transfer plasmids carrying crispr guides  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: Characterization of CRISPR–SaCas9 target site mutations in HIV breakthrough cultures. Supernatants from cultures exhibiting syncytia formation were collected at the peak of infection and used to infect fresh cells transduced with SaCas9/gRNA. After robust viral replication, cells were harvested, and total cellular DNA was extracted. The target region was amplified by PCR and analyzed by Sanger sequencing. Sequences were aligned to the LAI reference, with the wild-type sequence shown at the top and culture numbers indicated on the left. The target sequence is underlined, and the PAM sequence is highlighted in blue. Nucleotide substitutions are shown in red, predicted SaCas9 cleavage sites are indicated with black triangles, and corresponding amino acids are displayed on the right.

Article Snippet: gRNA spacers were designed using Benchling CRISPR guide design software ( https://www.benchling.com/ ), targeting the HIV-1 DNA genome of the primary LAI virus isolate.

Techniques: CRISPR, Infection, Transduction, Amplification, Sequencing

Journal: bioRxiv

Article Title: Plasmids weaponize conjugation to eliminate non-permissive recipients

doi: 10.64898/2026.02.10.705089

Figure Lengend Snippet: (A) Model of coercive assimilation. In non-permissive recipients (pink oval), intracellular defense systems (e.g., CRISPR-Cas9, teal) degrade the incoming plasmid, preventing TrbK expression. The unshielded recipient remains susceptible to repetitive, T4SS punctures and uncontrolled DNA delivery (lethal zygosis). (B) Restriction-Modification (defense). Left: Schematic of the RM competition assay. RM+ recipients (non-permissive, pink) and RM-recipients (permissive, yellow) compete in the presence of varying amounts of donor cells carrying RK2 plasmids (blue) that are either methylated (protected from digestion) or unmethylated (susceptible to digestion). Right: Competitive index of RM+ recipients relative to RM-recipients in the presence of donors with methylated (left facet) or unmethylated (right facet) RK2. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Different letters (a - b) indicate statistically significant differences within and between facets. When donors carried unmethylated RK2, RM+ recipients suffered significant fitness costs compared to the methylated plasmid treatment (p < 0.05) (C) CRISPR-Cas defense. Left: Schematic of the CRISPR competition assay. Recipients with Cas9 and an RK2-targeting gRNA (non-permissive, pink) or a non-targeting gRNA (permissive, yellow) were competed in the presence of varying amounts of RK2 donor. Right: Competitive index of targeting recipients relative to non-targeting recipients under uninduced or induced conditions. Different letters (a - b) indicate statistically significant differences (p < 0.05). Circles represent the competitive index of replicates, bars represent the mean (n = 3).

Article Snippet: CRISPR guide RNAs were designed to target RK2’s oriV using the Benchling guide design tool ( ).

Techniques: CRISPR, Plasmid Preparation, Expressing, Modification, Competitive Binding Assay, Methylation