april 2026 crispr cas9 vectors  (Addgene inc)

Journal: bioRxiv

Article Title: Baseline cellular state dictates the molecular impact of KRAS mutant variants in pancreatic cancer cells

doi: 10.64898/2026.03.10.710185

Figure Lengend Snippet: Generation of KRAS knockout clones. (A) Schematic of the CRISPR/Cas9 strategy used to generate KRAS knockout 8988T and KP4 cell lines by targeting exon 2 of KRAS . (B) Immunoblot of RAS - less mouse embryonic fibroblasts (MEFs) reconstituted with BRAF V600E , KRAS WT , HRAS WT , or NRAS WT , confirming specificity of the anti-KRAS antibody (clone 3B10-2F2). (C) Immunoblots showing absence of KRAS protein in 8988T (K275, K328) and KP4 (K22, K63) KRAS knockout clones and baseline PI3K (pAKT) and MAPK (pERK1/2) levels relative to parental cell lines. Loading control is HSP90. Images are representative of n = 3 biological replicates. (D) Bar graphs show quantified pERK/ERK and pAKT/AKT levels relative to the parental cell line (8988T or KP4) (mean ± SEM of n = 3 biological replicates) of immunoblots in ( C ). p -values of repeated measures one-way ANOVA with Tukey’s post hoc test are shown.

Article Snippet: A single-guide RNA (sgRNA) targeting KRAS exon 2 (sequence: 5’-AATTACTACTTGCTTCCTGT-3’) was cloned into the CRISPR-Cas9 expression vector pSpCas9(BB)-2A-GFP (PX458, Addgene plasmid #48138).

Techniques: Knock-Out, Clone Assay, CRISPR, Western Blot, Control

Journal: Nature

Article Title: The ubiquitin ligase KLHL6 drives resistance to CD8 + T cell dysfunction

doi: 10.1038/s41586-025-09926-8

Figure Lengend Snippet: a , Sanger sequencing of the Klhl6 locus in Cas9 + sgRNA + OT-I T cells transduced with sgCtrl , sgKlhl6 -1, or sgKlhl6 -2 in vitro. Primer sequences are listed in Supplementary Table . b , c , Mice were subcutaneously injected with 2×10 5 B16-OVA melanoma cells, followed by transfer of 3×10 Cas9 + CD8 + OT-I T cells expressing control sgRNA or sgKlhl6 (two targets) on day 9. Tumours were collected and analyzed 7 days later. Experimental design ( b ). The number of transferred Cas9 + OT-I T cells in the tumour ( c , n = 7 mice). d , e , Representative plots (left) and proportions (right) of LAG-3 + TIM-3 + TILs ( d ), and cell numbers of indicated subsets ( e ) among sgCtrl - or sgKlhl6 -transduced Cas9 + CD8 + OT-I TILs (n = 7 mice). f , g , Proportions of PD-1 + TIM-3 + ( f ) and (MTDR/MTG) lo ( g ) TILs among sgCtrl- or the indicated sgRNA- transduced Cas9 + CD8 + OT-I T cells from B16-OVA tumours (n = 6 mice). h , Schematic of CRISPR/Cas9-mediated generation of Klhl6 −/− mice. Guide RNA sequences are provided in Supplementary Table . i , Genotyping results for Klhl6 +/+ (WT) or Klhl6 −/− (KO) alleles. j , k , Percentages of thymocyte subsets: CD4 − CD8 − double-negative (DN), CD4 + CD8 + double-positive (DP), CD4 + single-positive (CD4SP) and CD8 + single-positive (CD8SP) ( j ), and CD44 + single-positive (DN1), CD44 + CD25 + double-positive (DN2), CD25 + single-positive (DN3) and CD44 − CD25 − double-negative (DN4) subpopulations ( k ) in DN cells of WT and KO mice (n = 6 mice). l , Numbers of CD19 + , CD4 + , and CD8 + cells in spleens of WT and KO mice (n = 6 mice). m , Percentages of naïve (CD44 lo CD62L hi ), central memory (CD44 hi CD62L hi ; CM) and effector/effector memory (CD44 hi CD62L lo , EFF/EM) CD8 + and CD4 + T cells in spleens of WT and KO mice (n = 6 mice). n , o , Proliferation of naïve WT and KO CD8 + T cells following anti-CD3/CD28 activation measured by CFSE dilution ( n ), and expression of activation markers (CD44, CD69 and CD25) at days 1-2 in vitro ( o ). p , Experimental design related to Fig. . q - s , Percentages of transferred CD45.1/2 + OT-I T cells in dLN and spleen ( q ), and quantification of PD-1, TIM-3, LAG-3, and TOX expression OT-I TILs ( r , s ) at day 14 post-ACT (n = 14 mice). t , Survival curves of tumour-bearing mice after transfer of 5×10 CD45.1/2 + WT and KO OT-I T cells (n = 13 mice). Mice with tumour volumes >1500 mm 3 were euthanized and defined as death. Diagram in b created in BioRender. Li, G. (2025) https://BioRender.com/aicmpdn . Diagram in p created in BioRender. Li, G. (2025) https://BioRender.com/0feebot . Data are presented as mean ± s.e.m. Statistical analyses were determined by unpaired two-tailed Student’s t -test ( q - s ), two-way ANOVA with Tukey’s multiple-comparisons test ( c - g ), two-way ANOVA with Sidak’s multiple-comparisons test ( j - m ), and Log-rank (Mantel-Cox) test ( t ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ns, not significant.

Article Snippet: In this study, CRISPR–Cas9 sgRNA was expressed using pSL21-Thy1.1 or pSL21-mCherry (Addgene, 164410) . sgRNAs were generated by annealing two DNA oligos and then ligated into the pSL21-Thy1.1 or pSL21-mCherry vector after digestion with BbsI.

Techniques: Sequencing, Transduction, In Vitro, Injection, Expressing, Control, CRISPR, Activation Assay, Two Tailed Test