Journal: Nature Biomedical Engineering

Article Title: Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs

doi: 10.1038/s41551-023-01011-7

Figure Lengend Snippet: a , Indel patterns were analyzed using Cdh1-P2A 2 -AIMS in mESCs ( n = 3, independent experiments). The spacerless-PX459 plasmid (p:CP, 250 ng) was co-transfected with different amounts of the P2A 2 -sgRNA4 expression plasmid (p:R). The total number of colonies analyzed is shown in each column. b , Construction of [0 C]–[30 C]sgRNA expressing all-in-one plasmids. Linkers were inserted into the BpiI site of the PX459 plasmid. Adenine (A, blue) was inserted at the third position from the 3’-end of the cytosine extension to create an overhang sequence for the insertion of spacer sequences with CCAC overhang. The [5 C]–[30 C]sgRNA-expressing all-in-one plasmids were produced by inserting a standard 18–20-bp spacer linker between two BpiI sites. c , Relationship between Cas9 activity (AIMS[P]) and mosaic frequency before and after passage of puromycin-resistant primary colonies, assessed using different [C]sgRNAs in mESCs with Cdh1-P2A1-AIMS. Data are means ± SEMs for three independent experiments performed at different times. Statistical significance was assessed using two-way analysis of variance (ANOVA), followed by a post hoc Tukey–Kramer test. d , Table shows percentages of the two types of mono-allelic indel patterns in mESCs. Totals indicate the means of all data ( n = 73), which are shown in Fig. . Data are means ± SEMs.

Article Snippet: To construct all-in-one AsCpf1 plasmids enabling puromycin selection, a synthetic DNA fragment encoding U6 promoter and two BpiI sites (AZENTA) (Supplementary Table ) was inserted into a PX459 plasmid while removing a U6-gRNA cassette using PciI and XbaI sites.

Techniques: Plasmid Preparation, Transfection, Expressing, Sequencing, Produced, Activity Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR-Cas9 homology-independent targeted integration of exons 1–19 restores full-length dystrophin in mice

doi: 10.1016/j.omtm.2023.08.009

Figure Lengend Snippet: Design of a homology-independent targeted integration-based system for correction of mutations in the first 19 exons of DMD (A) The HITI-based gene editing system is delivered via a pair of AAV vectors, one of which encodes the S. aureus Cas9 enzyme, and the other which contains a U6-gRNA expression cassette and a donor fragment, flanked by Cas9 target sites in the reverse orientation relative to genomic DNA. The donor itself consists of the first 19 exons of DMD , under the control of the MHCK7 promoter. (B) Cas9 is expressed from the AAV vector and cleaves both the donor vector and genomic DNA. The ends are repaired by non-homologous end-joining. Integration of the HITI donor in the reverse orientation reconstitutes the Cas9 target sites and allows re-cleavage until correct integration is achieved.

Article Snippet: A DNA fragment encoding the HITI donor construct and a U6-promoter-driven gRNA targeting intron 19 was synthesized with NotI restriction sites (Twist Bioscience) and cloned between the NotI sites in the plasmid.

Techniques: Expressing, Plasmid Preparation, Non-Homologous End Joining

Journal: bioRxiv

Article Title: Knockout of the tomato HAIRY MERISTEM 4 alters phloem-characteristics and impairs development

doi: 10.1101/2024.08.02.606343

Figure Lengend Snippet: (A) Principal components analysis of all expressed genes showing three distinct groups. (B) Total number of DEGs in slham4 CRΔ4 mutant and heterozygous ( slham4 CRΔ4(-/+) ) ovaries compared to wild-type. (C) Venn diagram displaying specific and overlapping DEGs between slham4 CRΔ4 and slham4 CRΔ4(-/+) data sets. (D) Expression profile of cluster 24 cDEGs in fruit pericarp tissues based on TEA database data ( Fernandez-Pozo et al ., 2017 ). Cluster-wide average expression is plotted with solid lines, first and third quartiles by dashed lines, and maximum and minimum by dotted lines. The SlHAM4 specific profile is plotted with red solid line. (E and F) Overlap between DEGs and cDEGs (E) or Arabidopsis orthologs of cDEGs (F) with indicated published vascular datasets. To compensate for dataset size, the numbers of up and down DEGs and cDEGs were normalized relative to the total number of genes in the largest group dataset (Supplementary Table S1F). Asterisks indicate statistical significance of overlap as calculated by http://nemates.org/MA/progs/overlap_stats.cgi . Ph, phloem; Vi, vascular initials; Xy, xylem.

Article Snippet: Then, a construct containing the two sgRNAs in tandem each driven by the synthetic Arabidopsis U6 promoter delimited by 5’- Mlu I and 3’- Hind III was artificially synthesized (GeneWiz, USA) and cloned into pUC57 to generate pUC57-U6::sgRNA1- U6::sgRNA2.

Techniques: Mutagenesis, Expressing