Journal: PLoS ONE

Article Title: Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

doi: 10.1371/journal.pone.0216674

Figure Lengend Snippet: (A) Schematic representation of the mouse and human Tyr loci and the CRISPR/Cas9 experimental design of the two RNA guides are represented in the exon 1 sequence. SDE-sgRNAs match the splice site between exon 1 and intron 1–2. IE-sgRNAs target a central position at the coding sequence of exon 1. (B) Schematic representation of the mouse and human ATM loci and the CRISPR/Cas9 experimental design the two RNA guides are represented in the exon 10 sequence. SDE-sgRNAs match the splice site between exon 10 and intron 10–11, and IE-sgRNAs target a coding sequence of exon 10. (C) Schematic representation of the human ABL-1 locus and the CRISPR/Cas9 experimental design the two RNA guides. SDE-sgRNAs match the splice site between exon 4 and intron 4–5, and IE-sgRNAs target a coding sequence of exon 6. Sequences of each SDE-sgRNA are represented (blue line) and its expected cleavage point (blue arrowhead) at the splice donor sequence (red dotted box). Also, several candidates to SDE-sgRNAs are listed with its respective scores (red box correspond to selected sgRNAs).

Article Snippet: pX458 (Addgene plasmid # 48138)[ ], which contains the coding sequence of Cas9 nuclease and GFP, and a cloning site for sgRNA sequence, was digested with BpiI (NEB).

Techniques: CRISPR, Sequencing

Journal: PLoS ONE

Article Title: Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

doi: 10.1371/journal.pone.0216674

Figure Lengend Snippet: (A) Fluorescent microscopy of cells electroporated with empty px480 vector (controls) and carrying each RNA guides. (B) Sequences of CRISPR/Cas9 edited cells through IE-sgRNA (red box) and SDE-sgRNA (blue box). Edited cells showed a mixture of sequences around the expected cleavage point for each sgRNA.

Article Snippet: pX458 (Addgene plasmid # 48138)[ ], which contains the coding sequence of Cas9 nuclease and GFP, and a cloning site for sgRNA sequence, was digested with BpiI (NEB).

Techniques: Microscopy, Plasmid Preparation, CRISPR

Journal: PLoS ONE

Article Title: Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

doi: 10.1371/journal.pone.0216674

Figure Lengend Snippet: Graphic NGS analysis of CRISPR/Cas9-mediated edition of Tyr locus in mouse blastocysts. Genotyping of embryos microinjected with sgRNAs targeting Tyr gene, by NGS, revealed that only 67.57% of edited sequences from embryos microinjected with IE-m Tyr sgRNA correspond to null alleles, while 100% SDE-m Tyr sgRNA-modified alleles gave rise to null alleles. Black and gray circles correspond to null and functional alleles, respectively, while the background indicates the type of mutation (dark blue: splice donor site in-frame and/or frameshift; light blue: frameshift).

Article Snippet: pX458 (Addgene plasmid # 48138)[ ], which contains the coding sequence of Cas9 nuclease and GFP, and a cloning site for sgRNA sequence, was digested with BpiI (NEB).

Techniques: CRISPR, Modification, Functional Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

doi: 10.1093/nar/gkaa561

Figure Lengend Snippet: Delivery of the first generation of the two-component lentivector nanoparticles carrying the Cas9 nuclease protein and a template for the U6-sgRNA expression cassette (VECTR-Cas(sgGFP)) to human HEK293-EGFP cells. ( A ) Design of the constructs to generate the lentivector particles. Cas9 was fused to the C-terminus of Vpr containing an authentic HIV-1 protease cleavage site (CTLNF/PISPI; Vpr.Prot.Cas9). The U6-sgRNA expression cassette was incorporated into a lentiviral expression vector (Lenti(sgRNA)). The packaging construct (psPAX2) encodes the structural and enzymatic components of virions. The VSV.G envelope protein was used to pseudotype and stabilize viral particles (pHCMV-G). Efficient nuclear export and colocalization of mRNA for translation were supported by adding the Rev-responsive element (RRE) to the constructs and by overexpressing Rev during virion production (pRSV-Rev). Gag-Pol subunits: matrix (MA), capsid (CA), nucleocapsid (NC), p6, protease (PR), reverse transcriptase (RT) and integrase (IN). Packaging signal (ψ); promoters (CMV, CAG, RSV, U6 and EFS), polyadenylation signal (pA), posttranscriptional regulatory element (WPRE). ( B ) The EGFP gene disruption in HEK293-EGFP cells after transduction with the first generation of the two-component lentivector (VECTR-Cas(sgGFP); red entry) or a control LentiCRISPRv2(sgGFP) (blue entry).

Article Snippet: A pVpr.Prot.Cas9 plasmid was constructed by Gibson assembly of a gBlock ordered from IDT (containing Vpr-, protease cleavage site-, and SV40 nuclear localization signal–coding sequence) and two PCR products containing Cas9 coding sequence and Rev-responsive element (RRE), respectively.

Techniques: Expressing, Construct, Plasmid Preparation, Transduction

Journal: Nucleic Acids Research

Article Title: Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

doi: 10.1093/nar/gkaa561

Figure Lengend Snippet: Enhanced packaging of the Vpr.Prot.Cas9 fusion protein produced from the transcript containing the Rev responsive element (RRE). ( A ) Schematic representation of the expression cassettes encoding Vpr.Prot.Cas9 fusion protein. The transcripts encoding Cas9 contain either the Rev responsive element (RRE) or the constitutive transport element (CTE). ( B ) The constructs were co-transfected into HEK293T cells together with psPAX2 (encodes structural and enzymatic components of virions from transcript containing the RRE) and with the other plasmids required for virus production as described in the Figure . The presence of viral and heterologous proteins in the cell lysates (cell) and virions (virus) harvested from cell culture supernatant 48 h after transfection was determined by immunoblotting with antibodies as follows. The blot was probed with an antibody specific for the Cas9 protein. Next, the membrane was washed and re-probed with an antibody detecting the p24 (CA) and Pr55 (Gag) proteins to monitor the expression of the viral structural proteins in the lysates and to determine the amounts of virions released from transfected cells. Equivalent loading was confirmed by re-probing with an antibody directed against the HSP90 protein and by Coomassie blue staining of the SDS-PAGE gel after blotting. One representative example from three biological replicates performed in three different weeks is shown; 293T, untransfected cells; M, Color Prestained Standard NEB #P7712. ( C ) A model describing how nuclear export functions affect the packaging of heterologous proteins into virions. Retrovirus assembly and budding is a highly concerted process. It is mediated by numerous, largely undefined spatially and temporally regulated interactions between viral proteins and cellular factors. Previous reports showed that the regulation of the HIV-1 Gag assembly begins as soon as nuclear export factors are deposited onto the transcripts encoding the structural components of HIV-1. Here, we present a model whereby the selection of RNA export pathway modulates the cytosolic fate and function of the transcripts encoding heterologous proteins and facilitates the packaging of non-viral proteins into virions. The nuclear export of both viral and non-viral transcripts via the same pathway facilitates the cytoplasmic co-localization of the transcripts and their translation products. The close proximity of Gag and Vpr.Prot.Cas9 promotes the interaction between the two polyproteins that is required for encapsidation of the fusion protein into virions.

Article Snippet: A pVpr.Prot.Cas9 plasmid was constructed by Gibson assembly of a gBlock ordered from IDT (containing Vpr-, protease cleavage site-, and SV40 nuclear localization signal–coding sequence) and two PCR products containing Cas9 coding sequence and Rev-responsive element (RRE), respectively.

Techniques: Produced, Expressing, Construct, Transfection, Cell Culture, Western Blot, Staining, SDS Page, Selection

Journal: Nucleic Acids Research

Article Title: Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts

doi: 10.1093/nar/gkaa561

Figure Lengend Snippet: Delivery of the second (VECTRv2-Cas(sgGFP)) or third (VECTRv3-Cas(sgGFP)) generation of the two-component lentivector nanoparticles to human HEK293-EGFP cells. ( A ) The EGFP gene disruption in HEK293-EGFP cells after transduction with the second generation of the two-component lentivector (VECTRv2-Cas(sgGFP); red entries) or a control LentiCRISPRv2(sgGFP) (blue entry). ( B ) T7 endonuclease I (T7EI) assay to measure the indels in the EGFP gene resulting from transduction with the VECTRv2-Cas(sgGFP), the same vector lacking Vpr.Prot.Cas9 or a control vector pLentiCRISPRv2(sgGFP). The frequency of indel formation was calculated as described in the methods section. Please note that it may decrease the actual editing efficiency for highly efficient editing. ( C ) Mutant sequences at the EGFP locus and their frequencies, as determined by SYNTHEGO analysis of Sanger sequencing of a PCR product amplified from VECTRv2-Cas(sgGFP)-transduced HEK293-EGFP cells. The 20-nt target sequence is shown with a blue background. The protospacer adjacent motif (PAM) sequence is shown in blue. ( D ) Comparison of EGFP disruption after transduction with lentiviral particles containing integration-deficient (D64V; VECTRv3-Cas(sgGFP)) or integration-proficient (WT; VECTRv2-Cas(sgGFP)) integrase. ( E ) Time-course analysis of EGFP disruption mediated by the VECTRv3-Cas(sgGFP) or the gene-delivering LentiCRISPRv2(sgGFP). ( F ) A schematic representation of lentivector-mediated delivery of the Cas9 protein and viral RNA containing U6-sgRNA. Cas9 is packaged into virions as a Vpr.Prot.Cas9 fusion polyprotein that is proteolytically cleaved during virion maturation . Following virus entry into a recipient cell , the viral genome is reverse transcribed to DNA and translocated to the nucleus together with Cas9 , where the U6 promoter drives the expression of sgRNA . The nascent sgRNA associates with Cas9 and directs the nuclease to the target site in the genomic DNA (gDNA) for cleavage . (A, D) The mean activities of three replicates are shown. (E) The mean of two replicates are shown. (A, D, E) Error bars, mean ± s.e.m.

Article Snippet: A pVpr.Prot.Cas9 plasmid was constructed by Gibson assembly of a gBlock ordered from IDT (containing Vpr-, protease cleavage site-, and SV40 nuclear localization signal–coding sequence) and two PCR products containing Cas9 coding sequence and Rev-responsive element (RRE), respectively.

Techniques: Transduction, T7EI Assay, Plasmid Preparation, Mutagenesis, Sequencing, Amplification, Expressing

Journal: bioRxiv

Article Title: Enhancing gRNA Transcript levels by Reducing the Scaffold Poly-T Tract for Optimal SpCas9- and SaCas9-mediated Gene Editing

doi: 10.1101/2024.07.19.604224

Figure Lengend Snippet: The modified 3TC scaffold boosts SpCas9 gRNA expression levels, compared to the original 4T scaffold. (A) DNA sequence of the 4T and modified 3TC scaffolds. (B) Relative quantification (RQ) of mDmd gRNA delivered by nucleofection of PX459.V2 (4T) to C2C12 cells, by qRT-PCR. Mean Log 2 RQ ± 95% CI; n=3. One-way ANOVA with Dunnett’s multiple comparisons test performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001. (C, D, E) Comparison of the relative quantities of mDmd Sp gRNA, delivered by PX459.V2, pdg459.V2 (2x4T) and PX459.V3 (3TC), measured by qRT-PCR. Mean Log 2 RQ ± 95% CI; n=3. One-way ANOVA with Tukey’s multiple comparisons test performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: SaCas9-Puro.V2 was generated by replacing the SpCas9 coding sequences of PX459.V2 with SaCas9 coding sequences from PX601 (Addgene #61591).

Techniques: Modification, Expressing, Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Comparison

Journal: bioRxiv

Article Title: Enhancing gRNA Transcript levels by Reducing the Scaffold Poly-T Tract for Optimal SpCas9- and SaCas9-mediated Gene Editing

doi: 10.1101/2024.07.19.604224

Figure Lengend Snippet: Editing efficiencies of high-fidelity SpCas9s with the 3TC scaffold. Comparison of PX459.V2 SpCas9-HF1 (4T), PX459.V3 SpCas9-HF1 (3TC), PX459.V2 eSpCas9(1.1) (4T) and PX459.V3 eSpCas9(1.1) (3TC) plasmids delivered by lipofection at a (A) high and (B) low plasmid dose without puromycin selection in HEK239T cells, assessed by deep amplicon sequencing. Mean ± SEM; n=3. Two-way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) Editing efficiencies of hDMD-B in the G19 gRNA configuration with WT and high-fidelity Sp-Cas9 plasmids delivered by nucleofection with puromycin selection in HEK293Ts. Two-way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, ***p ≤ 0.001.

Article Snippet: SaCas9-Puro.V2 was generated by replacing the SpCas9 coding sequences of PX459.V2 with SaCas9 coding sequences from PX601 (Addgene #61591).

Techniques: Comparison, Plasmid Preparation, Selection, Amplification, Sequencing

Journal: bioRxiv

Article Title: The Drosophila ERG channel seizure plays a role in the neuronal homeostatic stress response

doi: 10.1101/521005

Figure Lengend Snippet: A ) Strategy for the generation of the seiGFP allele by using CRISPR/ Cas9 -dependent DNA editing. Stars represent single nucleotide substitutions in the PAMs of sgRNA sites. B-C) Behavior of seiGFP and wildtype flies in the acute heat assay. n=12. Data was analyzed using Student’s t -test and presented as mean ±SEM.

Article Snippet: Complementary oligos that correspond to each individual sgRNA (IDT) were cloned into the pDCC6 plasmid (a gift from Peter Duchek, Addgene plasmid # 59985), which also includes the coding sequence for Cas9 [ ], by using the BbsI restriction enzyme (NEB).

Techniques: CRISPR

Journal: Nature neuroscience

Article Title: Central role for PICALM in amyloid–β blood–brain barrier transcytosis and clearance

doi: 10.1038/nn.4025

Figure Lengend Snippet: a , qRT–PCR and western blot analysis of PICALM in control and AD endothelial monolayers. b , Diminished Aβ40 (1 nM) transcytosis across AD–derived endothelium and reversal by adenoviral–mediated Ad. PICALM re–expression. Ad. mLRP1, LRP1 minigene. Mean ± s.e.m., from 8 isolates in triplicate for control and AD monolayers. c , Diagram of CRISPR/Cas9–based generation of isogenic iPSC lines homozygous for the protective (A) or non–protective (G) allele of rs3851179 . gRNA = guide RNA. d , SspI restriction digest of PCR products from iPSC genomic DNA at rs3851179 region. *denotes the CRISPR–Cas9 modified iPSC line. e , Sanger sequencing of iPSCs at rs3851179 confirming independent isogenic lines homozygous for either the G or A variant. f , FACS dot plot showing 15.7% of iPSC–derived endothelial cells via embryoid body (EB) formation are positive for endothelial markers CD31 and VE–Cadherin. g , iPSC–derived endothelial cells co–cultured with pericyte conditioned media form monolayer in vitro with ZO–1 positive tight junctions (green). Bar=100 µm. h–i , qRT–PCR and western blot analysis of PICALM ( h ) and Aβ40 (1 nM) transcytosis ( i ) in human iPSC–derived endothelial monolayers carrying the protective rs3851179 (AA) variant and the non–protective rs3851179 (GG) variant. In h–i , means ± s.e.m., from 6 cultures for each rs3851179 variant in triplicates.

Article Snippet: These cells were suspended with Nucleofector solution and Supplement (Lonza) and mixed with gRNA expression vector, Cas9 coding pSpCas9(BB)-2A-Puro (Addgene) and double-strand plasmid DNA for donor.

Techniques: Quantitative RT-PCR, Western Blot, Control, Derivative Assay, Expressing, CRISPR, Modification, Sequencing, Variant Assay, Cell Culture, In Vitro