragment (Genscript) containing a 5’ EcoNI restriction site, T7 promoter, lac operator, and ribozyme-binding sequence in addition to the sequence encoding the 10×His:MBP:TEV tag followed by an inverted BbsI site incorporating a sequence that upon digestion would yield a compatible NcoI overhang was digested with EcoNI and BbsI and column purified (Qiagen). The two purified fragments were then joined using T4 DNA ligase (NEB), transformed into One Shot TOP10 E. coli cells (Invitrogen), and constructs confirmed by Sanger sequencing. For AsCas12f1, a similar strategy was used except EcoNI and AvaI restriction enzymes (NEB) were used. Links to the plasmid sequences (pMBP-SpCas12f1 and pMBP-AsCas12f1) are provided in Supplementary Table 1.
Expression and purification of Cas12f1-RNA complexes and Cas12f1 proteins
To obtain Cas12f1-RNA complexes, pMBP-SpCas12f1 and pMBP-AsCas12f1 plasmid-borne CRISPR systems encoding both nuclease and gRNA were transformed into E. coli cells (Arctic Express (DE3)). Cultures were grown in LB broth supplemented with ampicillin (100 µg/ml) and gentamicin (10 µg/ml) at 37 °C to an OD600 of 0.6–0.8. At this point, the temperature was decreased to 16 °C and expression induced with 0.5 mM IPTG. After 16 h, cells were pelleted, re-suspended in loading buffer (20 mM Tris-HCl (pH 8.0 at 25 °C), 250 mM NaCl, 5 mM 2-mercaptoethanol, 25 mM imidazole, 2 mM PMSF, 5% (v/v) glycerol), and disrupted by sonication. After removing cell debris by centrifugation, the supernatant was loaded on Ni2+-charged HiTrap chelating HP column (GE Healthcare) and eluted with a linear gradient of increasing imidazole concentration (from 25 to 500 mM) in 20 mM Tris-HCl (pH 8.0 at 25 °C), 250 mM NaCl, 5 mM 2-mercaptoethanol, and 5% (v/v) glycerol. The fractions with Cas12f1-RNA complexes were then dialyzed against 20 mM Tris-HCl (pH 8.0 at 25 °C), 250 mM NaCl, 2 mM dithiothreitol (DTT), and 50% (v/v) glycerol and stored at −20 °C.
SpCas12f1 and AsCas12f1 proteins (without gRNAs) were also expressed and purified using pMBP-SpCas12f1 and pMBP-AsCas12f1. For experimentation requiring dead (d) or nuclease-inactivated Cas12f1 protein, pMBP-SpCas12f1 and pMBP-AsCas12f1 were further modified introducing D228A and D225A encoding codons into SpCas12f1 and AsCas12f1 genes, respectively, using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific). E. coli cells were grown in LB broth supplemented with ampicillin (100 µg/ml) and gentamicin (10 µg/ml) at 37 °C. After culturing to an OD600 of 0.6–0.8, temperature was decreased to 16 °C and protein expression induced with 0.5 mM IPTG. After 16 h, cells were pelleted, re-suspended in loading buffer (20 mM Tris-HCl (pH 8.0 at 25 °C), 1.5 M NaCl, 5 mM 2-mercaptoethanol, 25 mM imidazole, 2 mM PMSF, 5% (v/v) glycerol), and disrupted by sonication. Cell debris was removed by centrifugation. The supernatant was loaded on Ni2+-charged HiTrap chelating HP column (GE Healthcare) and eluted with a linear gradient of increasing imidazole concentration (from 25 to 500 mM) in 20 mM Tris-HCl (pH 8.0 at 25 °C), 0.5 M NaCl, and 5 mM 2-mercaptoethanol. The fractions containing Cas12f1 protein were pooled and subsequently loaded on HiTrap heparin HP column (GE Healthcare). Linear gradient of increasing NaCl concentration (from 0.2 to 1.0 M) was used for elution. The fractions containing the protein of interest were pooled and the 10×His:MBP:TEV tag was cleaved by incubating overnight with TEV protease at 4 °C. To remove the cleaved 10×His:MBP:TEV tag and TEV protease, reaction mixtures were loaded onto a HiTrap heparin HP 5 column (GE Healthcare), and a linear gradient of increasing NaCl concentration (from 0.2 to 1.0 M) was used for elution. The collected fractions with Cas12f1 were then dialyzed against 20 mM Tris-HCl (pH 8.0 at 25 °C), 500 mM NaCl, 2 mM DTT, and 50% (v/v) glycerol and stored at −20 °C. The sequences of the Cas12f1 proteins are listed in Supplementary Table 2.
RNA purification from Cas12f1-RNA complex
To isolate Cas12f1-bound RNA species, SpCas12f1 and AsCas12f1 RNP complexes (250 μl) were incubated with 5 μl (20 mg/ml) of Proteinase K (Thermo Fisher Scientific) for 45 min at 37 °C in 1 ml of 10 mM Tris-HCl (pH 7.5 at 37 °C), 1 mM EDTA, 1 mM DTT, 100 mM NaCl, and 5 mM MgCl2 buffer. Furthermore, DNA was removed by incubation for 45 min at 37 °C with 10 μl of DNase I (Thermo Fisher Scientific). The RNA was purified using a GeneJet PCR Purification column (Thermo Fisher Scientific) and eluted in nuclease-free water. RNA concentration and purity were measured by NanoDrop spectrophotometer and RNA integrity was visualized by separating reaction products on TBE-Urea (8 M) 15% denaturing polyacrylamide gel with 0.5×TBE (Tris-borate-EDTA) buffer (Thermo Fisher Scientific) and staining with SYBR Gold (Thermo Fisher Scientific).
RNA sequencing and analysis
Purified RNA was prepared for sequencing using a TruSeq Small RNA Library Preparation Kit (Illumina) according to the manufacturer’s instruction except that an expanded size selection was performed allowing RNA species ~30–300 nts in length to be captured. After library preparation, 150 nt paired-end sequencing was performed on a MiSeq System (Illumina). The resulting data were post-processed trimming to a Phred quality score of 13, adapters hard-clipped using Cutadapt v2.10, and mapped to the reference using Bowtie2 v2.4.237. Coverage data were then viewed in IGV38 and crRNA and tracrRNA species were identified from the resulting read pileups.
RNA synthesis
Templates for T7 transcription of Cas12f1 single gRNAs were generated by PCR using overlapping oligonucleotides, altogether containing a T7 promoter followed by the gRNA sequence. RNAs were produced by in vitro transcription using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) and purified using the GeneJET RNA Purification Kit (Thermo Fisher Scientific). Sequences of the gRNAs used in our study are available in Supplementary Table 3.
DNA substrate generation
Complementary oligonucleotides (Metabion) containing target and PAM sequences were annealed and cloned into pUC18 plasmid over HindIII (Thermo Fisher Scientific) and EcoRI (Thermo Fisher Scientific) restriction sites. The links to the plasmid sequences are provided in Supplementary Table 1.
The 5’-ends of oligonucleotides were first radiolabeled using T4 PNK (Thermo Fisher Scientific) and [γ-32P]ATP (PerkinElmer). Then DNA substrates were generated by annealing two oligonucleotides with complementary sequences of whom one already had a radioactive label introduced at the 5’-end. Annealing was performed at 95 °C following slow cooling to room temperature. The sequences of the oligoduplexes are provided in Supplementary Table 4.
Cas12f1-gRNA complex assembly for in vitro DNA cleavage
In all, 1 µM of purified Cas12f1 protein was combined with its corresponding gRNA in 1:1 molar ratio in complex assembly buffer (10 mM Tris-HCl (pH 7.5 at 37 °C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT) and allowed to incubate at 37 °C for 30 min.
DNA cleavage assays
Reaction mixtures of 3 nM plasmid DNA, 100 nM Cas12f1 RNP complex in 10 mM Tris-HCl (pH 7.5 at 37 °C), 1 mM EDTA, 1 mM DTT, 10 mM MgCl2, and 200 or 100 mM NaCl buffer for SpCas12f1 and AsCas12f1, respectively, were incubated at 45 °C or as specified. The reaction was initiated by addition of Cas12f1 RNP complexes and was quenched at timed intervals (60 min if not indicated differently) by mixing with 3× loading dye solution (0.01% Bromophenol Blue and 75 mM EDTA in 50% (v/v) glycerol)). Reaction products were analyzed by agarose gel electrophoresis and ethidium bromide staining.
Reactions with oligoduplexes or ssDNA oligonucleotides were typically carried out by mixing labeled DNA samples with Cas12f1 RNP complex and incubating at 45 °C. Reaction mixtures contained 1 nM labeled duplex, 100 nM Cas12f1 RNP complex, 10 mM Tris-HCl (pH 7.5 at 37 °C), 1 mM EDTA, 1 mM DTT, 10 mM MgCl2, and 200 or 100 mM NaCl for SpCas12f1 and AsCas12f1, respectively, in a 100 µl final volume. Aliquots of 6 μl were removed from the reaction mixture at timed intervals (0, 5, 15, 30, and 60 min for SpCas12f1 or 0, 1, 5, 15, and 30 min for AsCas12f1) and quenched with 10 μl of a loading dye (95% (v/v) formamide, 0.01% Bromophenol Blue, and 25 mM EDTA). Reaction products were analyzed by denaturing gel electrophoresis (20% polyacrylamide containing 8.5 M urea in 0.5× TBE buffer), which were dried and visualized by phosphorimaging.
DNA-binding assay
Binding assays were performed by incubating different amounts of Cas12f1 RNP complexes (0, 10, 50, 100, and 250 nM) with 1 nM of 32P-5′-labeled ssDNA or dsDNA substrates (Supplementary Table 4) in binding buffer (40 mM Tris, 20 mM acetic acid (pH 8.4 at 25 °C), 1 mM EDTA, 0.1 mg/ml bovine serum albumin, 10% (v/v) glycerol, and 5 mM Mg(C2H3O2)2). All reactions were incubated for 30 min at room temperature (or as indicated) prior to electrophoresis on a native 8% (w/v) polyacrylamide gel. Electrophoresis was carried out at room temperature for 3 h at 110 V using 40 mM Tris, 20 mM acetic acid (pH 8.4 at 25 °C), 1 mM EDTA, and 5 mM Mg(C2H3O2)2 as the running buffer. Gels were dried and visualized by phosphorimaging.
Molecular weight measurements by mass photometry
Measurements were performed on an OneMP mass photometer (Refeyn Ltd). To prepare the measurements, coverslips (No. 1.5 H, 24 × 50 mm, Marienfeld) were cleaned by sequential sonication for 5 min in Milli-Q-water, isopropanol and Milli-Q-water. Coverslips were then dried using a clean stream of nitrogen. Measurement stock solutions of Cas12f1 RNP complex were prepared freshly before each measurement by mixing Cas12f1 protein (1 µM) and gRNA (500 nM) in complex assembly buffer (10 mM Tris-HCl (pH 7.5 at 37 °C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT) followed by incubation at 37 °C for 30 min. To prepare measurement stock solutions for pure Cas12f1 protein, gRNA and DNA samples of the respective stock solutions were diluted to 500 nM concentration in complex assembly buffer and incubated for 30 min at 37 °C. For DNA-binding experiments, 200 nM Cas12f1 RNP complex and 25 nM DNA were mixed in binding buffer (40 mM Tris-HAc (pH 8.4 at 25 °C), 5 mM Mg(CH3COO)2) and incubated for 30 min at 45 °C. After incubation, all samples were diluted by 1:10 in the respective sample buffer just before the measurement. Prior to the measurements, a cleaned coverslip was mounted onto the mass photometer and a gasket (CultureWell™ Reusable Gasket, Grace Bio-Labs) was placed on top. A gasket well was filled with 10 µl of the corresponding sample buffer, 10 µl of the diluted sample were added, and the adsorption of biomolecules was monitored for 120 s using the AcquireMP software (Refeyn Ltd, Version 2.3.0). For converting the measured ratiometric contrast into molecular mass, Un1Cas12f1 and its oligomers ranging from 60 to 250 kDa (monomer to tetramer) were used for calibration. All mass photometry movies were analyzed using DiscoverMP (Refeyn Ltd, Version 2.3.0). All samples were measured in triplicates.
M13 cleavage assay
M13 ssDNA cleavage reactions were initiated by mixing M13 ssDNA (New England Biolabs) with/or without DNA activator and Cas12f1 RNP complex at 45 °C. In all, 10 mM Tris-HCl (pH 7.5 at 37 °C), 1 mM EDTA, 1 mM DTT, 10 mM MgCl2, and 200 or 100 mM NaCl buffers were used, respectively, for SpCas12f1 and AsCas12f1. The final reaction mixture consisted of 3 nM M13 ssDNA, 100 nM ssDNA or dsDNA activator, or no activator and 100 nM Cas12f1 RNP. After initiating the reaction by adding Cas12f1 RNP, the samples were collected at timed intervals (0, 5, 15, 30, 60 min) by mixing with 3× loading dye solution (0.01% Bromophenol Blue and 75 mM EDTA in 50% (v/v) glycerol). Reaction products were separated on an agarose gel and stained with SYBR Gold (Thermo Fisher Scientific). The sequences of the activators are listed in Supplementary Table 4.
Human cell culture and transfection
HEK293T cells were purchased from ATCC (catalog number CRL-3216) and cultivated using Dulbecco’s Modified Eagle Medium (DMEM) supplied with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) (Thermo Fisher Scientific). Cells were first seeded in a 24-well plate at a density of 1.4 × 105 cells/well. After approximately 1 day of growth, a transfection mixture was prepared by diluting 1 µg of nuclease and its gRNA-containing plasmid (listed in Supplementary Table 1) in 100 µl serum-free DMEM and 2 µl of TurboFect transfection reagent was added (Thermo Fisher Scientific). After a 15 min incubation at room temperature, the transfection mixture was then added dropwise to each well containing the prepared cells. Transfected cells were then grown for 72 h at 37 °C and 5% CO2.
Z. mays transformation
First, 0.6 µM (average size) gold particles were coated with SpCas12f1 expression cassettes (Supplementary Table 1) using TransIT-2020, pelleted by centrifugation, washed with ethanol, and resuspended using sonication. Ten microliters of the DNA-linked gold particles were then loaded onto a microcarrier and allowed to air dry. Using a PDS-1000/He gun (Bio-Rad), particles were next bombarded into 9–10-day-old immature maize embryos (genotype PH1V69) with a 425 lb/in2 rupture disc. For transient assays, a gene encoding a yellow fluorescent protein, ZsYELLOW1 N139, was also delivered to aid in the selection of evenly transformed embryos 3 days after transformation. To produce T0 plants, post-bombardment culture, selection, and plant regeneration were performed using methods described previously40 except that bbm and wus2 genes were expressed with non-constitutive promoters, maize phospholipid transferase protein (Zm-PLTP), and maize auxin-inducible (Zm-Axig1) promoters, respectively41.
Human and Z. mays genome editing assay
Transfected HEK293T cells were collected by trypsinization and their genomic DNA was extracted using QuickExtract solution (Lucigen). For transient Z. mays assays, immature embryos were harvested 3 days post transformation, lyophilized, finely ground, and their total DNA extracted using the Synergy 2.0 Plant DNA Extraction Kit (Ops Diagnostics). To ensure chimeric and germ line edits could be distinguished in T0 plants, two leaf punches were taken from different leaves (V2 or V3). The fresh tissue was then ground, and DNA was extracted using PB buffer (Qiagen) in combination with a glass fiber 96-well microfilter plate (Agilent). PCR was then performed in two rounds to amplify the DNA region surrounding each target site and add on the sequences required for Illumina sequencing and indexing20,42. Briefly, 1–4 µl of DNA (10–200 ng) was used in a primary PCR with primers specific to the genomic locus that were 5’ tailed with Illumina sequences in a final volume of 20–50 µl (Supplementary Table S5). To ensure a balanced read composition within the initial cycles of sequencing, a mixture of four forward primers were used (see F1–F4 in Supplementary Table S5). Each of these primers was identical except for a 6 nt region immediately 3’ of the Illumina sequencing primer-binding site (Supplementary Table S5). Primary PCR was followed by a second round of PCR using 1 µl of the initial reaction as a template and primers specific to the Illumina sequences added in the primary PCR that also encoded the remaining sequences needed for Illumina bridge amplification, sequencing, and data deconvolution (in a 20–50 µl final volume). All primers and targets can be found in Supplementary Tables 5 and 6, respectively. Both rounds of PCR were allowed to proceed for 20 cycles and were carried out using NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB), Phusion High-Fidelity PCR Master Mix with GC Buffer (ThermoFisher Scientific), or Platinum SuperFi II Master Mix with Green Dye (ThermoFisher Scientific) according to the manufacturer’s instruction. After PCR, 5–10 µl were separated on a 1–2% agarose gel, stained with RedSafe (iNtRON) or ethidium bromide (Sigma), and visualized relative to DNA molecular weight standards to be the correct size. DNA was then purified using a Monarch PCR purification column (NEB) or Zymoclean Gel DNA Recovery Kit (Zymo Research), combined in an equimolar fashion, and sequenced on a MiSeq System (Illumina) with custom sequence primers, one for the amplicon and the second for the index (Supplementary Table 5). Sequences were trimmed to a Phred quality score of 13 and evaluated using a custom script43 for detection of insertion or deletion (indel) mutations that occurred within the expected cut-site. To be considered as true evidence of DSB repair, indel types were grouped, counted, and required to be at least 30 times greater in frequency than that found in the negative controls. The frequency of mutant reads was calculated by dividing the total number of mutant reads by the total number of wild-type reads. Mutant reads were visualized by aligning them against the wild-type reference highlighting the differences in contrasting colors. Percentage of edited plants (Fig. 3g) was calculated by dividing the number of plants with the specific mutation by the total number of plants with targeted modification.
SpCas12f1 specificity
Potential off-target sites were identified using Cas-OFFinder44. Molecular inversion probes (MIPs) were designed within a 50–400 bp window spanning all sites with up to four mismatches and two bulges and synthesized as a high-density pool from LC Sciences (Houston, TX). Altogether, 20 T0 plants that contained an on-target alteration classified as either heterozygous or homozygous, were sampled. This included five plants from the ms26 1 × 45 °C, ms26 3 × 45 °C, waxy 1 × 45 °C, and waxy 3 × 45 °C experiments. For use as a negative control, seven wild-type PH1V69 plants were also sampled. MIP targeting and sequence pools were prepared, and indexed amplicons were generated as described earlier45. Following Ampure XP PCR purification (Beckman Coulter Inc.), libraries were sequenced on an Illumina NextSeq, producing 150 nt paired end reads. If needed, targeted next-generation sequencing was used to supplement MIP sequence coverage for sites more closely related to the on-target (up to three SNPs and two SNPs in combination with two bulges). After sequencing, low-quality reads (phred quality score <30) were discarded and mapped to the PH1V69 reference genome with Bowtie237. For a given target site to be considered, at least five reads were required to be uniquely mapped to the locus harboring the off-site. The alignment files were next processed with a custom python script to characterize the edits46. Alterations that occurred in a 12 base pair window centered over the expected cleavage site were flagged and examined further. If the total number of mutant reads were >20% and not present in negative controls, the site was designated as a putative edit and manually verified by visualizing the reads with IGV38. All MIP sequences, targeted sequencing primers, and associated amplicons can be found in Supplementary Data 4.
Statistics and reproducibility
All statistical analyses were performed using GraphPad Prism (v.8.4.3). The exact replication numbers are indicated in the figure legends. The findings in all the figures of the gel images were successfully reproduced in similar experimental conditions at least three times independently.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
