Journal: STAR Protocols

Article Title: Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons

doi: 10.1016/j.xpro.2025.103945

Figure Lengend Snippet: Schematic of CRISPR KI vector design and key elements hU6: Human U6 promoter, amplified from the pMJ117 plasmid (Addgene plasmid #85997; a gift from Jonathan Weissman), drives robust expression of the single guide RNA (sgRNA) in mammalian cells. gRNA Insertion Site: Site cleaved by the BbsI restriction enzyme to allow insertion of the sgRNA sequence of interest. Scaffold cr1: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ114 plasmid (Addgene plasmid #85995; a gift from Jonathan Weissman), forms the structural component of the sgRNA and facilitates Cas9 binding. mU6: Mouse U6 promoter, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman), drives expression of a second sgRNA cassette. gRNA (DRSR2): sgRNA sequence targeting the donor recognition sequence R2 (DRSR2): 5′-GCGATCGTAATCACCCGAGT-3′,used for homology-independent targeted integration.Scaffold cr2: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman, used in conjunction with the DRSR2-targeting gRNA to support Cas9 function. DRSR2: Target site recognized by the gRNA: DRSR2, 5′-GCGATCGTAATCACCCGAGTGGG-3′ to enable targeted cleavage and integration of the KI cassette. Linker: Flexible amino acid linker (N-GGGGSGGGGSGGGGS-C) inserted between the targeted protein and oScarlet to minimize steric hindrance and preserve protein function. Sequence Between the Linker and oScarlet: Distinct sequences were inserted between the linker and oScarlet to preserve the correct reading frame in each construct. The sequences are as follows: ORF-0, 5′-CCTCGA-3’; ORF-1, 5′-CTCGA-3’; ORF-2, 5′-CGCTCGA-3’.oScarlet: Codon-optimized red fluorescent protein (RFP) used as a KI tag, It enables visualization of the tagged endogenous protein in live or fixed cells, amplified from the pAAV-Ef1a-oScarlet plasmid (Addgene plasmid #137135; gift from Karl Deisseroth). X: Sequence containing multiple stop codons in all three reading frames to ensure translational termination where appropriate.

Article Snippet: Schematic of CRISPR KI vector design and key elements hU6: Human U6 promoter, amplified from the pMJ117 plasmid (Addgene plasmid #85997; a gift from Jonathan Weissman), drives robust expression of the single guide RNA (sgRNA) in mammalian cells. gRNA Insertion Site: Site cleaved by the BbsI restriction enzyme to allow insertion of the sgRNA sequence of interest.

Techniques: CRISPR, Plasmid Preparation, Amplification, Expressing, Sequencing, Binding Assay, Construct

Journal: Science advances

Article Title: EIF3D safeguards the homeostasis of key signaling pathways in human primed pluripotency.

doi: 10.1126/sciadv.adq5484

Figure Lengend Snippet: Fig. 1. EIF3D is essential for maintaining primed pluripotency. (A) Rank plot from CRISPRi screening. Red and blue dots represent genes significantly increased or decreased (±1 SD) following 8 and 16 days of KD, respectively. n = 3. The full list is provided in data S1 and S2. (B) Protein expression in undifferentiated and differentiating PSCs (10 and 20 days post-FGF withdrawal) and HDFs. Vinculin (VCL) was used as a loading control. (C) Representative images of control and EIF3D KD iPSCs, 5 days post- KD induction. Scale bars, 100 μm. (D) Cell counts on days 3 (P = 4.70 × 10−4) and 5 (P = 3.53 × 10−7) post-KD induction (mean ± SD, n = 6). P values determined via unpaired t test. (E) Cell cycle phase distribution (mean ± SD, n = 3). G1: P = 2.14 × 10−3; S: P = 4.43 × 10−7; G2/M: P = 1.99 × 10−4, calculated by unpaired t test. (F) RNA expression of pluripotency and p53-related genes during EIF3D KD. n = 3. (G) Expression of pluripotency and p53-associated proteins during EIF3D KD. d, days; FC, fold change.

Article Snippet: Genome- wide CRISPRi screens Ten micrograms of the genome- wide CRISPRi library hCRISPRi- v2 (courtesy of J. Weissman; Addgene, #83969) which contains five sgRNA for each of 18,905 mRNAs along with 3.75 μg of psPAX2 (courtesy of D. Trono; Addgene, #12260) and 1.25 μg of pMD2.G (courtesy of D. Trono; Addgene, #12259) were transfected into 293T/17 cells (passage 27) using TransIT- Lenti Transfection Reagent (Mirus).

Techniques: Expressing, Control, RNA Expression

Journal: STAR Protocols

Article Title: Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons

doi: 10.1016/j.xpro.2025.103945

Figure Lengend Snippet: Schematic of CRISPR KI vector design and key elements hU6: Human U6 promoter, amplified from the pMJ117 plasmid (Addgene plasmid #85997; a gift from Jonathan Weissman), drives robust expression of the single guide RNA (sgRNA) in mammalian cells. gRNA Insertion Site: Site cleaved by the BbsI restriction enzyme to allow insertion of the sgRNA sequence of interest. Scaffold cr1: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ114 plasmid (Addgene plasmid #85995; a gift from Jonathan Weissman), forms the structural component of the sgRNA and facilitates Cas9 binding. mU6: Mouse U6 promoter, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman), drives expression of a second sgRNA cassette. gRNA (DRSR2): sgRNA sequence targeting the donor recognition sequence R2 (DRSR2): 5′-GCGATCGTAATCACCCGAGT-3′,used for homology-independent targeted integration.Scaffold cr2: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman, used in conjunction with the DRSR2-targeting gRNA to support Cas9 function. DRSR2: Target site recognized by the gRNA: DRSR2, 5′-GCGATCGTAATCACCCGAGTGGG-3′ to enable targeted cleavage and integration of the KI cassette. Linker: Flexible amino acid linker (N-GGGGSGGGGSGGGGS-C) inserted between the targeted protein and oScarlet to minimize steric hindrance and preserve protein function. Sequence Between the Linker and oScarlet: Distinct sequences were inserted between the linker and oScarlet to preserve the correct reading frame in each construct. The sequences are as follows: ORF-0, 5′-CCTCGA-3’; ORF-1, 5′-CTCGA-3’; ORF-2, 5′-CGCTCGA-3’.oScarlet: Codon-optimized red fluorescent protein (RFP) used as a KI tag, It enables visualization of the tagged endogenous protein in live or fixed cells, amplified from the pAAV-Ef1a-oScarlet plasmid (Addgene plasmid #137135; gift from Karl Deisseroth). X: Sequence containing multiple stop codons in all three reading frames to ensure translational termination where appropriate.

Article Snippet: Scaffold cr1: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ114 plasmid (Addgene plasmid #85995; a gift from Jonathan Weissman), forms the structural component of the sgRNA and facilitates Cas9 binding. mU6: Mouse U6 promoter, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman), drives expression of a second sgRNA cassette. gRNA (DRSR2): sgRNA sequence targeting the donor recognition sequence R2 (DRSR2): 5′-GCGATCGTAATCACCCGAGT-3′,used for homology-independent targeted integration.Scaffold cr2: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman, used in conjunction with the DRSR2-targeting gRNA to support Cas9 function.

Techniques: CRISPR, Plasmid Preparation, Amplification, Expressing, Sequencing, Binding Assay, Construct

Journal: PLOS Genetics

Article Title: CRISPRi screen of long non-coding RNAs identifies LINC03045 regulating glioblastoma invasion

doi: 10.1371/journal.pgen.1011314

Figure Lengend Snippet: A) Schematic of the screening protocol. The CRISPRi library was packaged into lentivirus and transduced into U87 cells stably expressing dCas9-KRAB, selected for to obtain 1000 fold coverage/sgRNA, and seeded onto Matrigel Boyden chambers. After 24 hours, cells in the top chamber were harvested and processed for deep sequencing. Created by Biorender.com . B) Volcano plot of invasion phenotype and negative log 10 (Mann-Whitney p-value). Screen replicates were averaged and the top 3 sgRNAs for each lncRNA compared to non-targeting controls were used to determine screen hits. The dashed lines represent thresholds to determine screen hits.

Article Snippet: The U87-specific CRISPR inhibition (CRISPRi) library, derived from the human CRISPRi non-coding library (CRinCL) (Addgene #86542, a gift from Jonathan Weissman) by selecting sub-libraries that targeted all expressed lncRNAs in U87 [ ], was used to generate the sgRNA pool used for screening.

Techniques: Stable Transfection, Expressing, Sequencing, MANN-WHITNEY

Journal: PLOS Genetics

Article Title: CRISPRi screen of long non-coding RNAs identifies LINC03045 regulating glioblastoma invasion

doi: 10.1371/journal.pgen.1011314

Figure Lengend Snippet: A) qPCR of CRISPRi knockdown (KD) of the top two sgRNAs for LINC03045 in U87 cells. All expression levels were normalized to the housekeeping gene RPLP0 . KD cells were compared to the non-targeting sgRNA control. Data are expressed as mean ± SD for replicates. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 B) An MTT assay showing no change in proliferation after KD of LINC03045 by CRISPRi in U87 cells. KD cells were compared to the non-targeting sgRNA control. Data are expressed as mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. C) Representative images of invasion assay through a Matrigel-coated Boyden chamber after KD of LINC03045 by CRISPRi in U87 cells. The bar graph represents the number of invaded cells per field counted, with KD cells compared to the non-targeting sgRNA control. Data are expressed as mean ± SEM of three independent experiments with 9 fields imaged per experiment. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: The U87-specific CRISPR inhibition (CRISPRi) library, derived from the human CRISPRi non-coding library (CRinCL) (Addgene #86542, a gift from Jonathan Weissman) by selecting sub-libraries that targeted all expressed lncRNAs in U87 [ ], was used to generate the sgRNA pool used for screening.

Techniques: Knockdown, Expressing, Control, MTT Assay, Invasion Assay

Journal: STAR Protocols

Article Title: Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons

doi: 10.1016/j.xpro.2025.103945

Figure Lengend Snippet: Schematic of CRISPR KI vector design and key elements hU6: Human U6 promoter, amplified from the pMJ117 plasmid (Addgene plasmid #85997; a gift from Jonathan Weissman), drives robust expression of the single guide RNA (sgRNA) in mammalian cells. gRNA Insertion Site: Site cleaved by the BbsI restriction enzyme to allow insertion of the sgRNA sequence of interest. Scaffold cr1: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ114 plasmid (Addgene plasmid #85995; a gift from Jonathan Weissman), forms the structural component of the sgRNA and facilitates Cas9 binding. mU6: Mouse U6 promoter, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman), drives expression of a second sgRNA cassette. gRNA (DRSR2): sgRNA sequence targeting the donor recognition sequence R2 (DRSR2): 5′-GCGATCGTAATCACCCGAGT-3′,used for homology-independent targeted integration.Scaffold cr2: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman, used in conjunction with the DRSR2-targeting gRNA to support Cas9 function. DRSR2: Target site recognized by the gRNA: DRSR2, 5′-GCGATCGTAATCACCCGAGTGGG-3′ to enable targeted cleavage and integration of the KI cassette. Linker: Flexible amino acid linker (N-GGGGSGGGGSGGGGS-C) inserted between the targeted protein and oScarlet to minimize steric hindrance and preserve protein function. Sequence Between the Linker and oScarlet: Distinct sequences were inserted between the linker and oScarlet to preserve the correct reading frame in each construct. The sequences are as follows: ORF-0, 5′-CCTCGA-3’; ORF-1, 5′-CTCGA-3’; ORF-2, 5′-CGCTCGA-3’.oScarlet: Codon-optimized red fluorescent protein (RFP) used as a KI tag, It enables visualization of the tagged endogenous protein in live or fixed cells, amplified from the pAAV-Ef1a-oScarlet plasmid (Addgene plasmid #137135; gift from Karl Deisseroth). X: Sequence containing multiple stop codons in all three reading frames to ensure translational termination where appropriate.

Article Snippet: Scaffold cr1: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ114 plasmid (Addgene plasmid #85995; a gift from Jonathan Weissman), forms the structural component of the sgRNA and facilitates Cas9 binding. mU6: Mouse U6 promoter, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman), drives expression of a second sgRNA cassette. gRNA (DRSR2): sgRNA sequence targeting the donor recognition sequence R2 (DRSR2): 5′-GCGATCGTAATCACCCGAGT-3′,used for homology-independent targeted integration.Scaffold cr2: Codon-optimized sgRNA scaffold sequence, amplified from the pMJ179 plasmid (Addgene plasmid #85996; a gift from Jonathan Weissman, used in conjunction with the DRSR2-targeting gRNA to support Cas9 function.

Techniques: CRISPR, Plasmid Preparation, Amplification, Expressing, Sequencing, Binding Assay, Construct