Journal: Nucleic Acids Research

Article Title: Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency

doi: 10.1093/nar/gkad456

Figure Lengend Snippet: Small RNA-seq analysis of different pegRNA species bound to the Prime editor in HEK293T cells. ( A ) Schematic for the small RNA-seq library preparation. Briefly, HEK293T cells were transfected with plasmids encoding one of two effectors (SpCas9 or PEmax), and one guide RNA (sgRNA, pegRNA or epegRNA). Cells were harvested after 2 days, crosslinked and then lysed for total RNA isolation. To sequence the bound pegRNA or epegRNA population, the SpCas9 or PEmax protein (containing 3xHA-tag) was immunoprecipitated then crosslinking was reversed to purify the bound RNA. This was followed by 3’ DNA adapter ligation (3’ adapter contains 15 bp UMI sequence) to the purified RNA, cDNA synthesis and two rounds of PCR to add sequencing adapters. The final library was deep sequenced and analyzed. A detailed protocol is present in the methods section. ( B ) Bulk or effector-bound RNA species present from each treatment group. ‘Bulk’ indicates sequencing of the sgRNA/pegRNA present in the cell without IP pulldown to examine the sgRNA/pegRNA 3’ sequence lengths irrespective of whether it is bound to SpCas9 or PEmax. The length of the PBS in the pegRNA (7 or 13 nt) is indicated in the name. Small RNAs were categorized into six species based on the length of 3’ truncation: full-length pegRNA, pegRNA with truncated but potentially functional PBS (≥7 nt remaining), pegRNA with truncated likely insufficient PBS (<7 nt), pegRNA with truncated RTT, and pegRNA with truncated sgRNA scaffold. Abundance of each RNA species was calculated based on UMIs incorporated into the 3’ adaptor from the small RNA-seq library (see for IGV plots). ( C ) RNP-mediated PE3 editing efficiencies in mCherry reporter cell line with different ratio of pegRNA:nicking sgRNA. The amount of PEmax protein (50 pmol) and pegRNA (200 pmol; IDT) was held constant while increasing the amount of nicking sgRNA (IDT) delivered by electroporation. Frequency of mCherry positive cells was quantified by flow cytometry 72 h following treatment. One-way ANOVA was used to compare all the groups for each graph, PE2 was used as a control column for multiple comparisons. ns stands for P > 0.05, * indicates P ≤ 0.05,** indicates P ≤ 0.01, and **** indicates P ≤ 0.0001 (also see Supplementary table). (D, E) RNP-mediated PE3 editing efficiencies at the specified positions for ( D ) FANCF (+5 G to T) and ( E ) HEK4 (+5 G to T) loci in HEK293T cells. The amount of PEmax protein (50 pmol) and pegRNA (200 pmol; IDT) was held constant while increasing the amount of nicking sgRNA (from IDT) delivered by electroporation. Editing efficiency reflects the frequency of sequencing reads from amplicon deep sequencing that contain the intended edit or others (indels and imprecise prime editing) among all sequencing reads. Values and error bars reflect mean ± s.d. of n = 3 independent biological replicates. One-way ANOVA was used to compare the intended edit across all the groups for each graph, PE2 was used as a control column for multiple comparisons. ns indicates P > 0.05, * indicates P ≤ 0.05,** indicates P ≤ 0.01, and **** indicates P ≤ 0.0001 (also see Supplementary table).

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene (#122089).

Techniques: RNA Sequencing, Transfection, Isolation, Sequencing, Immunoprecipitation, Adapter Ligation, Purification, cDNA Synthesis, Functional Assay, Electroporation, Flow Cytometry, Control, Amplification

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