MATERIALS AND METHODSAll sequences for CRISPR gRNA spacers, C-Check oligonucleotides, PCR primers, PCR amplification conditions and genotyping/haplotyping of the TTR mutant clones can be found in Supplementary Tables S1–S5.SaCas9, SpCas9, xCas9 and Cas12a asPAM SNPsTo generate the asPAM SNP and CRISPR database, first, all SNPs with a heterozygous frequency >30% were retrieved from the 1000 human genome database. Next, only those SNPs giving rise to opposing PAM activities were included. The reference and alternative alleles of each SNP must be located in the conserved PAM motif (letters in bold) -NNGRRN for SaCas9, spacer-NGG for SpCas9, spacer-NGK for xCas9 and TTTV-spacer for Cas12a and give rise to either active or dead PAM, respectively. For all the corresponding asPAM CRISPR gRNA spacers, we have aligned them against the human reference genome with bowtie (version 1.1.2) and calculated the number of genomic loci with up to three mismatches. Finally, all asPAM SNPs and CRISPRs were incorporated in the CRISPR database. This database has been made publicly available, and detail of information and instructions on how to use the database can be found on our website (www.crispratlas.com/knockout).CRISPR asPAM database and HTT and TTR asPAM SNP outputThe asPAM-SNP and asPAM CRISPR databases were integrated into our CRISPR Atlas website (www.crispratlas.com), allowing the search of SNPs and CRISPRs for any gene of interest (GOI). To use the database, simply select ‘asPAM CRISPR editing’ from the webtools then output the asPAM-SNPs information of the GOI following the four guided steps in the opened webpage: (i) select GRCh37/h19 as the reference genome; (ii) select the CRISPR and PAM; (iii) select ‘chromosome interval’ as search method; (iv) input the start and end positions of a genome region including the GOI locus. In our test cases, the TTR locus is chr18: 29136875–29202208 and HTT locus is chr4: 3042475–3250766. Alternatively, an Excel sheet containing all the asPAM SNPs and CRISPRs can be requested from the corresponding authors.In this study, a genome region including a GOI locus comprises GOI DNA sequences and its context excluding the first upstream and downstream genes. Taking the TTR locus as an example, with the first upstream gene DSG-AS1 and downstream gene B4GALT6, we obtain end and start positions as chr18: 29136874 and chr18: 29202209, respectively. Thus, the chromosome interval range used for TTR locus input is chr18: 29136874 + 1 to chr18: 29202209 – 1, which actually is chr18: 29136875–29202208.Oligonucleotides and plasmidsAll DNA oligonucleotides in this study are ordered from either BGI-Qingdao, China (Chromosome1, TTR) or Sigma (HTT). Sequences of these oligonucleotides can be found in the Supplementary Tables. The CRISPR plasmids used in this research are: LentiCRISPRv2 (Addgene plasmid #52961), pUC19 (Addgene plasmid #50005), C-Check vector (Addgene plasmid #66817), pX601-AAV-CMV:NLS-SaCas9-NLS-3xHA-bGHpA;U6:BsaI-sgRNA (Addgene plasmid #61591).CRISPR gRNA design and vector constructionIn this study, as an example, we tested the asPAM CRISPRs and universal gRNAs using the SaCas9 system, for which the spacer length of gRNA is 21 nt. Other CRISPRs used for eccDNA generation and CRISPR-hapC were based on the SpCas9 system as described earlier (16). However, it should be noted that we have also tested the SaCas9 system for eccDNA generation. This also works efficiently.All the CRISPR gRNA spacers were designed utilizing the online webtools: CRISPOR (http://crispor.tefor.net) and the asPAM CRISPR database. Synthesized gRNA oligonucleotides were annealed and introduced into AAV-SaCas9 or LentiCRISPRv2 (SpCas9) vectors by Golden Gate Assembly (GGA) as described previously (17). Briefly, 1 μl sense strand (SS) and anti-sense strand (AS) gRNA oligonucleotides (100M) were mixed in 2 μl 10 × NEB Buffer 2.1, with a supplement of ddH2O to a final volume of 20 μl. The annealing program was 95°C for 5 min and ramped down to 25°C at a rate of −5°C/min. Then 1 μl annealed gRNA was added into a GGA reaction system that contains 1l T4 ligase (NEB), 2 l 10× T4 ligase buffer, 1l digest enzyme (Esp3I for lentiCRISPRv2 and Eco31I for AAV-saCas9, ThermoFisher Scientific) and ddH2O in a total volume of 20 l. The GGA program was performed in a thermocycler as: 10 cycles of 37°C for 5 min and 22°C for 10 min; 1 cycle of 37°C for 30 min; 1 cycle of 75°C for 15 min. Then 1 l of the GGA product was used for transformation of competent cells, and colony PCR screening was conducted to select positive colonies carrying the gRNA spacer. All CRISPR plasmids were further validated by Sanger sequencing.For C-Check (CC) vector construction in this study, oligonucleotides including target sequences (protospacer) and active PAM or dead PAM sequences were synthesized and inserted into CC vectors as described previously (18). All the CC oligonucleotides can be found in Supplementary Tables. The construction steps for CC vectors were the same as that for gRNA ligation, which was also conducted by GGA. CC vectors containing active PAM target sites were named as CC-aPAM and those containing dead PAM sequences were denoted CC-dPAM.Cell culture and transfectionCell lines used in this study include human embryonic kidney 239T (HEK293T, ATCC® CRL-3216), liver hepatocellular carcinoma cell line (HepG2, ATCC® HB-8065), human osteorsarcoma cell line (U2OS, ATCC® HTB-96), Hela cells, human ovarian adenocarcinoma cell line (Skov3, ATCC® HTB-77), human lung carcinoma epithelial cell line (A549, ATCC® CCL-185), human fibroblasts (BJ fibroblasts), human bone osteorsarcoma cell line (Saos-2, ATCC® HTB-85), human myelogenous leukemia cell line (K562, ATCC® CCL-243) and human breast cancer cell line (MCF-7, ATCC® HTB-22). All the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (LONZA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMAX (Gibco) and penicillin/streptomycin (100 units penicillin and 0.1 mg streptomycin/ml) in a 37°C incubator with 5% CO2 atmosphere and maximum humidity. Cells were re-seeded every 2–3 days when the confluence reached up to 80–90%.Transfection was conducted with Lipofectamine 2000 transfection reagent (Invitrogen) or X-tremeGene 9 (Roche) in 24-well plates according to the manufacturer’s protocol. Briefly, 60 000 cells/per well were seeded in 24-well plates and the medium was changed when the cells reached 50–70% confluency before transfection (typically 24 h after seeding). For each transfection, 500 ng total plasmid DNA and 1.5 μl Lipofectamine 2000 was diluted separately in Opti-MEM (Gibco) to a total volume of 25 μl. The diluted DNA was added to the diluted Lipofectamine and mixed gently. After 15-min incubation at room temperature, the transfection mixture was homogeneously added to the adherent cells in a dropwise manner. We changed medium 24 h after transfection and harvested cells 48 h later for subsequent assays. For all the co-transfection experiments in the research, including C-Check efficiency test, eccDNA generation and allele-specific knockout, the plasmids co-transfection ratio was 1:1.Flow cytometry (FCM) analysisCells in 24-well plates, dissociated with 100 μl 0.5% trypsin–EDTA, were suspended in 100 μl 5% FBS–PBS and transferred to a 96 deep-well plate on ice. Cells were spun down at 2000 rpm for 5 min and the supernatant was removed. Then the cell pellets were re-suspended in 600 μl PBS and immediately subjected to FCM analysis. FCM was performed using a BD LSRFortessa (supported by the FACS CORE facility, Department of Biomedicine, Aarhus University and FACS CORE, BGI-Qingdao) with at least 30 000 events collected for each sample in triplicate. TTR mutation cell model establishmentIn order to model the TTR mutation genotype that occurs in TTR-FAP patients, we established TTR mutated HepG2 cell line using CRISPR/spCas9. A gRNA targeting exon 2 (near the location of V30M mutation site) of TTR gene was transfected into HepG2 cells. Transfected cells were cultured in selection medium with 1 μg/ml puromycin in 10-cm dishes for 2 weeks, and cell colonies were manually picked and genotyped by PCR and Sanger sequencing. A cell colony (#21) with heterozygous mutation in exon 2 was selected as a model cell line for asPAM CRISPR editing of TTR.Genomic DNA extraction, PCR and TA cloningGenomic DNA was extracted using the TIANamp Genomic DNA Kit (for the TTR gene editing, TIANGEN, China) or by cell lysis (for the HTT gene) in accordance with the manufacturer’s instructions. The PCR were conducted using high-fidelity platinum Pfx polymerase in the presence of 2 × enhancer solution (#11708013, Thermo Fisher Scientific). All the primers for SNP validation and eccDNA detection can be found in Supplementary Tables. ECC PCR products were sub-cloned into the pMD-19T vector utilizing TOPO TA Cloning Kit (TaKaRa) according to the manufacturer’s instructions and analyzed by Sanger sequencing.Generation of eccDNA by CRISPRA detail protocol for the generation of eccDNA by CRISPR and haplotyping by eccDNA was provided in the Supplementary Methods.Data analysisThe webtool asPAM CRISPR editing (http://www.crispratlas.com/knockout) was used for GOI SNP-PAM output. Flowjo software was used to gate and output FCM data. Prism 7 was used to analyze FCM data and plot histograms. Sanger sequencing results were deciphered by Snapgene Viewer, and the webtool ICE analysis (https://ice.synthego.com/#/) was used for genotype percentage analysis with Sanger data.StatisticsUnless stated elsewhere, all experiments were performed in triplicate. The Student’s paired T test was used for statistical analysis, with a P value <0.05 considered as statistically significant.
Article TitleHaplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing
Allele-specificprotospaceradjacentmotif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of theHTTCAG expansion allele andTTRexon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.