Materials and Methods

Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

MATERIALS AND METHODSPreparation of the CRISPR–Cas12a recombinant protein and chimeric guidesFor purification of Cas12a recombinant protein, pET28a–Cas12a (Acidaminococcus sp. (As) Cas12a, Lachnospiraceae bacterium (Lb) Cas12a, Francisella novicida (Fn) Cas12a) bacterial expression vectors were introduced into Escherichia coli BL21 (DE3) species and transformed, and then cultured at 37°C until the O.D. reached 0.6. After 48 h of IPTG inoculation, bacterial cells were precipitated to remove the culture medium, and the remaining cell pellet was resuspended in lysis buffer 20 mM Tris–HCl (pH8.0), 300 mM NaCl, 10 mM β-mercaptoethanol, 1% TritonX-100, 1 mM PMSF. Then, bacterial cell membranes were broken by sonication (ice, 3 min), and the cell lysate was harvested by centrifugation (5000 rpm, 10 min). For single-step (using 6xHis at N-terminus of Cas12a) purification, Ni-NTA resin pre-washed with wash buffer 20 mM Tris–HCl (pH8.0), 300 nM NaCl and the ultrasonically disrupted intracellular solution were mixed and stirred for 1 h 30 min in a cold room (4°C). After bacterial cell precipitation, non-specific binding components were removed by washing with buffer B 20 mM Tris–HCl (pH 8.0), 300 nM NaCl at 10 times volume, elution buffer 20 mM Tris–HCl (pH 8.0), 300 nM NaCl, 200 mM Imidazole was used to elute the AsCas12a protein. The buffer which is used for protein elution was exchanged to storage buffer 200 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM DTT, 40% glycerol using a centricon (Amicon Ultra) and stored at –80°C. For two-step purification, eluted proteins were further incubated with anti-FLAG resin for 1 h 30 min in a cold room (4°C). Non-specific binding components were washed out with wash buffer 20 mM Tris–HCl (pH8.0), 300 nM NaCl again. After washing, elution buffer 20 mM Tris–HCl (pH 8.0), 300 nM NaCl, 3xFLAG peptide (5 mg/ml) was used to elute the Cpf12a protein. The eluted buffer is exchanged against to storage buffer 200 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM DTT, 40% glycerol by using centricon (Amicon Ultra,) and stored at –80°C. Chimeric DNA–RNA guides (bioneer) were batch synthesized according to the target sequences in each target gene (Supplementary Table S1).In-vitro transcription and purification of the guide RNA for Cas12a and Cas9For in vitro transcription, each sense and anti-sense DNA oligo containing the target (cr)RNA sequence (Supplementary Table S1) was purchased (Macrogen). The annealed DNA template was mixed with T7 RNA polymerase (NEB) and the reaction mixture (50 mM MgCl2, 100 mM rNTP (Jena Bio, NU-1014), 10× RNA polymerase reaction buffer, RNase Inhibitor Murine, 100 mM DTT, DEPC). (cr)RNA was synthesized by incubation for 8 h at 37°C. After synthesis, the DNA template was completely removed by incubation at 37°C for 1 h with DNase, and only the RNA was separated through the column (MP Biomedicals, GENECLEAN® Turbo Kit). Purified RNA was concentrated through lyophilization (2000 rpm, 1 h, −55°C, 25°C). In-vitro DNA cleavage assay and calculation of the DNA cleavage efficiencyOn-/off-target PCR amplicons are obtained from purified genomic DNA (HEK293FT) using DNA primers (Supplementary Table S2) corresponding to each target gene (DNMT1, HPRT1, RPL32P3, CCR5, FANCF, GRIN2B, EMX1). To cleave the amplicons, purified recombinant Cas12a protein and synthesized chimeric (cr)RNA–DNA (purchased from Bioneer) or purified (cr)RNA corresponding to each locus were premixed and incubated at 37°C for 1 h in cleavage buffer (NEB3, 10 μl volume). Then, the reaction was stopped by adding a stop buffer (100 mM EDTA, 1.2% SDS). DNA cleavage was checked by 2% agarose gel electrophoresis. DNA cleavage efficiency was determined by calculating the image pattern according to the formula (Intensity of the cleaved fragment/total sum of the fragment intensity × 100 = %) measured using ImageJ software(NIH).Cell sub-culture and transfectionHEK293FT cell line (ATCC) was passaged in DMEM media (DMEM (Gibco) with 10% FBS (Gibco)) every 48 h at 37°C, 5% CO2 to maintain a confluency of 70%. For efficient endogenous locus editing, we used single transfection method with electroporation (Lonza, V4XC-2032) or sequential transfection with electroporation and lipofection method. For single transfection with electroporation, 105 cells were mixed with Cas12a-chimeric guide pre-mixed complex (Cas12a: 60 pmol, (cr)RNA: 240 pmol) and followed by electric shock (program: CM-130) in electroporation buffer (manufacturer's guide). Subsequently, transfer the transfected cells into pre-incubated (37°C and 5% CO2) DMEM media solution (500 ul) of a 24-well plate, and incubate at the same conditions (37°C and 5% CO2) for 72 h. In case of the sequential transfection, HEK293FT cells were nucleofected with vector mixture (human codon optimized) for AsCpf1 (500ng), d/n-SpCas9 (D10A, H840A for dead / D10A for nickase, 100 ng), crRNA (150 pmol), and sgRNA (15 pmol) expression. After 12 h, same amount of crRNA and sgRNA were transfected with 2.8 μl of lipofectamine (ThermoFisher) and 2.0 μl of P3000 reagent twice at the same interval. Seventy two hours after transfection, genomic DNA was extracted from HEK293FT cells and analyzed by targeted amplicon sequencing (illumina, SY-420-1001). To induce the mutation on plasmid target sequence, target plasmids (DNMT1, GRIN2B on-/off-target plasmid, 1μg) were co-transfected with Cas12a–crRNA pre-mixture and extracted target plasmids were further analyzed by targeted amplicon sequencing (illumina, SY-420-1001).Genomic DNA purificationForty eight hours after the delivery of the chimeric (cr)RNA–DNA and the recombinant Cas12a protein complex into cells (HEK293FT), genomic DNA was isolated using a genomic DNA purification kit (Qiagen, DNeasy Blood & Tissue Kit) according to the manufacturer's protocol. In the case of plasmid delivery, genomic DNAs were extracted after 72 h transfection.Targeted amplicon sequencing and data analysisPCR amplicons (DNMT1, HPRT1, RPL32P3, CCR5, FANCF, GRIN2B, EMX1) were prepared by using DNA primer (Supplementary Table S2) corresponding to the target locus. Then, nested PCR (denaturation: 98°C – 30 s, primer annealing: 58°C – 30 s, elongation: 72°C – 30 s, 35 cycles) was performed to insert the adapter and index sequences into both 5′ and 3′-end of the amplicon (denaturation: 98°C – 30 s, primer annealing: 62°C – 15 s, elongation: 72°C – 15 s, 35 cycles). Thereafter, the tagged amplicon mixture was loaded onto a mini-SEQ analyzer (Illumina, SY-420-1001) according to the manufacturer's guidelines and subjected to targeted deep sequencing. The saved Fastq files were analyzed with Cas-Analyzer (24) and the indel efficiency was calculated.

Article TitleEnhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

Abstract

Targeted deep sequencing data are available at NCBI Sequence Read Archive (SRA) under accession number SRP247270. CRISPR RGEN Tools is an open-source collaborative initiative available in the repository (http://www.rgenome.net/).


Login or Signup to leave a comment
Find your community. Ask questions. Science is better when we troubleshoot together.
Find your community. Ask questions. Science is better when we troubleshoot together.

Have a question?

Contact support@scifind.net or check out our support page.