Clontech Gesicles Improve CRISPR/Cas9 Performance
Clontech has launched the Guide-it CRISPR/Cas9 Gesicle Production System, a new technology that improves CRISPR/Cas9 delivery and reduces its footprint. Cas9 endonucleases and target-specific sgRNAs are encapsulated in cell-derived nanovesicles that can target different cell types via surface glycoproteins. The new system avoids constant Cas9 expression, avoiding off-target effects, and enhances Cas9 delivery to all sorts of cells.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a bacterial immune system that cuts foreign DNA in pieces that are integrated at the CRISPR loci. After transcription, the CRISPR RNA (crRNA) binds Cas endonucleases and targets them to cleave complementary DNA. This system has been adapted to become an eukaryotic gene-editing technique where crRNA targets Cas9 to specific genes. CRISPR/Cas9 is a major breakthrough in biotechnology, but it still has some shortcomings: delivery efficiency to different kinds of cells could be improved; and constant expression of the introduced Cas9 gene can lead to off-target effects.
Gesicles encapsulate Cas9-sgRNA ribonucleoprotein complexes
To solve this problems, Clontech has developed the Guide-it CRISPR/Cas9 Gesicle Production System. First, Cas9 is produced in 293T cells that contain the Cas9 gene. Then, these producer cells are transfected with a vector that includes the target-specific sgRNA and glycoproteins that induce nanovesicle formation. When the gesicles are formed with Cas9 proteins inside, the iDimerize system connects a Cas9 domain with membrane fluorescent proteins that allow to identify gesicles that contain the endonuclease. The gesicles can then be collected from the supernatant and stored for one year at -70 ºC or applied to a wide variety of cells. When gesicles fuse with the target cells, these become transiently red, until Cas9 is directed to the nucleus -thanks to its nuclear localization signal (NLS)- and the dimer with the fluorescent protein is disassembled. Once inside the nucleus, the sgRNA-Cas9 ribonucleoprotein can edit its target gene, and shortly after is degraded by endogenous mechanisms, avoiding off-target effects.
Source: Takara Clontech