A New GFP CRISPR for Primary and Non-Dividing Cells

Use of antibiotic resistance markers with CRISPR is an effective tool for cells that proliferate.  However, for primary cells and non-dividing cells, all-in-one sgRNA-Cas9 lentiviral vector that contains a fluorescence marker (GFP-CRISPR) could be a better option.

Our standard, Single-Vector CRISPR-Cas9 System contains a puromycin selection that can be used to isolate transduced cells. In cases where you want to knockout a gene in cytostatic populations, substitution of the puromycin selection for a fluorescent marker enables selection using FACS. Hence, a fluorescent marker such as GFP also allows easy visualization and can expedite your workflow.

Ideally, the most effective approach would be to include both the fluorescent and antibiotic genes in the vector, as we normally do in our shRNA constructs, this design is not an option in our lentiviral based single-vector CRISPR system due to size constraints imposed by the large 4 kb Cas9 nuclease gene, which is required for CRISPR, to be effectively packaged into lentiviral particles. As a result, only one selection marker can be included in Cas9-containing lentiviral vectors.

For genome-wide knockout screens, we recommend using a two-vector CRISPR. With this configuration, both the fluorescent and antibiotic markers are included.

Vector map of the GFP CRISPR construct from Cellecta
Vector map of the GFP CRISPR construct from Cellecta



Also in Cellecta Blog & News

Gene Expression Profiling of Single-Cell Samples: DriverMap Targeted Expression Profiling vs SMART Technology

Single-cell expression analysis provides insights about gene expression and cell heterogeneity at the single-cell level. It enables the elucidation of intracellular gene regulatory networks and intracellular pathways that would otherwise be masked in bulk analysis (Massaia et al., 2018). The DriverMap™ Targeted Gene Expression Profiling (TXP) assay combines highly multiplexed RT-PCR amplification with the depth and precision of Next-Generation Sequencing (NGS) to quantitatively measure gene expression of up to 19,000 target genes in a single assay–even down to the single-cell level.
Read More
Comparing DNA vs. RNA Samples for Immune Repertoire Profiling

Adaptive immunity relies on B and T cells that recognize foreign antigens via hypervariable B cell and T cell receptors (BCRs and TCRs). Diversity among B cell and T cell receptors is primarily produced by V(D)J recombination, which involves the shuffling and joining of the variable (V), diversity (D), joining (J), and constant region (C) gene segments. This results in a diverse repertoire called the adaptive immune repertoire (AIR) that comprises multiple individual clonotypes (sequence) for particular receptor chains.
Read More
Inducible Cas9 Expression in a Single Lentiviral Vector

Introducing Inducible Cas9 Expression in a Single Lentiviral Vector to make cells capable of high Cas9 expression for a limited time during which CRISPR-mediated targeted rearrangements can occur, and then shut off Cas9 expression for downstream assays with the modified cells.
Read More