Materials and Methods

qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites

MATERIALS AND METHODSPlasmidsThe sgRNA sequences E4.1 and E4.2, which are specific for target sequences within the TP53 gene, and sgRNAs specific for the VEGFA (VEGFA), EMX1 (EMX1), and CCR5 (CCR5.1, CCR5.6, and CCR5.7) genes have been described previously (31–33). To generate the Cas9_E4.1, Cas9_E4.2, Cas9_VEGFA, Cas9_EMX1, Cas9_CCR5.1, Cas9_CCR5.6 and Cas9_CCR5.7 plasmids, sense and antisense DNA strands of sgRNAs were synthesized (IBB, Warsaw, Poland), annealed to each other, and ligated into the FastDigest BsmBI (Thermo Fisher Scientific, Waltham, MA, USA) digested pSpCas9(BB)-2A-GFP (PX458) (Addgene, Cambridge, MA, USA) plasmid. Chemically competent Escherichia coli GT116 cells (InvivoGen, San Diego, CA, USA) were transformed with the ligated plasmids, plated onto ampicillin selection plates (100 μg/ml ampicillin) and incubated overnight at 37°C. The plasmids were isolated using the Gene JET Plasmid Miniprep kit (Thermo Fisher Scientific) and analyzed by Sanger sequencing with the U6-Fwd primer. The same strategy was used to generate plasmids encoding Cas9 and three sgRNAs specific for the HTT gene (34): Cas9_HTT.sg1, Cas9_HTT.sg3 and Cas9_HTT.sg4. Two HTT sgRNAs were also used with the nickase version of Cas9 (Cas9n; D10A mutant; pSpCas9n(BB)-2A-GFP (PX461)): Cas9n_HTT.sg1 and Cas9n_HTT.sg4. The oligonucleotide sequences are presented in Supplementary Table S1.Cell culture and transfectionHuman colon cancer cells (HCT116), human embryonic kidney cells (HEK293T), human myeloid leukemia cells (K562) and HeLa cells were grown in Dulbecco's modified Eagle's medium (Lonza; Basel, Switzerland) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Saint Louis, MS, USA), antibiotics (Sigma-Aldrich) and l-glutamine (Sigma-Aldrich). HCT116 and HeLa transfections were performed using Lipofectamine LTX (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Plasmids were transfected with a concentration of 0.25 μg to 2 μg/12-well plate. The transfection efficiency (GFP positive cells) was determined by flow cytometry (BD Accuri™ C6, BD Biosciences, Franklin Lakes, NJ, USA). HEK293T were transfected using the calcium phosphate method with 10 μg of plasmid DNA or 5 μg of each plasmid from a Cas9n_HTT.sg1 and Cas9n_HTT.sg4 pair for 3 × 105 cells/plate. HCT116 and K562 cells were electroporated with the Neon™ Transfection System (Invitrogen, Carlsbad, CA, USA). Briefly, 0.5–1 × 105 cells were harvested, resuspended in Buffer R and electroporated with 1 μg of plasmid DNA in 10 μl tips using the following parameters: 1130 V, 30 ms, 2 pulses or 1450 V, 10 ms, 3 pulses for HCT116 and K562 cells, respectively. In multiplex analysis, the concentration of each plasmid DNA was either 200 ng (0.6 μg in total) or 330 ng (1 μg in total). In HDR experiments, the HEK293T cells were electroporated with 125 ng of plasmid DNA (62.5 ng of each Cas9n_HTT.sg1 and Cas9n_HTT.sg4 plasmids) and 0.5 μl of 10 μM oligodeoxynucleotide (ssODN) as a donor template (IDT, Skokie, IL, USA) (Supplementary Table S1) using the following parameters: 1150 V, 20 ms, 2 pulses. The cells were cultured for 48 hours, after which genomic DNA was isolated with the Cells and Tissue DNA Isolation Kit (Norgen, Biotek Corp., Schmon Pkwy, ON, Canada) according to the manufacturer's instructions.In vitro T7 transcription of sgRNA and RNP complex deliveryR_1s and R_1a oligonucleotides corresponding to HTT_sg.1 (IBB, Warsaw) (Supplementary Table S1) were annealed and ligated into the FastDigest BpiI (Thermo Fisher Scientific) digested p31 vector, which contains a T7 RNA polymerase promoter and Cas9 gRNA scaffold sequence. Chemically competent E. coli GT116 cells (InvivoGen) were transformed with the ligated plasmids, plated onto ampicillin selection plates (100 μg/ml ampicillin) and incubated overnight at 37°C. The plasmids were isolated using the Gene JET Plasmid Miniprep kit (Thermo Fisher Scientific) and analyzed by Sanger sequencing with the WSF6 primer. The sgRNA expression vectors were digested with FastDigest DraI (Thermo Fisher Scientific), and the sgRNA was synthesized using an AmpliScribe T7-Flash Transcription Kit (Epicentre, Madison, WI, USA). The synthesized sgRNA was purified by phenol–chloroform–isoamyl alcohol (PanReac AppliChem, Barcelona, Spain) extraction and its integrity was checked by electrophoresis on a 10% PAA/urea/1× TBE gel.The RNP complex was produced by mixing recombinant NLS-SpCas9-NLS nuclease (VBCF Protein Technologies facility http://www.vbcf.ac.at) and in vitro transcribed sgRNA. Cas9 RNPs were prepared immediately before electroporation by incubating 2.5, 5 and 10 μg of Cas9 protein with 6, 12 and 24 μg of sgRNA transcript in an appropriate buffer containing 200 mM HEPES (pH 7.5), 1.5 M KCl, 5 mM DTT and 1 mM EDTA at 37°C for 10 min. HEK293T cells were electroporated with a Neon transfection system (Invitrogen) according to the manufacturer's instruction.Fluorescence-activated cell sortingCells were sorted using the BD FACSAria™ III (BD Biosciences) flow cytometer (cell sorter) 48 h post-electroporation. The configuration of the flow cytometer was as follows: a 100-μm nozzle and 20 psi (0.138 MPa) of sheath fluid pressure. Cells were characterized by two non-fluorescent parameters, forward scatter (FSC) and side scatter (SSC), and one fluorescent parameter, which was green fluorescence from GFP collected using the 530/30 bandpass filter (FITC detector). Data were acquired in a four-decade logarithmic scale as area signals (FSC-A, SSC-A, and FITC-A) and analyzed with FACS DIVA software (BD Biosciences). GFP-positive cells were divided into three fractions based on the fluorescence intensity. Each fraction contained ∼1–2 × 104 cells that were seeded onto a 6-well plate, maintained until confluence and collected for genomic DNA extraction.Single-cell clonesTo obtain single-cell clones, the HCT116 cells were transfected with the Cas9_E4.1 or Cas9_E4.2 plasmids, sorted by FACS and plated onto 150-mm cell plates to form single-cell clones. Individual colonies were picked with the use of cloning rings and transferred into 48-well plates according to the manufacturer's instructions (Promega, Madison, WI, USA). DNA was isolated from cells using 0.5 × Direct-Lyse buffer as described by Ramlee et al (30). PCR was performed using the MK024 and MK025 primers, and single-cell clones were genotyped by Sanger sequencing with the MK024 primer (Supplementary Table S1).T7E1 mutation detection assayFor the T7E1 mismatch assay, genomic DNA was amplified using Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific) with primers (MK024 and MK025) as described in the paper by Ramlee et al (30). The PCR amplification conditions were as follows: initial denaturation for 3 min at 95°C; 30 cycles of 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s; and a final elongation at 72°C for 5 min. PCR products were purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific). Next, 400 ng of purified PCR product was used in an annealing reaction and enzymatic digestion by the T7E1 enzyme (New England Biolabs, Ipswich, MA, USA). The DNA fragments were separated on a 1.3% agarose gel and visualized by EB staining. The DNA band intensity was quantified using G:BOX (Syngene, Cambridge, UK) and analyzed with GelPro software (Media Cybernetics, Rockville, MD, USA). The INDEL frequency was estimated by comparing the digested band intensities to all bands.The qEva-CRISPR assay generationThe qEva-CRISPR analysis was performed using the following custom-designed assays (probe mixes) with 7–12 MLPA probes: qEva_E4.1, qEva_E4.1_delA, qEva_E4.1_A/T, qEva_E4.2, qEva_HTT.sg1, qEva_HTT.sg3, qEva_HTT.sg4, qEva_HTT.edit, qEva_SNP, qEva_VEGFA, qEva_EMX1, qEva_CCR5.1, qEva_CCR5.6, qEva_CCR5.7 and qEva_multiplex. In most cases, the assay IDs corresponded to the IDs of the tested sgRNA/targets (Supplementary Table S2). Most assays consisted of one target specific (TS) probe and one to three off-target specific (OS) probes. The qEva_HTT.edit assay consisted of two probes (TS_HTT.sg1 and TS_HTT.sg4) specific for targets of HTT.sg1 and HTT.sg4 as well as one probe (TS_HTT.HDR) specific for sequence overlapping the ssODN sequence (inserted to genome by HDR) and the genomic sequence flanking the incorporated ssODN sequence. Additionally, the 5′ half-probe of TS_HTT.sg1 and the 3′ half-probe of TS_HTT.sg4 formed a new probe (TS_HTT.sg1/4) that is specific to a new sequence created after rejoining of free-ends created after the excision of the sequence between the HTT.sg1 and HTT.sg4 cuts. In addition to the TS and OS probes, each assay consisted of up to eight control probes. The sequences, genomic positions, and detailed characteristics of the probes used in this study are presented in Supplementary Table S3. The qEva-CRISPR probes were designed according to a strategy previously proposed for MLPA probes (27,28). Three three-oligonucleotide probes (TS_HTT.HDR, TS_CCR5.1 and OS_CCR5.1) were designed according to a strategy adopted from (35,36). All probes were synthesized by IDT, Skokie, IL, USA. All reagents, except for the probe mixes, were purchased from MRC-Holland, Amsterdam, The Netherlands. The MLPA reactions were performed according to the manufacturer's general recommendations (http://www.mlpa.com). Briefly, 5 μl of genomic DNA (at a concentration of approximately 20 ng/μl) were incubated at 98°C for 5 min, cooled to room temperature and mixed with 1.5 μl of an appropriate probe mixture as well as 1.5 μl of SALSA hybridization buffer. The reaction was then denatured at 95°C for 2 min and hybridized at 60°C for 16 h. The hybridized probes were ligated at 54°C for 15 min by the addition of 32 μl of the ligation mixture. Following heat inactivation, the ligation reaction was cooled to room temperature, mixed with 10 μl of a PCR mixture (polymerase, dNTPs, and universal primers, one of which was labeled with fluorescein) and subjected to PCR amplification for 35 cycles.The products of the MLPA reactions were diluted 20x in HiDi formamide containing GS Liz600, which was used as a DNA sizing standard, and separated via capillary electrophoresis (POP7 polymer) on an ABI Prism 3130XL apparatus (Applied Biosystems, Foster City, CA, USA). The obtained electropherograms were analyzed using GeneMarker software v2.4.0 (SoftGenetics, State College, PA, USA). The signal intensities (peak heights) were retrieved and transferred to prepared Excel sheets (available upon request). For each individual sample, the signal intensity of each probe was divided by the average signal intensity of the control probes to normalize the obtained values and to equalize the run-to-run variation. To calculate the relative signal (RS), the normalized signal of each probe was divided by the corresponding value of a reference (untreated) sample (or by the averaged value of the reference samples).

Article TitleqEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites

Abstract

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting theTP53, VEGFA, CCR5, EMX1andHTTgenes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in ‘difficult’ genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.


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