Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

doi: 10.1111/gtc.12379

Figure Lengend Snippet: Figure 4 Genotyping of founders with different phenotypes with RFLP analyses. (A) A schematic of restriction enzyme-RFLP (RE-RFLP) and RNA-guided engineered nuclease-RFLP (RGEN-RFLP) analysis. (B) Estimation of somatic mutation rates of the target alleles in each phenotype. PCR products of tyr and ltk were cleaved by Cas9 protein with sgRNAs in vitro. PCR prod- ucts of slc45a2 were cleaved by BfaI. Cleaved fragments were analyzed by electrophoresis (MultiNA system; Shimadzu). Upper and lower images show Cas9 protein or BfaI (+) and () gel images converted from electropherograms. PCR products of target regions are shown by black arrowheads. Colored arrowheads indicate cleaved products by Cas9 or BfaI. Some extra bands were observed and thought to be large deletions and insertions. The mutation rates were calculated from the molarity of the uncleaved bands (black arrowheads and other extra bands) and the larger cleaved fragments (blue arrowheads). Wt, wild type; W, weak phe- notype; M, moderate phenotype; S, severe phenotype; PS, severe phenotype by Cas9 protein injection; H, half phenotype; F, full phenotype. Full scan images of RFLP analyses are shown in Figs S5–S8 in Supporting Information.

Article Snippet: Preparation and injection of Cas9 and sgRNA Humanized codon hspCas9 cDNA from pX330 (Addgene, #42230; Cong et al. 2013) containing two NLSs was subcloned into a modified pCS2+ vector (pCS2+-T7/hspCas9; Fig. S12 in Supporting Information).

Techniques: Mutagenesis, In Vitro, Electrophoresis, Injection

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

doi: 10.1111/gtc.12379

Figure Lengend Snippet: Figure 7 Example of target gene analysis according to our workflow. We designed an sgRNA targeting activin receptor-like kinase 2 (alk2). (A) Representative phenotypes produced by Cas9 mRNA and sgRNA (upper panel). Most phenotypes exhibited a partial loss of ventral fin tissue accompanied by the hypertrophic anus. Normal means no alteration in the ventral fin or anus. The total numbers of individuals are shown at the top of each graph. In vitro fertilization (IVF) indicates control embryos without injection. Frequencies of alk2-disrupted phenotypes from three independent experiments are shown in under panel. (B) RGEN-RFLP analy- sis of somatic mutation rates. (C) Sanger DNA sequencing of somatic mutation alleles from five phenotypes. In total, 107 clones were sequenced. (D) Off-target analysis using heteroduplex mobility assay (HMA). Wt, wild type; P, phenotype.

Article Snippet: Preparation and injection of Cas9 and sgRNA Humanized codon hspCas9 cDNA from pX330 (Addgene, #42230; Cong et al. 2013) containing two NLSs was subcloned into a modified pCS2+ vector (pCS2+-T7/hspCas9; Fig. S12 in Supporting Information).

Techniques: Produced, In Vitro, Control, Injection, Mutagenesis, DNA Sequencing, Clone Assay

Journal: BIO-PROTOCOL

Article Title: Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1

doi: 10.21769/bioprotoc.5108

Figure Lengend Snippet: Figure 4. AsCpf1-mediated mutation formation in hPSCs. A. Representative fluorescent micrographs of hPSCs electroporated with the corresponding constructs on the left. B. Disruption of tdTomato expression in H1-tdT cells by CAGGS promoter driven AsCpf1 with a tdT crRNA. A 4-nucleotide deletion was identified in cells that lost tdTomato expression but not in control cells. Scale bar: 100 μm. G. Knockin of hPSC and multiplex genome editing

Article Snippet: Anneal the tdT-targeting crRNA oligos and ligate to CAGGS-AsCpf1-2A-GFP-U6-crRNA-cloning vector (Addgene, #159281) based on the procedures described in section B, giving rise to the CAGGS-AsCpf12A-GFP-U6-tdT-crRNA plasmid (Addgene, #194724).

Techniques: Mutagenesis, Construct, Disruption, Expressing, Control, Knock-In, Multiplex Assay

Journal: BIO-PROTOCOL

Article Title: Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1

doi: 10.21769/bioprotoc.5108

Figure Lengend Snippet: Figure 7. AsCpf1-mediated targeting in the INS locus in hPSCs enables monitoring of INS expression. A. The design of an INS targeting crRNA. The PAM sequence is highlighted in magenta, and the guide sequence is highlighted in yellow. B. The targeting strategy for generating INS luciferase and tdTomato reporter INS- luciferase-tdT allele by knocking the 2A-luciferase-2A-tdt cassette to the 3′ end of the INS coding sequence. C. Southern blots with internal probe (left panel) and external probe (right panel) to identify clones with a single copy of the 2A-luciferase-2A-tdt cassette inserted into the INS locus. Correct clones were labeled in green. The double bands in the control samples for the external probe (right panels) correspond to two wild-type INS alleles of the H1-OCT4-GFP cells. D. Expression of tdTomato in cells during the differentiation of the INS-luciferase- tdT reporter hPSCs toward pancreatic β-like cells. E. Representative confocal micrographs of INS-luciferase- tdT hPSC differentiated β-like cells with anti-tdTomato staining (red) and anti-C-peptide staining (green). F.

Article Snippet: Anneal the tdT-targeting crRNA oligos and ligate to CAGGS-AsCpf1-2A-GFP-U6-crRNA-cloning vector (Addgene, #159281) based on the procedures described in section B, giving rise to the CAGGS-AsCpf12A-GFP-U6-tdT-crRNA plasmid (Addgene, #194724).

Techniques: Expressing, Sequencing, Luciferase, Clone Assay, Labeling, Control, Staining

Journal: Nature Communications

Article Title: Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

doi: 10.1038/s41467-021-22295-w

Figure Lengend Snippet: a Installation (via G•C-to-A•T) of the pathogenic SERPINA1 E342K mutation in HEK293T cells using PE2, PE2*, and Sa KKH PE2*. Editing efficiencies reflect sequencing reads which contain the desired edit. The presence of sgRNAs to promote nicking of the complementary strand is indicated on the x -axis. Results were obtained from three independent experiments and are presented as mean ± SD. b pegRNA used for correction (via A•T-to-G•C) of the E342K mutation includes a spacer sequence, a sgRNA scaffold, an RT template including edited bases (red), and a primer-binding site (PBS). A PAM mutation (AGG to AAG) was introduced to reduce re-cutting of the locus that results in a synonymous codon change. c Evaluating PE expression and subcellular distribution in mouse liver. FVB mice were injected with PE2 or PE2* expression plasmids containing a 3xHA-tag. IHC was performed with an HA-tag antibody. Scale bars: 100 µm (×20 lens). d Average percentage of HA-tag signal from the nucleus. Each dot is the average calculated signal intensity within the nucleus relative to the whole cell from all positive cells in a microscopic image. Numbers are mean ± sem ( n = 20 total images from 3 mice). e Schematic overview of correction strategy of the SERPINA1 E342K mutation in PiZ transgenic mouse model of AATD. Prime editor, pegRNA, and nicking sgRNA plasmid were delivered by hydrodynamic tail-vein injection. f Comparison of the efficiency of K342E correction and indels in mouse livers in PE2 or PE2* treatment groups. Precise editing is defined as the fraction of sequencing reads with both A to G prime editing and synonymous PAM modification. Results were obtained from three mice and presented as mean ± SD. **P < 0.01, * **P < 0.001, * ***P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene: backbone plasmids used for pegRNA and sgRNA cloning are addgene #122089 and 122090.

Techniques: Mutagenesis, Sequencing, Binding Assay, Expressing, Injection, Transgenic Assay, Plasmid Preparation, Comparison, Modification

Journal: Nature Communications

Article Title: Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

doi: 10.1038/s41467-021-22295-w

Figure Lengend Snippet: a pegRNA used for installation (via C•G-to-T•A) of the oncogenic S45F in Ctnnb1 in mouse liver. b Schematic overview of the somatic cell editing strategy to drive tumor formation. Prime editor (PE2 or PE2*), pegRNA for Ctnnb1 S45F, and nicking sgRNA plasmids were delivered by hydrodynamic tail-vein injection along with the MYC transposon and transposase plasmids. c Representative images of tumor burden in mouse liver with PE2 or PE2*. d Tumor numbers in the livers of mice 25 days after injection with PE2 or PE2*. The Control group was pegRNA only. Results were obtained from 4 mice and presented as mean ± SD. e Sanger sequencing from normal liver and representative tumors. The dashed box denotes C to T editing in tumors. *P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. f Schematic of Ctnnb1 S45 deletion strategy using PE2* (S45del). pegRNA used for 3 bp deletion (TCC) is shown. g PE2* treatment leads to oncogenic activation of Ctnnb1 . Prime editor (PE2*), pegRNA ( Ctnnb1 S45del or SERPINA1), and nicking sgRNA plasmids were delivered by hydrodynamic tail-vein injection along with the MYC transposon and transposase plasmids. Mice treated with the peg Ctnnb1 S45del ( n = 4) displayed a large number of liver tumors whereas mice treated with pegSERPINA1 as a control displayed no noticeable oncogenic lesions. beta-Catenin (CTNNB1) IHC staining was performed. Scale bars: 100 µm (×20 lens). h Prime editing efficiency and indels determined by targeted deep sequencing in control liver and representative tumors. Results were obtained from 3 tumors in each group and presented as mean ± SD.

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene: backbone plasmids used for pegRNA and sgRNA cloning are addgene #122089 and 122090.

Techniques: Injection, Control, Sequencing, Activation Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

doi: 10.1038/s41467-021-22295-w

Figure Lengend Snippet: a Schematic of split-intein dual AAV prime editor. Full-length primer editor (original PE2 ) was reconstituted from two PE2 fragments employing the Npu DNAE split intein . C, carboxy-terminal; N, amino-terminal. b Schematic of the in vivo experiments. Dual AAV8 split-intein prime editor (2 × 10 11 vg total) was delivered to six-week-old PiZ mice by tail-vein injection. Livers were harvested at 2 ( n = 2), 4 ( n = 3), and 10 ( n = 3) weeks after injection and the genomic DNA was isolated for sequencing. c Prime editing efficiency of K342E correction and indels determined by targeted deep sequencing in mouse livers of dual AAV-treated mice. Precise editing is defined as the fraction of sequencing reads with both A to G prime editing and synonymous PAM modification. Results were obtained from two (2 weeks) or three mice (6 and 10 weeks) and presented as mean ± SD. **P < 0.01, * **P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. d Composition of edited alleles at SERPINA1 by UDiTaS analysis. The circle plot shows the fraction of edits that are precise (intended base conversion), small indels (<50 bp) or substitution, deletions between pegRNA and nicking sgRNA sites (<100 bp), large deletions (>100 bp), and AAV fragment insertion. Numbers are average of 3 mice in 10 weeks treated cohort. e The statistically significant large deletion sequences detected by UDiTaS in the 10 weeks treated cohort are displayed as bars spanning the sequence that is deleted (a representative liver of n = 3 mice). Positions of the pegRNA and nicking sgRNA are indicated by dotted lines and the approximate positions of the locus-specific UDiTaS primers are indicated by arrows below the bar chart. The deletion size and number of UMIs associated with each deletion are indicated to the right of each bar. Statistical significance was calculated as a Benjamini–Hochberg adjusted p -value with a cut-off of 0.05.

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene: backbone plasmids used for pegRNA and sgRNA cloning are addgene #122089 and 122090.

Techniques: In Vivo, Injection, Isolation, Sequencing, Modification

Journal: Molecular cell

Article Title: CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons

doi: 10.1016/j.molcel.2017.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: B270 (containing sgRNA targeting ATP1A1 + empty sgRNA-expressing cassette) , This paper , Available in Addgene.

Techniques: Virus, Subcloning, Recombinant, Transfection, DNA Extraction, PCR Cloning, Cloning, Plasmid Preparation, Software

Journal: Science translational medicine

Article Title: Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy

doi: 10.1126/scitranslmed.ade1633

Figure Lengend Snippet: (A) ABE correction of the R636Q mutation using sgRNA (blue) and ABEmax-VRQR-SpCas9. On-target: A6 (red). Bystander: A14 and A20 (green). Silent: A4, A13, and A19 (brown). (B) Percentage of adenine-to-guanine editing determined by deep sequencing of cDNA from hearts of R636Q/R636Q mice (6 weeks after ABE correction). Data are expressed as means ± SEM (n = 3). (C) Fractional shortening at 4 and 8 weeks after ABE correction in WT, R636Q/+, R636Q/R636Q, and corrected mice. Data are expressed as means ± SEM (n = 6 per group). Two-way ANOVA with Tukey’s multiple comparisons test was performed. ****P < 0.0001. (D) H&E staining of four-chamber hearts (12 weeks after ABE correction). Scale bar, 1 mm. LV, left ventricle. (E) Kaplan-Meier survival curve of all groups (n = 16 per group). Log-rank (Mantel-Cox) test was performed. ****P < 0.0001 for R636Q/R636Q versus other groups. (F) Immunohistochemistry showing the translocation of RBM20 in CMs of WT, R636Q/+, R636Q/R636Q, and corrected mice (12 weeks after ABE correction). cTnT (red), RBM20 (green), and DAPI (blue). Scale bar, 10 μm. (G) Quantification of RBM20 localization before and after correction in R636Q/R636Q mice. Data are expressed as means ± SEM (n = 4 per genotype). Two-way ANOVA with Bonferroni’s multiple comparisons test was performed. ****P < 0.0001.

Article Snippet: The N-terminal and C-terminal regions of ABEmax-VRQR-SpCas9 were extracted from CMV_Npu-ABEmax N-terminal (Addgene plasmid no. 137173) ( 52 ) and hu6 HGPS sgRNA expression and ABE7.10max VRQR C-terminal AAV vectors (Addgene plasmid no. 154430) ( 28 ) from D. Liu’s laboratory, respectively.

Techniques: Mutagenesis, Sequencing, Staining, Immunohistochemistry, Translocation Assay