pspcas13b crrna backbone  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells

doi: 10.1093/nar/gkag112

Figure Lengend Snippet: Use uSpyCLIP to identify the off-target binding sites of the dPspCas13b-gRNA and dRfxCas13d-gRNA complexes. ( A ) Schematic representation of the strategy for identifying the off-target binding sites of dCas13-gRNA complex. Take dCas13d as an example. ( B ) Schematic for constructs expressing Cas13 effectors and corresponding gRNA cassettes. ( C ) Western blot of protein extracts from Lenti-X 293T cells stably expressing HA- and Spy-tagged dPspCas13b or dRfxCas13d and their catalytically active counterparts. ( D ) Immunocytochemistry of dPspCas13b and dRfxCas13d proteins showing localization and expression. ( E ) RNA knockdown activity of endogenous EZH2 or TP53 mRNA in Lenti-X 293T cells expressing HA- and Spy-tagged PspCas13b or dPspCas13b. ( F ) RNA knockdown activity of endogenous EZH2 or TP53 mRNA in Lenti-X 293T cells expressing HA- and Spy-tagged RfxCas13d or dRfxCas13d. gNT, non-targeting gRNA; EZH2-g1 and EZH2-g2, two independent gRNAs targeting EZH2 mRNA; TP53-g1, gRNA targeting TP53 mRNA. Values are shown as mean ± SD with n = 3.

Article Snippet: For PspCas13b gRNA constructs, individual gRNA spacers were cloned into the PspCas13b crRNA backbone (Addgene #103854, a gift from Feng Zhang), which contains a PspCas13b crRNA direct repeat sequence and two BbsI restriction sites for cloning of the spacer sequence.

Techniques: Binding Assay, Construct, Expressing, Western Blot, Stable Transfection, Immunocytochemistry, Knockdown, Activity Assay

Journal: Nucleic Acids Research

Article Title: Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells

doi: 10.1093/nar/gkag112

Figure Lengend Snippet: dPspCas13b preferentially binds to RNA sites with long stem-loop structures. ( A ) Top HOMER motif identified for all dPspCas13b binding sites or the top 500 dPspCas13b binding sites. ( B ) The frequency of base pairing for all dPspCas13b binding sites or the top 500 dPspCas13b binding sites. RBFOX2 and SLBP were used as controls. ( C ) Schematic of the PspCas13b crRNA direct repeat region. ( D ) The sequence composition and secondary structure of three representative gRNA-independent off-target sites bound by dPspCas13b. The secondary structure was calculated using RNAfold. ( E ) EMSA assays examining binding of His-tagged dPspCas13b to Cy5-labeled RNA oligonucleotides corresponding to the identified off-target sites and a scrambled control.

Article Snippet: For PspCas13b gRNA constructs, individual gRNA spacers were cloned into the PspCas13b crRNA backbone (Addgene #103854, a gift from Feng Zhang), which contains a PspCas13b crRNA direct repeat sequence and two BbsI restriction sites for cloning of the spacer sequence.

Techniques: Binding Assay, Sequencing, Labeling, Control

Journal: Nucleic Acids Research

Article Title: Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells

doi: 10.1093/nar/gkag112

Figure Lengend Snippet: Binding and cleavage specificity of Cas13-EZH2-g2 complexes. ( A ) EMSA analysis of 50-nt ssRNA oligonucleotides corresponding to the on-target site of PspCas13b EZH2-g2 (30 consecutive complementary base pairs), an off-target site containing 10 consecutive complementary base pairs, and a negative control lacking complementarity. ( B ) Denaturing gel analysis showing efficient cleavage of the on-target RNA substrate by the PspCas13b-EZH2-g2 complex, whereas no detectable cleavage is observed for the off-target or negative-control substrates. ( C ) EMSA analysis of 50-nt ssRNA substrates corresponding to the on-target site of RfxCas13d EZH2-g2 (30 consecutive complementary base pairs), an off-target site containing seven consecutive complementary base pairs, and a negative control lacking complementarity. ( D ) Denaturing gel analysis demonstrating specific cleavage of the on-target RNA by the RfxCas13d-EZH2-g2 complex, with no cleavage detected for the off-target or negative-control substrate.

Article Snippet: For PspCas13b gRNA constructs, individual gRNA spacers were cloned into the PspCas13b crRNA backbone (Addgene #103854, a gift from Feng Zhang), which contains a PspCas13b crRNA direct repeat sequence and two BbsI restriction sites for cloning of the spacer sequence.

Techniques: Binding Assay, Negative Control

Journal: Nucleic Acids Research

Article Title: Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells

doi: 10.1093/nar/gkag112

Figure Lengend Snippet: gRNA-dependent off-target binding alters the expression of essential genes and induces substantial alterations in cell proliferation. ( A – D ) RT-qPCR and western blot analyses of on-target EZH2 and three gRNA-dependent off-target genes bound by the PspCas13b-EZH2-g2 complex, with comparisons between non-targeting and EZH2-g2 targeting conditions. ( E – H ) RT-qPCR and western blot analyses of on-target EZH2 and three gRNA-dependent off-target genes bound by the RfxCas13d-EZH2-g2 complex, with comparisons between non-targeting and EZH2-g2 targeting conditions. RT-qPCR data are presented as mean ± SD ( n = 3). Relative changes in protein abundance were quantified by densitometric analysis and are indicated as red numbers at the bottom of each panel. ( I ) Proliferation of cells expressing PspCas13b (left panel) or RfxCas13d (right panel) with either targeting gRNAs or a non-targeting control gRNA (gNT), measured by CCK-8 assay. Proliferation rates were measured by absorbance at days 1–5 and normalized to that at day 1. Data are presented as mean ± SD ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001. ns, not significant. ( J ) Apoptosis analysis of cells expressing PspCas13b (left panel) or RfxCas13d (right panel) with either targeting gRNAs or non-targeting control gRNA (gNT). Data are presented as mean ± SD ( n = 3). ns, not significant.

Article Snippet: For PspCas13b gRNA constructs, individual gRNA spacers were cloned into the PspCas13b crRNA backbone (Addgene #103854, a gift from Feng Zhang), which contains a PspCas13b crRNA direct repeat sequence and two BbsI restriction sites for cloning of the spacer sequence.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, CCK-8 Assay