Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic for one-vector lentiviral constructs expressing Cas13 effector and corresponding sgRNA cassette. (B) RNA knockdown activity of endogenous EZH2 mRNA in HEK293FT cells by transient transfection of indicated one-vector lentiviral Cas13 plasmids, each with an empty vector, two independent crRNAs and a non-targeting (NT) crRNA. Values shown as mean ± SD with n = 3. ** p < 0.01. (C) Surviving cell number after puromycin selection for A549 cells infected by indicated one-vector Cas13 lentiviruses, mean ± SEM with n = 3. (D-E) Knockdown activity of EZH2 in A549 cells (D) or T47D cells (E) by one-vector Cas13d lentiviruses, mean ± SD with n = 3. * p < 0.05, ** p < 0.01. (F) Schematic of evaluation assays for one-vector lentiviral Cas13 systems. (G) Virus titer of one-vector Cas13a/b/d lentiviruses, mean ± SEM with n = 3. (H) Surviving cell number against puromycin selection for A549 cells infected with indicated lentiviruses with high MOI, mean ± SEM with n = 3. Mock, uninfected cell group. (I) Lentiviral RNA expression of infected A549 cells by detecting NLS (the common tag for different Cas13 constructs) or PuroR elements, mean ± SD with n = 3. (J) Integrated lentiviral DNA levels from genomic DNA of infected A549 cells, mean ± SD with n = 3. (K-L) Time course examination of lentiviral RNA (K) or integrated DNA (L) post lentiviral infection in A549 cells, mean ± SD with n = 3. See also and Table S1.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, Construct, Expressing, Activity Assay, Transfection, Selection, Infection, RNA Expression

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Western blot of protein extracts from HEK293FT cells transiently expressing different Cas13 effectors in one-vector format. (B) Immunocytochemistry of Cas13 proteins showing localization and expression. Scale bar, 20 µm. (C-D) Surviving cell number after puromycin selection for T47D (C) or LNCaP (D) cells infected by indicated one-vector Cas13 lentiviruses, mean ± SEM with n = 3. (E) Cell viability analysis for HEK293FT cells with transient transfection of indicated one-vector lentiviral constructs, mean ± SEM with n = 3. Vector: lentiv2-w/o Cas9. (F) Cell viability analysis for HEK293FT cells during lentivirus production by transient transfection of one-vector Cas13 along with packaging plasmids, mean ± SEM with n = 3. (G-I) Assessment of surviving cell number (G), lentiviral RNA (H) and integrated lentiviral DNA (I) in A549 cells infected with indicated lentiviruses at low MOI. (J-L) Assessment of surviving cell number (J), lentiviral RNA (K) and integrated lentiviral DNA (L) in A549 cells infected with indicated lentiviruses containing different crRNAs. (M) Evaluation of lentiviral defect for one-vector Cas13 systems under pHAGE-EF1α-puro plasmid backbone.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Western Blot, Expressing, Plasmid Preparation, Immunocytochemistry, Selection, Infection, Transfection, Construct

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic of two-vector lentiviral Cas13 constructs. (B) Schematic of evaluation assays for two-vector lentiviral Cas13 systems. (C-E) Assessment of surviving cell number (C), lentiviral RNA (D) and integrated lentiviral DNA (E) in A549 cells infected with Cas13-only lentiviruses at high MOI. (F-G) Surviving cell number (F) and lentiviral RNA level (G) after puromycin selection for A549 cells infected by Cas13DR lentiviruses of two-vector systems. (H) Schematic of one-vector lentiviral constructs containing Cas13b along with different Cas13DR. No spacer was inserted. (I-K) Assessment of surviving cell number (I), lentiviral RNA (J) and integrated lentiviral DNA (K) in A549 cells infected with indicated lentiviruses. (L) Western blot of protein extracts from A549 cells of untreated (Mock) or infected with indicated lentiviruses. See also and Table S1.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, Construct, Infection, Selection, Western Blot

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A-C) Assessment of surviving cell number (A), lentiviral RNA (B) and integrated lentiviral DNA (C) in A549 cells infected with indicated lentiviruses at low MOI. (D) Effect on cell survival after puromycin selection for A549 cells infected with high or low MOI of Cas13-only lentiviruses of two-vector system under pHAGE-EF1α-puro vector backbone, mean ± SEM with n = 3. (E) Assessment of surviving cell number, lentiviral RNA and integrated lentiviral DNA levels in A549 cells infected with indicated lentiviruses at high or low MOI. (F) Schematic of co-infection assay using indicated lentiviruses to evaluate lentiviral defect. Red arrowheads indicate detection region by qPCR at RNA and DNA levels. (G-J) Assessment of surviving cell number (G) and lentiviral RNA levels using Cas13 (H), Cas13DR (I) or PuroR (J) elements in A549 cells co-infected with indicated lentiviruses. (K-L) Cycloheximide (CHX) chase analysis for determining Cas13 protein stability in HEK293FT cells. Western blot showing the protein levels at indicated time points (K) and quantified band intensity was plotted in (L).

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Infection, Selection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic of one-vector lentiviral Cas13a-WT and different RNase-deficient variant constructs. No spacer was inserted. (B) Western blot of protein extracts from HEK293FT cells transiently transfected with indicated constructs. (C) Virus titer of indicated lentiviruses, mean ± SEM with n = 3. (D-F) Assessment of surviving cell number (D), lentiviral RNA (E) and integrated lentiviral DNA (F) in A549 cells infected with indicated lentiviruses at high or low MOI. (G) Heatmap of differentially expressed genes in Cas13a variant-expressing samples. (H) Viral process-related GO categories enriched among up-regulated genes by Cas13a variants. (I) Heatmap of representative up-regulated or de-repressed genes in RNase-deficient Cas13a variants compared with Cas13a-WT. Viral process-related genes are marked by red asterisk. (J) Schematic for evaluating lentiviral capacity of HSPA6 knockdown by siRNA in HEK293FT cells. Lentiviruses produced in either control (siCtrl) or HSPA6 knockdown HEK293FT cells were used to infect A549 cells and lentiviral RNA or DNA level was determined. (K) Assessment of HSPA6 knockdown efficiency in HEK293FT cells (left), lentiviral DNA (center) and lentiviral RNA level (right) in A549 cells by measuring a region spanning NLS and FLAG tag in lentiCRISPR v2 vector, mean ± SD with n = 3. See also , Tables S1 and S3.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, Variant Assay, Construct, Western Blot, Transfection, Infection, Expressing, Produced, FLAG-tag

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic of lentiviral vectors in one-vector or two-vector format for CRISPR-Cas13 screens. (B) Schematic of procedures for Cas13-based cell fitness screens. (C) The distribution of crRNA abundance across samples of different time points compared to plasmid pool for one-vector or two-vector Cas13d screen. (D) The β score distribution of core essential genes versus other genes at early (Day 5) or end point (Day 33) of the screens. (E) Scatter plot of β score change for target transcripts between two biological replicates in Cas13d-null or Cas13d-expressing conditions of two-vector Cas13d screens. Red dots indicate core essential genes. (F) Rank-ordered presentation of targeted protein-coding genes for their essentiality according to β score. The top selected genes were highlighted. (G) Rank-ordered presentation of targeted lncRNA genes for their essentiality according to β score. The top selected lncRNAs were highlighted. (H) Schematic of lasso-based machine learning model to determine crRNA-specific features underlying lentiviral defect. (I) A logo for highly represented sequence features embedded in early depleted crRNAs. (J) Receiver Operator Characteristic (ROC) curves showing the predictive power of lasso model using 5-fold cross validation. See also ; Table S4 and S5.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, CRISPR, Expressing, Sequencing

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic for one-vector lentiviral constructs expressing Cas13 effector and corresponding sgRNA cassette. (B) RNA knockdown activity of endogenous EZH2 mRNA in HEK293FT cells by transient transfection of indicated one-vector lentiviral Cas13 plasmids, each with an empty vector, two independent crRNAs and a non-targeting (NT) crRNA. Values shown as mean ± SD with n = 3. ** p < 0.01. (C) Surviving cell number after puromycin selection for A549 cells infected by indicated one-vector Cas13 lentiviruses, mean ± SEM with n = 3. (D-E) Knockdown activity of EZH2 in A549 cells (D) or T47D cells (E) by one-vector Cas13d lentiviruses, mean ± SD with n = 3. * p < 0.05, ** p < 0.01. (F) Schematic of evaluation assays for one-vector lentiviral Cas13 systems. (G) Virus titer of one-vector Cas13a/b/d lentiviruses, mean ± SEM with n = 3. (H) Surviving cell number against puromycin selection for A549 cells infected with indicated lentiviruses with high MOI, mean ± SEM with n = 3. Mock, uninfected cell group. (I) Lentiviral RNA expression of infected A549 cells by detecting NLS (the common tag for different Cas13 constructs) or PuroR elements, mean ± SD with n = 3. (J) Integrated lentiviral DNA levels from genomic DNA of infected A549 cells, mean ± SD with n = 3. (K-L) Time course examination of lentiviral RNA (K) or integrated DNA (L) post lentiviral infection in A549 cells, mean ± SD with n = 3. See also and Table S1.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, Construct, Expressing, Activity Assay, Transfection, Selection, Infection, RNA Expression

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Western blot of protein extracts from HEK293FT cells transiently expressing different Cas13 effectors in one-vector format. (B) Immunocytochemistry of Cas13 proteins showing localization and expression. Scale bar, 20 µm. (C-D) Surviving cell number after puromycin selection for T47D (C) or LNCaP (D) cells infected by indicated one-vector Cas13 lentiviruses, mean ± SEM with n = 3. (E) Cell viability analysis for HEK293FT cells with transient transfection of indicated one-vector lentiviral constructs, mean ± SEM with n = 3. Vector: lentiv2-w/o Cas9. (F) Cell viability analysis for HEK293FT cells during lentivirus production by transient transfection of one-vector Cas13 along with packaging plasmids, mean ± SEM with n = 3. (G-I) Assessment of surviving cell number (G), lentiviral RNA (H) and integrated lentiviral DNA (I) in A549 cells infected with indicated lentiviruses at low MOI. (J-L) Assessment of surviving cell number (J), lentiviral RNA (K) and integrated lentiviral DNA (L) in A549 cells infected with indicated lentiviruses containing different crRNAs. (M) Evaluation of lentiviral defect for one-vector Cas13 systems under pHAGE-EF1α-puro plasmid backbone.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Western Blot, Expressing, Plasmid Preparation, Immunocytochemistry, Selection, Infection, Transfection, Construct

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic of two-vector lentiviral Cas13 constructs. (B) Schematic of evaluation assays for two-vector lentiviral Cas13 systems. (C-E) Assessment of surviving cell number (C), lentiviral RNA (D) and integrated lentiviral DNA (E) in A549 cells infected with Cas13-only lentiviruses at high MOI. (F-G) Surviving cell number (F) and lentiviral RNA level (G) after puromycin selection for A549 cells infected by Cas13DR lentiviruses of two-vector systems. (H) Schematic of one-vector lentiviral constructs containing Cas13b along with different Cas13DR. No spacer was inserted. (I-K) Assessment of surviving cell number (I), lentiviral RNA (J) and integrated lentiviral DNA (K) in A549 cells infected with indicated lentiviruses. (L) Western blot of protein extracts from A549 cells of untreated (Mock) or infected with indicated lentiviruses. See also and Table S1.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, Construct, Infection, Selection, Western Blot

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A-C) Assessment of surviving cell number (A), lentiviral RNA (B) and integrated lentiviral DNA (C) in A549 cells infected with indicated lentiviruses at low MOI. (D) Effect on cell survival after puromycin selection for A549 cells infected with high or low MOI of Cas13-only lentiviruses of two-vector system under pHAGE-EF1α-puro vector backbone, mean ± SEM with n = 3. (E) Assessment of surviving cell number, lentiviral RNA and integrated lentiviral DNA levels in A549 cells infected with indicated lentiviruses at high or low MOI. (F) Schematic of co-infection assay using indicated lentiviruses to evaluate lentiviral defect. Red arrowheads indicate detection region by qPCR at RNA and DNA levels. (G-J) Assessment of surviving cell number (G) and lentiviral RNA levels using Cas13 (H), Cas13DR (I) or PuroR (J) elements in A549 cells co-infected with indicated lentiviruses. (K-L) Cycloheximide (CHX) chase analysis for determining Cas13 protein stability in HEK293FT cells. Western blot showing the protein levels at indicated time points (K) and quantified band intensity was plotted in (L).

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Infection, Selection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic of one-vector lentiviral Cas13a-WT and different RNase-deficient variant constructs. No spacer was inserted. (B) Western blot of protein extracts from HEK293FT cells transiently transfected with indicated constructs. (C) Virus titer of indicated lentiviruses, mean ± SEM with n = 3. (D-F) Assessment of surviving cell number (D), lentiviral RNA (E) and integrated lentiviral DNA (F) in A549 cells infected with indicated lentiviruses at high or low MOI. (G) Heatmap of differentially expressed genes in Cas13a variant-expressing samples. (H) Viral process-related GO categories enriched among up-regulated genes by Cas13a variants. (I) Heatmap of representative up-regulated or de-repressed genes in RNase-deficient Cas13a variants compared with Cas13a-WT. Viral process-related genes are marked by red asterisk. (J) Schematic for evaluating lentiviral capacity of HSPA6 knockdown by siRNA in HEK293FT cells. Lentiviruses produced in either control (siCtrl) or HSPA6 knockdown HEK293FT cells were used to infect A549 cells and lentiviral RNA or DNA level was determined. (K) Assessment of HSPA6 knockdown efficiency in HEK293FT cells (left), lentiviral DNA (center) and lentiviral RNA level (right) in A549 cells by measuring a region spanning NLS and FLAG tag in lentiCRISPR v2 vector, mean ± SD with n = 3. See also , Tables S1 and S3.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, Variant Assay, Construct, Western Blot, Transfection, Infection, Expressing, Produced, FLAG-tag

Journal: bioRxiv

Article Title: Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

doi: 10.1101/2022.05.14.491940

Figure Lengend Snippet: (A) Schematic of lentiviral vectors in one-vector or two-vector format for CRISPR-Cas13 screens. (B) Schematic of procedures for Cas13-based cell fitness screens. (C) The distribution of crRNA abundance across samples of different time points compared to plasmid pool for one-vector or two-vector Cas13d screen. (D) The β score distribution of core essential genes versus other genes at early (Day 5) or end point (Day 33) of the screens. (E) Scatter plot of β score change for target transcripts between two biological replicates in Cas13d-null or Cas13d-expressing conditions of two-vector Cas13d screens. Red dots indicate core essential genes. (F) Rank-ordered presentation of targeted protein-coding genes for their essentiality according to β score. The top selected genes were highlighted. (G) Rank-ordered presentation of targeted lncRNA genes for their essentiality according to β score. The top selected lncRNAs were highlighted. (H) Schematic of lasso-based machine learning model to determine crRNA-specific features underlying lentiviral defect. (I) A logo for highly represented sequence features embedded in early depleted crRNAs. (J) Receiver Operator Characteristic (ROC) curves showing the predictive power of lasso model using 5-fold cross validation. See also ; Table S4 and S5.

Article Snippet: The following plasmid backbones were used in this study: lentiCRISPR v2 (lentiv2, Addgene #52961): a lentiviral vector backbone, U6 promoter expresses Cas9 sgRNA scaffold; EF1α promoter expresses Cas9 and PuroR (puromycin resistance) with P2A linker; lentiGuide-puro (Addgene #52963): an empty lentiviral vector expresses Cas9 sgRNA under U6 promoter with puromycin resistance; lenti-Cas9-blast (Addgene #52962): a lentiviral vector expresses Cas9 protein and blasticidin resistance under EF1α promoter. pHAGE-EF1α-puro: an empty lentiviral vector backbone, EF1α promoter drives expression of inserted cDNA; SV40 promoter expresses PuroR; pcDNA3.1: a derivative of pcDNA3.1-HA (Addgene #128034) vector, without HA tag; lentiv2-w/o Cas9: a derivative of the lentiCRISPR v2 vector with Cas9 open reading frame (ORF) removal; pET-28a(+): bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin site.

Techniques: Plasmid Preparation, CRISPR, Expressing, Sequencing