Journal: Molecular Therapy

Article Title: Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

doi: 10.1016/j.ymthe.2022.11.014

Figure Lengend Snippet: Characterization of HMD-G fusion to base editors in HEK293T cells (A) Comparison of the C-to-T base-editing efficiencies of BE4max and eeBE4max at 24 endogenous targets in HEK293T cells. (B) Comparison of the A-to-G base-editing efficiencies of ABEmax and eeABEmax at 15 endogenous targets in HEK293T cells. (C) Summary of the cumulative C-to-T base-editing efficiencies of multiple BE4max variants and multiple eeBE4max variants at endogenous targets for “NGG” PAM and “non-NGG” PAM in HEK293T cells. (D) Summary of the cumulative A-to-G base-editing efficiencies of multiple ABEmax variants and multiple eeABEmax variants at endogenous targets for “NGG” PAM and “non-NGG” PAM in HEK293T cells. (E) Frequency of indels by BE4max and eeBE4max. Each point represents the average indel frequency at a target site. (F) Frequency of indels by ABEmax and eeABEmax. Each point represents the average indel frequency at a target site. Data in (A) and (B) represent means ± SD (n = 3 independent experiments). The statistical analysis of (C), (D), (E), and (F) was performed by using paired two-tailed Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.

Article Snippet: 40 We first evaluated sgRNA-dependent off-target events of eeBE4max and eeABEmax on 73 previously reported off-target sites 41 , 42 and 28 web-predicted ( https://www.benchling.com/ ) off-target sites.

Techniques: Comparison, Two Tailed Test

Journal: Molecular Therapy

Article Title: Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

doi: 10.1016/j.ymthe.2022.11.014

Figure Lengend Snippet: Evaluation of the off-target effects of eeBE4max and eeABEmax in HEK293T cells (A) Comparison of on- and off-target efficiencies of BE4max or eeBE4max with sgRNA plasmid delivery, and BE4max or eeBE4max RNP delivery at EMX1-6, FANCF-2, FANCF-3, and HBB 02 sites. (B) Specificity metric of eeBE4max and BE4max with different delivery strategies in (A). (C) Specificity metric of eeABEmax and ABEmax for indicated target sites. (D) Evaluation of sgRNA-independent DNA off-target efficiencies of BE4max and eeBE4max via using an orthogonal R-loop assay. (E) Evaluation of sgRNA-independent DNA off-target efficiencies of ABEmax and eeABEmax using an orthogonal R-loop assay. In (B) and (C), the specificity metric is defined as on-target editing rate divided by the sum of all off-target editing rates. Data in (A), (D), and (E) represent means ± SD (n = 3 independent experiments).

Article Snippet: 40 We first evaluated sgRNA-dependent off-target events of eeBE4max and eeABEmax on 73 previously reported off-target sites 41 , 42 and 28 web-predicted ( https://www.benchling.com/ ) off-target sites.

Techniques: Comparison, Plasmid Preparation

Journal: Molecular Therapy

Article Title: Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

doi: 10.1016/j.ymthe.2022.11.014

Figure Lengend Snippet: Evaluation of the off-target effects of eeCas9 in HEK293T cells (A) gRNA-target mismatch tolerance experiment to evaluate the specificity of Cas9 and eeCas9. VEGF-targeting sgRNAs with single mismatch or two consecutive mismatches in the protospacer region were generated to evaluate gene-editing efficiencies by HTS analysis. The PAM sequences are shown in blue. The mismatched nucleotides are shown in red. n.c., not calculated because of no significance between eeCas9 and Cas9. (B) Comparison of on- and off-target efficiencies of Cas9 or eeCas9 with sgRNA plasmid delivery, Cas9 or eeCas9 with hp-sgRNA plasmid delivery, and Cas9 or eeCas9 RNP delivery at EMX1-6 site and FANCF-2 site. (C) Specificity metric of eeCas9 and Cas9 with different delivery strategies in (B). (D) On- and off-target efficiencies by high-fidelity Cas9 with or without HMG-D domain were measured by HTS analysis. (E) Specificity metric of high-fidelity Cas9 with or without HMG-D domain for indicated target sites. (F) Number of PEM-seq detected off-target sites for eeCas9 and Cas9. The values above the columns represent the number of detected off-target sites. In (C) and (E), the specificity metric is defined as on-target editing rate divided by the sum of all off-target editing rates. Data in (A) and (D) represent means ± SD (n = 3 independent experiments), and statistical analysis was performed by using unpaired two-tailed Student’s t test in (A). ∗∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.

Article Snippet: We first checked the top 20 web-predicted ( https://www.benchling.com/ ) off-target sites for sg1 or sg2 by HTS and detected no apparent (>0.1% efficiency) off-target event among those sites ( Figures S17 A and S17B).

Techniques: Generated, Comparison, Plasmid Preparation, Two Tailed Test

Journal: Molecular Therapy

Article Title: Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

doi: 10.1016/j.ymthe.2022.11.014

Figure Lengend Snippet: Evaluation of the off-target effects of eeBE4max and eeABEmax in HEK293T cells (A) Comparison of on- and off-target efficiencies of BE4max or eeBE4max with sgRNA plasmid delivery, and BE4max or eeBE4max RNP delivery at EMX1-6, FANCF-2, FANCF-3, and HBB 02 sites. (B) Specificity metric of eeBE4max and BE4max with different delivery strategies in (A). (C) Specificity metric of eeABEmax and ABEmax for indicated target sites. (D) Evaluation of sgRNA-independent DNA off-target efficiencies of BE4max and eeBE4max via using an orthogonal R-loop assay. (E) Evaluation of sgRNA-independent DNA off-target efficiencies of ABEmax and eeABEmax using an orthogonal R-loop assay. In (B) and (C), the specificity metric is defined as on-target editing rate divided by the sum of all off-target editing rates. Data in (A), (D), and (E) represent means ± SD (n = 3 independent experiments).

Article Snippet: We first checked the top 20 web-predicted ( https://www.benchling.com/ ) off-target sites for sg1 or sg2 by HTS and detected no apparent (>0.1% efficiency) off-target event among those sites ( Figures S17 A and S17B).

Techniques: Comparison, Plasmid Preparation

Journal: Molecular Therapy

Article Title: Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

doi: 10.1016/j.ymthe.2022.11.014

Figure Lengend Snippet: AAV delivery of eeCas9 for in vivo genome editing (A) Schematic depicting the experimental procedure to edit Pcsk9 gene in wild-type mice. Two sgRNAs (sg1 and sg2) were selected to target exon 3 and exon 5 of Pcsk9 gene, respectively. (B) sgRNA validation in NIH3T3cells via HTS analysis (n = 3 independent experiments). (C) Schematic diagram of dual AAV vector system for liver-directed gene editing. MiniCMV, minimal cytomegalovirus; short PolyA, short polyadenylation signal; ITR, inverted terminal repeat. (D) Time course of total serum cholesterol in animals treated with AAV8-Cas9 or AAV8-eeCas9. n = 6 for sg1, n = 3 for sg2. (E) Indels at Pcsk9-1(n = 6) and Pcsk9-2 (n = 3) targeting sites of Pcsk9 in mice. (F) Analysis of Pcsk9 protein expression in mouse liver by western blot. Psck9 level was determined by densitometry of western blot bands normalized by that of actin in each sample. (G) WGS analysis of potential off-target of two representative mice (1# and 2#) for Pcsk9-1 target. SNV, single-nucleotide variant; Indels, insertions and deletions. (H) PEM-seq analysis of the number of off-target sites of two representative mice (1# and 2#) for Pcsk9-1 target. Data represent means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: We first checked the top 20 web-predicted ( https://www.benchling.com/ ) off-target sites for sg1 or sg2 by HTS and detected no apparent (>0.1% efficiency) off-target event among those sites ( Figures S17 A and S17B).

Techniques: In Vivo, Biomarker Discovery, Plasmid Preparation, Expressing, Western Blot, Variant Assay

Journal: BMC Biotechnology

Article Title: Design of a generic CRISPR-Cas9 approach using the same sgRNA to perform gene editing at distinct loci

doi: 10.1186/s12896-019-0509-7

Figure Lengend Snippet: Main stages of the CRISPR-Cas9 targeting ‘bait’ DNA approach to perform gene-copy specific editing. The approach is based on the specific insertion by homologous recombination of a selection marker and a foreign DNA harboring the upstream (‘U’) and downstream (‘D’) regions surrounding the copy of interest. The star represents the various possible designs of the model DNA used to repair the lesion by homologous recombination. For clarity reasons, the scheme only represents a first recombination event occurring between U sequences but the homologous recombination could also take place between D sequences. Thereafter a genetic tool encoding Cas9 and a sgRNA specifically directed against foreign ‘bait’ DNA is introduced transitorily in the recombinant strain. The double strand break induced by this complex can be repaired by a second recombination event between either the U or D sequences, resulting in reversion or in the copy-specific editing, respectively. In absence of a second selection marker at the star position, the distinction between both situations can be achieved by PCR (arrows represent primer positions). The pictures show representative results of PCR performed without template (“Ø”), on the native genome (“WT”) or on the genome of scarless deleted mutants (“Δ”) obtained during this study. The PCR product expected sizes were 1106 pb and 815 pb for native and deleted lsr2A loci, and 729 pb and 481 pb for native and deleted lsr2B loci, respectively (see section for further details). The PCR product expected sizes for native and deleted loci are indicated (“L”: GeneRuler™1 kb DNA ladder, Thermo Scientific)

Article Snippet: The PCR product expected sizes for native and deleted loci are indicated (“L”: GeneRuler™1 kb DNA ladder, Thermo Scientific) We designed a sgRNA targeting this selection marker using CRISPRy-web site analysis (https://crispy.secondarymetabolites.org/#/input) [ ] and Cas-OFFinder ( http://www.rgenome.net/cas-offinder/ ) [ ] to select a 20-nucleotide target region that is predicted to have no off-targets within S. ambofaciens genome.

Techniques: CRISPR, Homologous Recombination, Selection, Marker, Recombinant