Takara Guide-it CRISPR/Cas9 Gesicle Production System \1 System

The Guide-it CRISPR/Cas9 Gesicle Production System is a system for producing high yields of target-specific CRISPR/Cas9 gesicles for gene editing. Gesicles are cell-derived nanovesicles used to deliver macromolecular cargoes to a broad range of target cells, including cells that are difficult to transfect with plasmids. The nanovesicles are produced in a Gesicle Producer 293T Cell Line (Cat. No. 632617) via co-overexpression of packaging mix components, which include a nanovesicle-inducing glycoprotein and a protein that is displayed on the cell surface that mediates binding and fusion with the cellular membrane of target cells. Simultaneous overexpression of additional macromolecular cargoes, in this case the Cas9 protein fromStreptococcus pyogenesand a target-specific guide RNA (sgRNA), results in incorporation of the Cas9/sgRNA complex within the gesicles. After the resulting Cas9/sgRNA gesicles are harvested and applied to your target cells in the presence of protamine sulfate, they will efficiently enter the cells and mediate gene editing. This system provides the components needed to clone and express your target-specific guide RNA, and packaging reagents to produce CRISPR/Cas9 gesicles. It is essential to use the pGuide-it-sgRNA1 Vector for expression and packaging of your guide RNA, since using other commonly used guide RNA vectors will not result in effective Cas9/sgRNA complexes.

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells.The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).

Cas9 protein delivery in culture cells.Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

The use of gesicles decreases off-target effects.HEK 293T cells were either simultaneously cotransfected with plasmids encoding Cas9 DNA and a sgRNA againstEMX1, or treated with gesicles loaded with Cas9-sgRNA ribonucleoprotein complexes. After 72 hr, theEMX1gene and a potential off-target locus (off-target 4) were analyzed using the Guide-it Mutation Detection Kit (Cat. # 631443). With the gesicles, no off-target effect could be detected (top panel). Sequencing data for the different clones were aligned with the underlined wild-type sequence, revealing a range of deletions and insertions (indels; highlighted in red). Gesicles correctly edited theEMX1gene with no off-target effects, whereas plasmid cotransfection resulted in indels in both the target site,EMX1, as well as off-target site 4 (bottom panel).

Editing efficiency is increased by an improved sgRNA scaffold design in gesicles.HT1080 cells containing an integrated fluorescent protein expression cassette were transfected with a plasmid encoding for Cas9 andAcGFP1-specific sgRNA or treated with gesicles. Both delivery methods were tested using either the traditional or an optimized, sgRNA scaffold targetingAcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. The optimized sgRNA scaffold had no effect on editing efficiency for plasmid-based delivery. However, the optimized sgRNA scaffold increased knockout efficiency by 36.4% for gesicles. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect.HT1080 cells containing an integrated fluorescent protein expression cassette were treated with 5 µl, 10 µl, 20 µl, or 30 µl of gesicles produced with either the traditional or an optimized, sgRNA scaffold targetingAcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. Editing efficiency using the traditional scaffold was low, regardless of gesicle dose. However, gesicles using the optimized scaffold demonstrated a dose-dependent increase in knockout up to 59%. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes.The cell-surface protein receptor CD81 was knocked out in Jurkat cells using either plasmid cotransfection of Cas9 DNA and sgRNA or gesicles preloaded with a Cas9-sgRNA ribonucleoprotein complex. The knockout efficiency was measured six days later via antibody labeling of the membrane receptor followed by flow cytometry analysis. Results for delivery via gesicles were significantly greater than results achieved with plasmid transfection.

Successful knockout ofCD81in hiPS cells.Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 (hiPSC ChiPSC18), cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated (control) cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative (left) and positive (right) labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr (left) and 24 hr (right) of gesicle treatment, labeled with anti-CD81 (FITC) antibodies.