sgrna library plasmid dna  (Thermo Fisher)

Journal: Cell metabolism

Article Title: ZBTB1 Regulates Asparagine Synthesis and Leukemia Cell Response to L-Asparaginase.

doi: 10.1016/j.cmet.2020.03.008

Figure Lengend Snippet: Figure 1. A CRISPR-Based Genetic Screen Identifies Transcription Machinery Essential for Proliferation under Serine and Asparagine Depri- vation (A) Schematic demonstrating the metabolic genes regulated by ATF4, the effector of the integrated stress response. (B) Schematic depicting pooled CRISPR screens under asparagine or serine deprivation using a transcription-focused sgRNA library. (C) Gene scores in Jurkat cells grown in complete versus serine-deficient media (left). The gene score is the median log2 fold change in the abundance of all sgRNAs targeting a given gene after 14 population doublings. Most genes, as well as non-targeting control sgRNAs, have similar scores in the presence or absence of serine. The top ten genes differentially required under serine deprivation are shown (right). (D) Gene scores in WT Jurkat cells grown in complete versus asparagine-deficient media (left). The top ten genes differentially required under asparagine deprivation are shown (right). Media was depleted of asparagine through the use of L-asparaginase (0.25 U/mL). (E) Changes in the abundance of individual ZBTB1 sgRNAs in complete media (black) or in the absence (gray) of asparagine (top) or serine (bottom). (F) Differential scores of genes required specifically under asparagine deprivation relative to serine deprivation. ZBTB1 is highlighted in blue. (G) Immunoblot analysis of parental, ZBTB1 knockout, and rescued ZBTB1 knockout Jurkat cells (top). b-actin was used as a loading control. (H) Fold change in cell number (log2) of parental (black), ZBTB1 knockout (blue), and rescued ZBTB1 knockout (gray) Jurkat cells after growth in media with indicated asparagine concentrations for 5 days (mean ± SD, n = 3). (I) Fold change in cell number (log2) of parental (black), ZBTB1 knockout (blue), and rescued ZBTB1 knockout (gray) Jurkat cells after treatment with indicated asparaginase concentrations for 5 days (mean ± SD, n = 3) (top). Representative bright-field micrographs of indicated cells after a 5-day asparaginase treatment are shown (bottom). Statistics: two-tailed unpaired t test. **p < 0.05, ***p < 0.01, ****p < 0.001.

Article Snippet: Experimental Models: Organisms/Strains Mouse: NOD scid gamma: IL2Rgammanull The Jackson Laboratory 005557 Oligonucleotides qPCR primers, gBlocks, sgRNA and EMSA oligonucleotides This study See Table S5 Recombinant DNA CRISPR Cas9 sgRNA library This study N/A pLenti CRISPR V2 Addgene 52961 (Continued on next page) Cell Metabolism 31, 852–861.e1–e6, April 7, 2020 e2

Techniques: CRISPR, Control, Western Blot, Knock-Out, Two Tailed Test