mrna reprogramming cocktail  (ReproCELL)

Journal: The CRISPR Journal

Article Title: Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing

doi: 10.1089/crispr.2023.0066

Figure Lengend Snippet: Prime editing (PE3) components and experimental process. (A) The prime editor (nCas9-RT) is directed to the target site by the pegRNA. nCas9-RT nicks the genomic strand containing the PAM sequence, allowing the pegRNA PBS sequence to hybridize with the newly released 3′ end of genomic strand. DNA synthesis occurs (not shown) at this untethered 3′ end of the nicked DNA strand using the RTT as a template. The sgRNA is usually positioned 40–90 bp from the pegRNA-induced nick and will introduce a second nick to promote repair of that strand. (B) The prime editing process includes design of pegRNA and sgRNA oligos to target candidate SNVs, transfection of iPSCs with combinations of prime editing components, expansion of transfected cells, pooled sequencing of the region spanning across the edit (1st Screen), and selection of top performing pools for single cell isolation and expansion to isolation isogenic lines (Single Cell Cloning inset). Clonal lines are sequenced (2nd Screen) to identify those with the correctly installed edit. Selected and expanded clones are sequenced to confirm the edited base and stained with cell surface markers to confirm pluripotency. gRNA, Guide RNA; nCas9, Cas9 nickase; PAM, protospacer adjacent motif; PBS, primer binding site; RT, reverse transcriptase; RTT, reverse transcriptase template; sgRNA, single-guide RNA; SNV, single nucleotide variant.

Article Snippet: HEK293T (ATCC crl-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX™ Supplement HEPES (Transfection System [TFS], Thermo Fisher Scientific), 10% fetal bovine serum (Biotechne), and 1 mM sodium pyruvate (TFS). iPSC lines derived from human fibroblast cells of consented study participants in the Finland–United States Investigation of NIDDM (FUSION) study (NIH protocol OH95-HG-N030; approved by NHGRI IRB) were generated at the New York Stem Cell Foundation Research Institute (New York, NY) using the Stemgent, #00–0071 mRNA transfection iPSC reprogramming kit.25.

Techniques: Sequencing, DNA Synthesis, Introduce, Transfection, Selection, Single-cell Isolation, Isolation, Clone Assay, Staining, Binding Assay, Variant Assay

Journal: The CRISPR Journal

Article Title: Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing

doi: 10.1089/crispr.2023.0066

Figure Lengend Snippet: Editing efficiencies predicted by pool sequencing are validated by single colony sequencing. Editing efficiencies predicted by pooled sequencing (X-axis) and editing efficiencies observed from subsequent single colony sequencing (Y-axis) across four T2D-associated SNVs near ABCC8, HNF4A, MTNR1B, and TCF7L2. The two sets of values were compared using the Pearson r2 statistic, which quantifies the degree to which single clone editing efficiency is approximated by pooled sequencing. The single colony sequencing efficiencies represent the proportion of edited alleles from all clones selected from one pool (transfection) and sequenced individually. T2D, type 2 diabetes.

Article Snippet: HEK293T (ATCC crl-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX™ Supplement HEPES (Transfection System [TFS], Thermo Fisher Scientific), 10% fetal bovine serum (Biotechne), and 1 mM sodium pyruvate (TFS). iPSC lines derived from human fibroblast cells of consented study participants in the Finland–United States Investigation of NIDDM (FUSION) study (NIH protocol OH95-HG-N030; approved by NHGRI IRB) were generated at the New York Stem Cell Foundation Research Institute (New York, NY) using the Stemgent, #00–0071 mRNA transfection iPSC reprogramming kit.25.

Techniques: Sequencing, Clone Assay, Transfection

Journal: The CRISPR Journal

Article Title: Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing

doi: 10.1089/crispr.2023.0066

Figure Lengend Snippet: pegRNA optimization with the PE3 system (A) transfection into HEK293T: plasmid-encoded prime editing components delivered into HEK293T included ABCC8 pegRNAs with variable primer binding site (PBS) and reverse transcriptase template (RTT) lengths; the prime editor Cas9 nickase-reverse transcriptase (RT); a pegRNA that targets the site for editing; and a single guide RNA (sgRNA) to target a 2nd nick on the nonedited strand, labeled with the distance of the sgRNA from the pegRNA-induced nick. Protospacer adjacent motif (PAM)-to-edit indicates the distance from the PAM site to the target nucleotide(s) being edited. PBS (underlined); (B) Transfection into induced pluripotent stem cell (iPSC): in vitro transcribed or synthetic RNA components delivered into iPSCs included an ABCC8 pegRNA (PD10) and a new guide PD15

Article Snippet: HEK293T (ATCC crl-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX™ Supplement HEPES (Transfection System [TFS], Thermo Fisher Scientific), 10% fetal bovine serum (Biotechne), and 1 mM sodium pyruvate (TFS). iPSC lines derived from human fibroblast cells of consented study participants in the Finland–United States Investigation of NIDDM (FUSION) study (NIH protocol OH95-HG-N030; approved by NHGRI IRB) were generated at the New York Stem Cell Foundation Research Institute (New York, NY) using the Stemgent, #00–0071 mRNA transfection iPSC reprogramming kit.25.

Techniques: Transfection, Plasmid Preparation, Binding Assay, Labeling, In Vitro, Sequencing, Next-Generation Sequencing

Journal: The CRISPR Journal

Article Title: Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing

doi: 10.1089/crispr.2023.0066

Figure Lengend Snippet: Editing efficiencies for different prime editing systems across seven targeted SNVs. (A–G) Different combinations of gRNA combinations (pegRNA and sgRNA) were transfected together with the prime editor (nCas9-RT). gRNA numbering is specific for each gene target. X-axis: prime editing complex systems represented by PE2 and PEmax in the presence or absence of modifications (epeg and DN). Y-axis: % editing efficiency as determined by read counts from sequencing of transfected cell pools. All SNVs except ADCY5 showed satisfactory efficiency. PD, pegRNAs; sg, sgRNAs, with distance from the pegRNA-induced nick represented by numeric values; epegRNA, modification on pegRNA; DN, MLH1dn; # rxns, number of transfections/data points in each plot.

Article Snippet: HEK293T (ATCC crl-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX™ Supplement HEPES (Transfection System [TFS], Thermo Fisher Scientific), 10% fetal bovine serum (Biotechne), and 1 mM sodium pyruvate (TFS). iPSC lines derived from human fibroblast cells of consented study participants in the Finland–United States Investigation of NIDDM (FUSION) study (NIH protocol OH95-HG-N030; approved by NHGRI IRB) were generated at the New York Stem Cell Foundation Research Institute (New York, NY) using the Stemgent, #00–0071 mRNA transfection iPSC reprogramming kit.25.

Techniques: Transfection, Sequencing, Modification

Journal: The CRISPR Journal

Article Title: Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing

doi: 10.1089/crispr.2023.0066

Figure Lengend Snippet: pegRNA design parameters affected editing efficiency. (A) Examples of pegRNAs with a constant PBS length but variable RTT length and PAM-to-edit distance. PBS; RTT; PAM-to-edit, distance between PAM sequence and edit site; RTT overhang, nucleotides after the installed edit encoded by in the RTT; red tick mark, nucleotide to be installed. (B–E) Editing efficiency for each target, based on PAM-to-edit distance, PBS length, RTT length, or RTT overhang. Each dot represents the average % editing of all replicate transfections of each pegRNA. Error bars show standard deviation of percent editing across multiple transfections of each pegRNA. Only a single point is shown for pegRNAs where editing efficiency was only measured for a single transfection. p-Values were generated by fitting a simple linear model with average editing efficiency as the outcome variable. See methods for additional details.

Article Snippet: HEK293T (ATCC crl-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX™ Supplement HEPES (Transfection System [TFS], Thermo Fisher Scientific), 10% fetal bovine serum (Biotechne), and 1 mM sodium pyruvate (TFS). iPSC lines derived from human fibroblast cells of consented study participants in the Finland–United States Investigation of NIDDM (FUSION) study (NIH protocol OH95-HG-N030; approved by NHGRI IRB) were generated at the New York Stem Cell Foundation Research Institute (New York, NY) using the Stemgent, #00–0071 mRNA transfection iPSC reprogramming kit.25.

Techniques: Sequencing, Transfection, Standard Deviation, Generated

Journal: The CRISPR Journal

Article Title: Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing

doi: 10.1089/crispr.2023.0066

Figure Lengend Snippet: Insertion–deletion rates. (A) Overall insertion–deletion rate within 800 bp for all clones for one target. Numbers at the top of the bars represent the number of clones sequenced for each target. (B) Rate across all transfections for each target with each dot representing one transfection. Dot sizes are on a continuous scale with the size increasing as the number of clones screened increases. The total number of clones screened for each target corresponds to those in panel A (HNF4A = 295, MTNR1B = 470, TCF7L2 = 778).

Article Snippet: HEK293T (ATCC crl-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX™ Supplement HEPES (Transfection System [TFS], Thermo Fisher Scientific), 10% fetal bovine serum (Biotechne), and 1 mM sodium pyruvate (TFS). iPSC lines derived from human fibroblast cells of consented study participants in the Finland–United States Investigation of NIDDM (FUSION) study (NIH protocol OH95-HG-N030; approved by NHGRI IRB) were generated at the New York Stem Cell Foundation Research Institute (New York, NY) using the Stemgent, #00–0071 mRNA transfection iPSC reprogramming kit.25.

Techniques: Clone Assay, Transfection