Journal: bioRxiv

Article Title: A high-density lineage tree reveals dynamics of expression differences accumulation in nondifferentiating clonal expansion

doi: 10.1101/2021.11.24.469964

Figure Lengend Snippet: ( A ) RT-PCR results for the RNA expression levels of the STADT barcode and Cas9 in cells growing in the presence of different concentrations of cumate. GAPDH was used as an internal control. The STADT barcode and Cas9 were amplified for 30 cycles, while GAPDH was amplified for 26 cycles. ( B ) RT-qPCR results for the expression levels of the STADT barcode relative to those of GAPDH. ( C ) RT-qPCR results for the expression levels of the Cas9 gene relative to those of GAPDH in cells cultured with different concentrations of cumate. ( D ) Western blot of the expression levels of Cas9 in cells cultured with different concentrations of cumate. GAPDH was used as an internal control. The small bands (*) may have resulted from the degradation of the Cas9 protein. ( E ) RT-PCR results showing the expression levels of sgRNA1 in the cells infected with different MOIs (0.1, 30) of the sgRNA lentivirus. U6 was used as an internal control. sgRNA1 was amplified for 35 cycles, while U6 was amplified for 30 cycles. ( F ) RT-qPCR result showing the expression level of sgRNA1 relative to that of U6 in the cell lines infected with different MOIs (0.1, 30) of the sgRNA lentivirus.

Article Snippet: For the sgRNAs, two DNA cassettes, U6>sgRNA1-U6>sgRNA2 and U6>sgRNA3-U6>sgRNA4, were synthesized and subsequently combined by Golden Gate ligation in the sgRNA vector pLV-U6>sgRNA1-U6>sgRNA2-U6>sgRNA3-U6>sgRNA4 (VectorBuilder, no. VB180515-1178wxn).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Amplification, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Infection

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ cells from three SCD patient donors were electroporated with ABE8e-NRCH mRNA or RNP using an sgRNA targeting the SCD mutant HBB codon. (a) The edited region of HBB with the target A at protospacer position 7 shown in blue along with potential bystander edits in green (silent), brown (silent), and red (non-silent). (b) Editing efficiencies by HTS at target and bystander adenines, and indels after 6 days in stem-cell culture media following electroporation. (c) Proportion of β-like globin proteins by HPLC of reticulocyte lysates after 18 days in differentiation media following electroporation. (d) Representative phase-contrast images of reticulocytes derived from unedited or edited donor HSPCs incubated 8 hours in 2% O2. Nine images of >50 cells each were collected per sample. Scale bar=50 μm. (e) Quantification of sickled reticulocytes from counting >300 randomly selected cells by a blinded observer from images as in (d). (f) Venn diagram showing candidate off-target sites nominated by Cas-OFFinder and CIRCLE-seq, and nominated sites for which off-target editing was observed by targeted DNA sequencing in SCD patient CD34+ cells electroporated with ABE8e-NRCH mRNA. (g) Predicted genomic features of validated off-target sites. TTS, ≤1 kb from the transcription termination site; UTR, untranslated region. (h) ABE8e-NRCH-treated HSPCs from two different SCD patient donors were sequenced at 697 potential off-target sites. The histogram shows the number of validated off-target base editing sites binned by average percentage of sequencing reads for each site with any A•T-to-G•C mutations in protospacer nucleotides 4-10. Bar values in (b), (c), and (e) and error bars reflect mean±SD of three independent biological replicates, with individual values shown as dots.

Article Snippet: The modified synthetic sgRNA contained 2′-O-methyl modifications in the first three and last three nucleotides, and phosphorothioate bonds between the first three and last three nucleotides 40 and was purchased from BioSpring.

Techniques: Mutagenesis, Stem Cell Culture, Electroporation, Derivative Assay, Incubation, DNA Sequencing, Sequencing

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: (a) Plasmids encoding the HBBS-targeting sgRNA and either ABE7.10-NRCH or ABE8e-NRCH were transfected by lipofection into HEK293T cells. Editing efficiency was measured after 3 days by high-throughput DNA sequencing (HTS). Unedited cells were not lipofected. (b) Two days after electroporation into human patient HSPCs of base editor mRNA and sgRNA, or electroporation of ribonucleoprotein (RNP), cell number and viability were measured using a Chemometec Nucleocounter-3000. Acridine orange was used to stain the total cell number and DAPI was used to stain dead, permeabilized cells. The percent viability was calculated as the DAPI stained cells divided by the acridine orange cells within each sample. The percent recovery was normalized to the cell count of the unedited sample. Unedited cells were not electroporated, (c) Six days after electroporation of SCD patient HSPCs, genomic DNA was extracted and the target HBB locus was PCR amplified and sequenced using an Illumina instrument. The sequencing analysis program CRIS.py was used to identify and quantify the resulting alleles. All alleles above a threshold of 0.2% frequency are shown. Below this threshold, variant alleles appear with greatest frequency in the untreated control sample, suggesting they do not arise from base editor treatment. Nucleotides altered from the endogenous sequence are shown in blue. Rare cytosine base editing was observed at <1% frequency, as has been previously described as a possible outcome from adenine base editing50. Bar values and error bars reflect mean±SD, n=3.

Article Snippet: The modified synthetic sgRNA contained 2′-O-methyl modifications in the first three and last three nucleotides, and phosphorothioate bonds between the first three and last three nucleotides 40 and was purchased from BioSpring.

Techniques: Transfection, High Throughput Screening Assay, DNA Sequencing, Electroporation, Staining, Cell Counting, Amplification, Sequencing, Variant Assay, Control