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