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: Viruses

Article Title: Validation of Candidate Host Cell Entry Factors for Bovine Herpes Virus Type-1 Based on a Genome-Wide CRISPR Knockout Screen

doi: 10.3390/v16020297

Figure Lengend Snippet: Multiplex CRISPRi against pro-viral candidate genes results in reduced viral titers. ( a ). CRISPR3i against a host gene in action by binding immediately downstream of TSS in tandem for synergistic gene repression. ( b ). The one-step cloning strategy to synthesize PiggyBac transposon vectors carrying three sgRNA expression cassettes in tandem to implement CRISPR3i by PB transposition. ( c ). Plaque formation assay results in cells with CRISPR3i expression intended to interfere with transcription of pro-viral genes involved in HS biosynthesis and other functions, as identified by the CRISPRko screen; CTRL represents virus titer from cells expressing three non-targeting sgRNAs (n >= 3). Results were ordered by p -value. ****: p < 0.0001; ***: p < 0.005; **: p < 0.01; *: p < 0.05; n.s. not significant with p > 0.05 based on one-way ANOVA followed by multiple comparisons between CTRL and CRISPR3i cells, error bars represent +/− 1 SD.

Article Snippet: The PiggyBac vector used to deliver CRISPRi, PB-U6g5_PGK_Puro2aBFP, was constructed by cutting out the segment containing the hU6 sgRNA expression cassette and the Puro2aBFP selection marker from pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (gift from Kosuke Yusa, Addgene # 67974) and cloning it into a PiggyBac backbone.

Techniques: Multiplex Assay, Binding Assay, Cloning, Expressing, Plaque Formation Assay, Virus