Genome editing with CRISPR/Cas9

CRISPR/Cas9 has become a very powerful and precise approach to genome engineering. It is based on the joint action of a gRNA molecule and Cas9 nuclease and it enables gene modifications through “molecular scissors” that can cut a sequence of DNA to eliminate a fragment and replace it. CRISPR/Cas9 has been proven to be easy to use, rapid, economic and very precise, so that it quickly gained great success.

RNP complex or plasmid vectors?

When the technology was first discovered by the Nobel prizes Emmanuelle Charpentier and Jennifer Anne Doudna, it was necessary to construct plasmid vectors to express Cas enzyme and the gRNA, because the long synthetic RNAs were very expensive and not widely available.

How did it work? gRNA combines two molecules, crRNA and tracrRNA, contains a sequence complementary to that of the DNA to be corrected. When guide RNA and Cas9 are introduced into a cell, they interact with the “target” sequence and correct it, for example, by removing or adding genetic material.

However, there are some potential complications that this method can entail:

•     Cells must be amenable to transfection or viral transduction
•     Appropriate promoters must be chosen for both Cas9 and gRNA expression
•     Plasmid DNA may be incorporated into the genome
•     Off-target effects can occur due to prolonged Cas9 expression
•     The requirement for Cas9 transcription and translation delays editing.[1]

Instead, the different approach of directly delivering the Cas protein and gRNA as a ribonucleoprotein (RNP) complex has been proved to be an efficient method that offers several advantages compared to plasmid expression vectors. RNP complexes, in fact, provide lower off-target editing because they are delivered directly to the cells as active complexes for high on-target editing, but do not persist in the cells to increase the risk of off-target editing.
 
 
IDT (Integrated DNA Technology), a world leader in oligo production and CRISPR gene editing, represents a great ally for your CRISPR experiments since it provides precise RNP components, tools and reagents to make your researches a success.

Let’s now analyze in detail which are the advantages of RNP over plasmid delivery of Cas enzymes and gRNAs:

1. Genome editing with plasmid is laborious
   IDT provides transfection-ready RNP components that simplify the process, otherwise very laborious: with plasmid delivery method you need to construct expression vectors, transform bacteria, select multiple clones, isolate DNA, perform restriction enzyme digests to identify positive clones, and sequence the positive clones to confirm their sequences
2. Plasmid delivery is inherently inconsistent
   Transfection efficiency differs between cell lines, and even varies from one experiment to the next. Plasmid delivery is particularly problematic for “hard-to-transfect” cell lines, such as primary cells, resulting in inefficient editing. The RNP complex can be delivered into cells with high efficiency by lipofection or electroporation, resulting in potent editing
3. Plasmids leave behind molecular footprints
   Researchers have demonstrated that when gRNAs are introduced as expression plasmids, not only multiple bases but also large fragments of plasmid vectors are inserted into the genome. RNP complex delivery produces fewer multiple-base insertions and avoids issues related to vector molecular footprint
4. Long-lasting expression from plasmid vectors increases off-target editing
   Plasmid-driven expression continues for a long time, which significantly increases off-target editing. In contrast, RNP delivery produces a “fast on, fast off” reaction that increases targeting accuracy and decreases the chance of off-target editing. Cas9 RNPs are, in fact, detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein degradation pathways.

To conclude, in the cases of DNA and RNA delivery, since one or both CRISPR components are produced by the cellular machinery, the result is an unpreventable latency between the availabilities of gRNA and Cas9. RNP delivery represents by now the most direct strategy to introduce the pre-assembled and immediately functional CRISPR/Cas9 system into cells, which generally results in higher genome editing efficiency compared to the other biomolecular formats.[2]

IDT solutions for your experiments with CRISPR/Cas9 gene editing

The Alt-R™ CRISPR-Cas9 System includes all the reagents needed for successful genome editing in your research applications based on the natural S. pyogenes CRISPR-Cas9 system.

Why choosing IDT Alt-R™ CRISPR-Cas9 System?

• Benefit from the latest improvements with on- and off-target design and chemical modifications
• Enjoy easy ordering of custom or predesigned guide RNAs
• Get desirable editing results with high on-target potency and reduced off-target activity with Alt-R HiFi CRISPR-Cas9 nuclease
• Expect precision control editing with efficient delivery of the RNP by lipofection or electroporation
• Protect cells from toxicity or innate immune response activation
• Design your HDR template & gRNA
• Order your custom HDR template.

What makes the IDT Alt-R™ CRISPR-Cas9 System so great?

• Exact length determination of the crRNAs for increased activity
• Chemical modifications to help increase nuclease resistance and reduce innate immune responses
• Knowledge that our native Cas9 and HiFi Cas9 variant has shown increased selectivity in our studies
• Two-part guide RNA (crRNA:tracrRNA) provides high activity at an affordable price
• Unique supporting reagents (electroporation enhancer, labeled tracrRNA, controls) provide successful editing
• Complete collection of user resources, including guides, protocols, and demonstrated protocols.

DISCOVER IDT PRODUCTS FOR GENE EDITING 

Citations:

[1] A., Hempstead, CRISPR 101: Ribonucleoprotein (RNP) Delivery, Addgene Blog, https://blog.addgene.org/crispr-101-ribonucleoprotein-rnp-delivery [Accessed on 6 February 2025]

[2] Y., Lin, E., Wagner and U., Lächelt, Non-viral delivery of the CRISPR/Cas system: DNA versus RNA versus RNP, Biomater. Sci., 2022, 10, 1166-1192, DOI: 10.1039/D1BM01658J