Journal: Nature

Article Title: Saturation Editing of Genomic Regions by Multiplex Homology-Directed Repair

doi: 10.1038/nature13695

Figure Lengend Snippet: (a) Experimental schematic. Cultured cells were co-transfected with a single Cas9-sgRNA construct (CRISPR) and a complex homology-directed repair (HDR) library containing an edited exon that harbors a random hexamer (blue, green, orange) and a fixed selective PCR site (red). CRISPR-induced cutting stimulated homologous recombination with the HDR library, inserting mutant exons into the genomes of many cells. At five days post-transfection, cells were harvested for gDNA and RNA. After reverse transcription, selective PCR was performed followed by sequencing of gDNA and cDNA derived amplicons. Hexamer enrichment scores were calculated by dividing cDNA counts normalized by gDNA counts. (b) Correlation of enrichment scores between biological replicates for hexamers observed in each experiment with positions of previously identified exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs) and stop codons indicated. (c) Rank-ordered plot of enrichment scores with positions of ESEs, ESSs, and stop codons indicated.

Article Snippet: A bicistronic Cas9-sgRNA vector designed to cleave within BRCA1 exon 18 (“pCas9-sgBRCA1x18”) was cloned according to a published protocol by ligating annealed oligonucleotides into a human codon-optimized S. pyogenous Cas9-sgRNA vector from the lab of Feng Zhang (pX330-U6-Chimeric_BB-CBh-hSpCas9; Addgene plasmid #42230).

Techniques: Cell Culture, Transfection, Construct, CRISPR, Random Hexamer Labeling, Homologous Recombination, Mutagenesis, Sequencing, Derivative Assay

Journal: bioRxiv

Article Title: CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

doi: 10.1101/2024.10.08.616922

Figure Lengend Snippet: a, Genome browser screenshot showing gene body enhancer of FMR1 and sgRNAs targeting FMR1 promoter and enhancer. b, Flow cytometry plots showing the significant downregulation of FMR1-mCherry expression after deleting FMR1 promoter and FMR1-E1 enhancer in both iPSCs and excitatory neurons. Positive controls (black line) are the FMR1-mCherry reporter cells. c, Genome browser screenshot showing identified enhancers of MECP2 and sgRNAs targeting MECP2 promoter and enhancers. d, Single clones of MECP2 promoter or enhancers deletion showing significant downregulation of MECP2-EGFP in both iPSCs and excitatory neurons. Positive controls (black line) are the MECP2-EGFP cells. C1 and C2 indicate two independent clones. e, RT-qPCR results showing the significant downregulation of MECP2 expression in each clone ( P < 0.05 for all the clones, two-tailed two-sample t -test; n = 2). Data are mean ± SEM. f, Flow cytometry plots showing the significant downregulation of APP-EGFP or APP-mCherry in APP promoter and APP-E3 deletion cells. Positive controls (black line) are the APP-EGFP/mCherry reporter cells. g, Flow cytometry plots showing the downregulation of SIN3A-EGFP or SIN3A-mCherry in SIN3A promoter and SIN3A-E4 deletion cells. Red dashed lines indicate the position of SIN3A-EGFP/mCherry double positive cells. h, The genome browser screenshot showing the CTCF ChIP-seq signal in SIN3A-E4 enhancer region in WTC11 iPSCs. The CTCF motif was obtained from JASPAR. Two sgRNAs were designed to target the CTCF motif. PAM sequences were in red. i, Flow cytometry plots showing the downregulation of SIN3A-EGFP or SIN3A-mCherry in sgRNA1 and sgRNA2 infected cells. j, The editing outcomes of sgRNA 1 in the cells of SIN3A-EGFP-/SIN3A-mCherry+. k, The enrichment of disease-associated CNVs in distal cCREs identified in diverse cell types in the human body. Heatmap shows the data from diseases with at least 10 CNVs and P value less than 1×10 -5 in at least one cell type.

Article Snippet: To clone the two sgRNAs into lentiCRISPRv2 vector (Addgene, #52961), we amplified the sgRNA scaffold and mouse U6 promoter using two oligos containing the designed sgRNA sequences, and inserted the amplified DNA fragments into the lentiCRISPR v2 vector (Addgene, #52961) using Gibson assembly (NEB, E2621L).

Techniques: Flow Cytometry, Expressing, Clone Assay, Quantitative RT-PCR, Two Tailed Test, ChIP-sequencing, Infection