Journal: bioRxiv

Article Title: Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

doi: 10.1101/2023.09.12.557474

Figure Lengend Snippet: (A) The exon-exon junction-targeting strategy and junction categories relating junction-level information to isoform-level information for a hypothetical gene. A common junction (gray exons) is shared across all isoforms, a fully unique junction (pink/purple exons) is specific to the 201 isoform, and a partially unique junction (pink/yellow exons) is present in a subset of isoforms, 202 and 203. For each junction, eight 23bp gRNAs are considered, with a starting position 15bp upstream of the junction and tiling every base pair to starting position of 8bp upstream of the junction. (B) Counts of theoretically targetable junctions and isoforms in GENCODE v41 (Basic Annotations, containing 18,568 protein-coding and 14,849 lncRNA genes), broken down by categories described in (A). (C) Left: the number of essential genes and their respective junctions that are targeted in the screen design. Right: the logic of the essentiality screen, where cells expressing gRNAs that knockdown essential genes are depleted over the 21 day time course. (D) Cumulative density function plot comparing Day 21 LFC between the re-defined Cas13d essential gene (n=27,804 gRNAs across 481 genes define by requiring more than 5% active gRNAs/gene) and Cas13d non-essential gene gRNAs (n=22,708 gRNAs across 356 genes) plus the two sets of control gRNAs for non-essential (NE, n=1,992 gRNAs) genes and non-targeting (NT, n=963 gRNAs). The dotted black lines mark the cutoff for active gRNAs defined as having LFC <0.01 quantile of NT gRNA normal distribution.

Article Snippet: To create the rtTA-sgRNA expressing piggyBac vector, the hU6_ Rfx Cas13d-DR_BsmBI was cloned from Addgene plasmid # 138151 ( ) in the piggyBac backbone from Addgene plasmid # 126028 ( ) by digestion with SfiI and BglII followed by Gibson Assembly (NEB).

Techniques: Expressing

Journal: bioRxiv

Article Title: Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

doi: 10.1101/2023.09.12.557474

Figure Lengend Snippet: (A) Counts of junction-level and isoform-level categories described in for the GENCODE mouse vM33 Basic Annotation (analogous to ). (B) Barplot (left) shows the number of gRNAs in our essentiality screen that target common and single isoform junctions (targets all isoforms a gene) and fully unique and partially unique junctions (targets a subset of isoforms). Cumulative density function plot (right) comparing Day 21 LFC of gRNAs targeting different junction types. Number of gRNAs per category is given in the barplot on the left. NT is used as a reference (gray line). (C) Pearson correlations of gene essentiality scores across different screening platforms in human A375 cells. We consider all genes, including the non-essential gene controls in our screen, shared across all screens (n = 1,165). Gene-level score from our screen is calculated as the median of the top 4 common guides per junction and then top 4 junctions per gene. (D) Pearson correlation of LFC for ∼258 gRNAs shared between a previous HEK293FT screen and our A375 screen. Left plot compares Day 21 LFC between biological replicates of our A375 screen and the right plot compares the average Day 21 LFC of the two replicates in our A375 screen to the average Day 30 LFC Wessels et al screen in HEK293FT. (E) LFC for 1,992 non-essential (NE) gene (n = 413 genes) control common junction gRNAs versus TPM gene expression in A375. Only a very slight reduction in LFC is seen for high TPM genes, indicating that Cas13d collateral activity is not a major concern.

Article Snippet: To create the rtTA-sgRNA expressing piggyBac vector, the hU6_ Rfx Cas13d-DR_BsmBI was cloned from Addgene plasmid # 138151 ( ) in the piggyBac backbone from Addgene plasmid # 126028 ( ) by digestion with SfiI and BglII followed by Gibson Assembly (NEB).

Techniques: Expressing, Activity Assay

Journal: Cell Genomics

Article Title: Multiple genes in a single GWAS risk locus synergistically mediate aberrant synaptic development and function in human neurons

doi: 10.1016/j.xgen.2023.100399

Figure Lengend Snippet:

Article Snippet: Single-strand shRNA oligos were cloned into BsmBI digested CROPseq-Guide-Puro (Addgene# 86708) vector using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) following vendor’s protocols.

Techniques: Scaffolding, Recombinant, Software