CRISPR/Cas9 Genome Editing: Transfection Methods
Overview of Genome Editing with CRISPR/Cas9
Transfection Tips Video: CRISPR/Cas9 Transfection
This video covers the basic mechanism of CRISPR/Cas9 genome editing and introduces the key points affecting researchers' decisions about whether to deliver Cas9 and guide RNA in plasmid, RNA, or RNP format.
CRISPR/Cas9 Genome Editing.
The Cas9 endonuclease (blue) is targeted to DNA by a guide RNA which can be supplied as a two-part system consisting of crRNA and tracrRNA or as a single guide RNA, where the crRNA and tracrRNA are connected by a linker (dotted line). Target recognition is facilitated by the protospacer-adjacent motif (PAM). Cleavage occurs on both strands (scissors) 3 bp upstream of the PAM.
- The use of CRISPR/Cas9 has emerged as a powerful method for genome editing that can be delivered in multiple formats. MoBiTec together with Mirus Bio provide solutions to address delivery of Cas9 and guide RNA in plasmid, RNA, or RNP format. To learn more about the basic mechanism of CRISPR/Cas9 genome editing and the key points affecting decisions about whether to deliver Cas9 and guide RNA in plasmid, RNA, or RNP format, please see a general overview below. For more details, see methods for delivery of CRISPR/Cas9 DNA, RNA, and RNP on the following pages:
- • pDNA + gRNA Transfection
- • mRNA + gRNA Transfection
- • Cas9/gRNA RNP Delivery
- • Request a FREE Sample for Your Genome Editing Research
- Bacteria and archaea exhibit chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR) that are part of an adaptive immune system that protects against invading viral and plasmid DNA. In Type II CRISPR systems, CRISPR RNAs (crRNAs) function with trans-activating crRNA (tracrRNA) and CRISPR-associated (Cas) proteins to introduce double-stranded breaks in target DNA. Target cleavage by Cas9 requires base pairing between the crRNA and tracrRNA as well as base pairing between the crRNA and the target DNA (see figure CRISPR/Cas9 Genome Editing). Target recognition is facilitated by the presence of a short sequence called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG.
- The bacterial CRISPR/Cas9 system has been adapted to serve as a versatile platform for RNA-directed genome editing in mammalian cells. The Cas9 endonuclease can be programmed by a dual RNA (crRNA and tracrRNA), or the core components of these RNAs can also be combined into a single hybrid guide RNA. Once Cas9 has cleaved the target DNA, two endogenous repair mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR), are triggered in response to the DNA break. The features of these DNA break repair pathways can be exploited to generate gene knock-outs or introduce defined modifications at the site of cleavage. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. HDR requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence flanked on either side by sequences bearing homology to the target.
- The simplicity of using a noncoding RNA guide to target DNA for site-specific cleavage provides a distinct advantage over alternative genome editing technologies such as ZFNs and TALENs. Using the CRISPR/Cas9 strategy, retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of DNA-binding proteins.
Multiple Approaches for Cas9 and Guide RNA Delivery
DNA, RNA, and Protein Formats for CRISPR Genome Editing.
TransIT-X2® Dynamic Delivery System was used to deliver Cas9 pDNA/gRNA and Cas9 protein/gRNA (RNP complex). TransIT®-mRNA was used to deliver Cas9 mRNA/gRNA. A T7E1 mismatch assay was used to measure cleavage efficiency at 48 hours post-transfection. For more details, see methods for delivery of CRISPR/Cas9 DNA, RNA, and RNP.
Comparison of Cas9 Formats: DNA, RNA, and Protein
Pros and Cons of DNA, RNA, and Protein Formats for Genome Editing.
Cas9 can be delivered as plasmid DNA for a simple, low-cost approach. Cas9 mRNA enables rapid gene expression, and eliminates the risk of insertional mutagenesis. Cas9/guide RNA ribonucleoprotein (RNP) complexes exhibit the most rapid pulse of genome editing activity and reduce the possibility of off-target cleavage events. Cas9 mRNA and RNP formats can also be efficiently delivered to cell types that are resistant to transfection with plasmid DNA.