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Efficient genomic correction methods in human iPS cells using CRISPR–Cas9 system

Li, Hongmei Lisa, Gee, Peter, Ishida, Kentaro, Hotta, Akitsu·2016·Methods
DOI 10.1016/j.ymeth.2015.10.015
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Fig. 1 Construction of CRISPR-sgRNAs. (A) Targeting sequence for Streptococcus pyogenes ( Sp ) CRISPR-sgRNA. The initial TSS (transcriptional start site) is better as ''G' or ''A' for transcription from H1 polIII promoter, and the last two nucleotides (position 22 and 23) must be ''GG' for PAM recognition. (B) Oligonucleotide primer sequences for cloning an Sp-sgRNA into pHL-H1-ccdB-EF1 a -RiH vector. Copy the initial 20 bp of the position 1-20 sequence from (A) into the forward (fwd) primer (indicated by blue and red ''Ns'). Reverse (rev) primer contains the scaffold from tracrRNA and a terminator signal and can be universally used for any target sequence. (C) Conduct PCR amplification of the sgRNA part using the two oligo primers from (B) and insert into the BamHI-EcoRI site of the pHL-H1-ccdB vector by In-Fusion reaction or standard ligation. Cloned sgRNA will be driven by H1 promoter, and mRFP expression can be used to monitor transfection efficiency. Hygromycin resistance gene can be used to enrich transfected cells.
Fig. 1 Construction of CRISPR-sgRNAs. (A) Targeting sequence for Streptococcus pyogenes ( Sp ) CRISPR-sgRNA. The initial TSS (transcriptional start site) is better as ''G' or ''A' for transcription from H1 polIII promoter, and the last two nucleotides (position 22 and 23) must be ''GG' for PAM recognition. (B) Oligonucleotide primer sequences for cloning an Sp-sgRNA into pHL-H1-ccdB-EF1 a -RiH vector. Copy the initial 20 bp of the position 1-20 sequence from (A) into the forward (fwd) primer (indicated by blue and red ''Ns'). Reverse (rev) primer contains the scaffold from tracrRNA and a terminator signal and can be universally used for any target sequence. (C) Conduct PCR amplification of the sgRNA part using the two oligo primers from (B) and insert into the BamHI-EcoRI site of the pHL-H1-ccdB vector by In-Fusion reaction or standard ligation. Cloned sgRNA will be driven by H1 promoter, and mRFP expression can be used to monitor transfection efficiency. Hygromycin resistance gene can be used to enrich transfected cells.CC-BY · From the PDF
Fig. 2 Testing cleavage activity of CRISPR-sgRNAs in 293T cells by SSA assay. (A) Examples of designed Sp-CRISPR-sgRNAs targeting exon 45 of the dystrophin gene on human X chromosome. sgRNA1-4 target the sense strand, whereas sgRNA5 targets the antisense strand relative to the dystrophin gene. (B) Principle of the SSA assay for detecting the DNA cleavage activity of CRISPR-sgRNAs. Luciferase cDNA is split by the target sequence of the five sgRNAs. Once DNA cleavage is induced, luciferase cDNA is restored via the single strand annealing (SSA) DNA repair pathway. (C) The SSA activities of CRISPR-sgRNAs measured in HEK293T cells. All five sgRNAs showed similar cleavage activities in this case. The values represent mean ± SD among technical replicates ( n = 3).
Fig. 2 Testing cleavage activity of CRISPR-sgRNAs in 293T cells by SSA assay. (A) Examples of designed Sp-CRISPR-sgRNAs targeting exon 45 of the dystrophin gene on human X chromosome. sgRNA1-4 target the sense strand, whereas sgRNA5 targets the antisense strand relative to the dystrophin gene. (B) Principle of the SSA assay for detecting the DNA cleavage activity of CRISPR-sgRNAs. Luciferase cDNA is split by the target sequence of the five sgRNAs. Once DNA cleavage is induced, luciferase cDNA is restored via the single strand annealing (SSA) DNA repair pathway. (C) The SSA activities of CRISPR-sgRNAs measured in HEK293T cells. All five sgRNAs showed similar cleavage activities in this case. The values represent mean ± SD among technical replicates ( n = 3).CC-BY · From the PDF
Fig. 3 Evaluation of CRISPR activity in human iPS cells by NEPA21 transfection. (A) Optimization of the transfection condition using NEPA electroporator in human iPS cell lines. Voltage and pulse width of the poring pulses were investigated. EGFP expressing plasmid was transfected, and the percentage of positive cells was analyzed by LSRFortessa (BD). (B) Schematic overview of the transfection procedures. (C) After the electroporation of Cas9 and sgRNAs into iPS cells, the cleavage activity of the endogenous dystrophin gene was assessed by the T7EI assay. Band intensities of the cleaved bands (indicated by red arrowhead) were detected by TapStation (Agilent), and values are indicated below the gel-electrogram image. (D) The same genomic DNA samples from (C) were also analyzed by the RFLP assay using the restriction enzyme XcmI (5 0 -CCANNNNN^NNNNTGG-3 0 ). Band intensities of the uncleaved band (red arrowhead) are indicated below. Since sgRNA5 cleaves far from the XcmI site, no cleavage activity was detected by the RFLP assay.
Fig. 3 Evaluation of CRISPR activity in human iPS cells by NEPA21 transfection. (A) Optimization of the transfection condition using NEPA electroporator in human iPS cell lines. Voltage and pulse width of the poring pulses were investigated. EGFP expressing plasmid was transfected, and the percentage of positive cells was analyzed by LSRFortessa (BD). (B) Schematic overview of the transfection procedures. (C) After the electroporation of Cas9 and sgRNAs into iPS cells, the cleavage activity of the endogenous dystrophin gene was assessed by the T7EI assay. Band intensities of the cleaved bands (indicated by red arrowhead) were detected by TapStation (Agilent), and values are indicated below the gel-electrogram image. (D) The same genomic DNA samples from (C) were also analyzed by the RFLP assay using the restriction enzyme XcmI (5 0 -CCANNNNN^NNNNTGG-3 0 ). Band intensities of the uncleaved band (red arrowhead) are indicated below. Since sgRNA5 cleaves far from the XcmI site, no cleavage activity was detected by the RFLP assay.CC-BY · From the PDF
Fig. 4 Flowchart for isolating genome-edited iPS cell clones. (A) Isolation of genome-edited iPS cell clones without drug selection. Once successful genome editing is confirmed in the bulk cell population by the T7EI or RFLP assay, spread the cells into two dishes; one dish for clump passaging and making frozen stocks and the other dish for dissociating into single cells and seeding onto feeder dishes at a density of 200-400 cells per 100 mm dish for clonal colony formation. After the formation of iPS cell colonies, scratch a part of the colony as a template for the PCR reaction and subsequent Sanger sequencing. The leftover cells in the same colony can be transferred into a well of a 24well plate for further expansion. (B) Isolation of genome-edited iPS cell clones with drug selection. We recommend starting the drug selection after passaging the transfected cells rather than right after the transfection to avoid extra stress on the damaged cells. It is important to apply the same drug selection to non-transfected cells as a control for the drug selection. Ideally, the concentration of antibiotic drug should be determined in advance. Once drug-resistant cells reach a sub-confluent state, split the cells into two dishes and one microcentrifuge tube. Then, extract genomic DNA for bulk PCR analysis (by using primers flanking the knock-in cassette) to roughly estimate the rate of knockin events. Colony isolation is the same as described in (A).
Fig. 4 Flowchart for isolating genome-edited iPS cell clones. (A) Isolation of genome-edited iPS cell clones without drug selection. Once successful genome editing is confirmed in the bulk cell population by the T7EI or RFLP assay, spread the cells into two dishes; one dish for clump passaging and making frozen stocks and the other dish for dissociating into single cells and seeding onto feeder dishes at a density of 200-400 cells per 100 mm dish for clonal colony formation. After the formation of iPS cell colonies, scratch a part of the colony as a template for the PCR reaction and subsequent Sanger sequencing. The leftover cells in the same colony can be transferred into a well of a 24well plate for further expansion. (B) Isolation of genome-edited iPS cell clones with drug selection. We recommend starting the drug selection after passaging the transfected cells rather than right after the transfection to avoid extra stress on the damaged cells. It is important to apply the same drug selection to non-transfected cells as a control for the drug selection. Ideally, the concentration of antibiotic drug should be determined in advance. Once drug-resistant cells reach a sub-confluent state, split the cells into two dishes and one microcentrifuge tube. Then, extract genomic DNA for bulk PCR analysis (by using primers flanking the knock-in cassette) to roughly estimate the rate of knockin events. Colony isolation is the same as described in (A).CC-BY · From the PDF

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Metadata

Authors
Li, H. L., Gee, P., Ishida, K., & Hotta, A.
Venue
Methods
Publisher
Elsevier BV
Published
2016-05
Volume
101
Pages
27-35
ISSN
1046-2023
DOI
10.1016/j.ymeth.2015.10.015
Cited by
62
References
6
Language
en
Licence
Funders
  • JSPS KAKENHI (15H05581)
  • AMED Research Center Network for Realization of Regenerative Medicine

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