human male ipsc line  (Jackson Laboratory)

Journal: Cell Reports Methods

Article Title: Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

doi: 10.1016/j.crmeth.2023.100672

Figure Lengend Snippet: Characterization of PB integrations and indel editing (A) Distribution of the number of distinct integrated gRNA genes per iPSC line and resultant indel editing. Bar heights indicate counts of lines containing from 0–8 different gRNA genes. Fill color within each bar signifies proportions of lines with indel editing at one (blue) or multiple (yellow, cyan, gray) corresponding target sites. Seven lines were excluded from the plot because they lacked confident gRNA gene integration genotypes. (B) gRNA representation and guide efficiencies for indel mutagenesis. Bar heights indicate counts of iPSC lines containing each gRNA gene, with guides ordered based on abundance. Fill color within each bar signifies the proportion of lines harboring each gRNA gene with indel editing at the corresponding target (red), lacking such editing (black), or having an uncertain genotype (maroon) due to poor MIP capture and low sequencing coverage across the target site. Dashed line shows expected gRNA representation based on sampling from a uniform distribution (1,000,000 simulations).

Article Snippet: male human iPSC line , Coriell Institute for Medical Research , GM08330.

Techniques: Mutagenesis, Sequencing, Sampling

Journal: Cell Reports Methods

Article Title: Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

doi: 10.1016/j.crmeth.2023.100672

Figure Lengend Snippet: PB excision (A) Plasmid containing PB transposon construct for simultaneous editing and excision is shown. TR, terminal repeat; TRE, tetracycline response element; UCOE, ubiquitous chromatin opening element; rtTA, reverse tetracycline-controlled transactivator. (B) FACS plots depict GFP fluorescence (y axis) and forward scatter area (x axis) from the 488 nm laser for live single cells (points) derived from two wells of transfected cells selected with puromycin, where one well was then treated with doxycycline for 4 days (bottom) and the other well was not treated (top). Gate encompasses cells deemed GFP negative (red), set based on analysis of naive cells from the same experiment. (C–E) Analyses of iPSC lines recovered from the excision experiment. (C) Distribution of the number of distinct integrated gRNA genes per iPSC line and resultant indel editing. Bar heights indicate counts of lines containing from 0–5 different gRNA genes. Fill color within each bar signifies proportions of lines with indel editing at one (blue) or multiple (yellow, cyan, gray, dark orange) corresponding target sites. Five lines were excluded from the plot because they had no detected gRNA gene integrations but were not deemed integration-free based on MIP data. (D) Distribution of the number of distinct gRNA targets (genes) edited per iPSC line. Bar heights indicate counts of lines harboring from 0–5 different mutated targets (genes). (E) Guide efficiencies for indel mutagenesis. Bar heights indicate counts of iPSC lines that acquired indel(s) at each gRNA target, with guides ordered based on editing frequency. Fill color within each bar signifies the proportion of lines with indel editing at each target exclusively (green) or in combination with indel formation at one or more additional targets (black).

Article Snippet: male human iPSC line , Coriell Institute for Medical Research , GM08330.

Techniques: Plasmid Preparation, Construct, Fluorescence, Derivative Assay, Transfection, Mutagenesis

Journal: Cell Reports Methods

Article Title: Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

doi: 10.1016/j.crmeth.2023.100672

Figure Lengend Snippet:

Article Snippet: male human iPSC line , Coriell Institute for Medical Research , GM08330.

Techniques: Virus, Recombinant, Plasmid Preparation, Sequencing, Derivative Assay, Software