CRISPR/Cas Systems: Biological Formats and Delivery Strategies for Genome Editing

In the previous article, “Unraveling the Mechanism of Gene Editing: A Primer on CRISPR Technology”, we provided a detailed introduction to the classification, mechanisms, and fundamental principles of the CRISPR system. In this article, we will elaborate on the biological formats and delivery technologies associated with CRISPR-based genome editing.

Biological Formats of CRISPR Technology

To achieve genome editing, the components of the CRISPR/Cas system must be delivered into the nucleus of target cells to exert their function. This can be accomplished through the delivery of Cas protein and guide RNA (gRNA) in three primary biological formats: plasmid DNA (pDNA), messenger RNA (mRNA), or Cas ribonucleoprotein (RNP) complexes.

1. pDNA Format

pDNA is commonly employed as a non-viral vector for DNA delivery. In this approach, the coding sequences for Cas9 and gRNA are incorporated into one or more pDNA vectors. Due to its ease of construction, operational simplicity, and cost-effectiveness, pDNA-mediated CRISPR technology has become a widely adopted gene editing strategy. The commonly used Cas9 plasmids typically contain two expression cassettes: one encoding a codon-optimized Cas9 and the other encoding a chimeric gRNA. Analogous to the central dogma of molecular biology, pDNA carrying CRISPR components must first enter the nucleus, where it is transcribed into mRNA, which is then exported to the cytoplasm for translation into functional Cas9 protein.

2. mRNA Format

mRNA-mediated CRISPR gene editing involves the co-delivery of Cas9 mRNA and gRNA into target cells. Compared to pDNA transfection, mRNA delivery enables faster protein expression. Unlike pDNA strategies, mRNA-based editing requires the simultaneous delivery of both Cas9 mRNA and gRNA. Cas9 mRNA is typically synthesized via in vitro transcription (IVT), followed by 5' capping and 3' polyadenylation to enhance stability and translational efficiency. The gRNA component exists in two structural forms: duplex and single-guide RNA (sgRNA). The duplex form consists of two RNA strands—CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA)—which must be annealed to form a stable complex. These components can be chemically synthesized separately and subsequently hybridized. In contrast, sgRNA is a single molecule in which crRNA and tracrRNA are fused, eliminating the need for annealing and enabling direct DNA cleavage activity. sgRNA can be produced either via IVT or chemical synthesis, with each method offering distinct advantages depending on experimental requirements.

IVT is widely used in research settings due to its high yield, cost efficiency, and technical simplicity. Chemical synthesis, on the other hand, leverages high-throughput platforms and allows for site-specific modifications, such as the incorporation of phosphorothioate and 2'-O-methyl modifications at the 5' and 3' ends of gRNA. These modifications enhance gRNA stability and reduce off-target effects, thereby improving the precision and safety of genome editing.

3. RNP Format

An alternative and highly efficient strategy involves the direct delivery of pre-assembled Cas9 protein and gRNA, bypassing the need for intracellular transcription and translation. This protein-based delivery method relies on the formation of a Cas9/gRNA ribonucleoprotein (RNP) complex, which carries a net negative charge. The RNP complex is formed in vitro by incubating purified Cas9 protein with gRNA and then delivered as a single entity into cells. This approach not only streamlines the editing process but also enhances both efficiency and specificity.

Figure 1. Three biological formats of CRISPR-Cas9^[1]

Comparative Analysis of Biological Formats

Each biological format possesses distinct physicochemical and biological characteristics.

1. Stability

pDNA exhibits the highest stability among the three formats. In contrast, mRNA is inherently less stable, although its stability can be improved through nucleotide modifications. The RNP format, composed of protein and RNA, is the most labile due to susceptibility to proteases and RNases, necessitating careful handling and storage.

2. Delivery and Mechanism of Action

Following cellular uptake, pDNA must enter the nucleus to undergo transcription and subsequent translation into Cas9 protein. mRNA, once delivered to the cytoplasm, can be directly translated into Cas9 protein, which then associates with gRNA to form the active complex. In contrast, RNP complexes can rapidly localize to the nucleus and initiate DNA cleavage without requiring prior gene expression, making this the most direct and efficient delivery strategy. Consequently, RNP delivery is often considered the optimal approach for precise genome editing.

3. Off-Target Effects

Prolonged intracellular persistence of CRISPR components increases the risk of off-target editing. RNP and mRNA formats exhibit transient expression, thereby minimizing the window of activity and reducing off-target effects. In contrast, pDNA can lead to sustained expression of Cas9, potentially increasing the likelihood of unintended genomic modifications.

4. Cas Protein Size

The commonly used Cas9, Cas12a, and Cas13a proteins are approximately 4 kb in coding sequence length. However, adeno-associated virus (AAV) vectors, frequently used for in vivo delivery, have a packaging capacity of ~4.7 kb. This constraint necessitates the development of compact Cas orthologs. Examples include Staphylococcus aureus Cas9 (SaCas9, >1000 amino acids), AaCas12b and BhCas12b (>1100 amino acids), Cas14 (also known as Cas12f1, ~500 amino acids), Cas12j (CasΦ, ~700 amino acids), and Cas13bt (~800 amino acids). The advent of these miniaturized Cas proteins marks the dawn of a "mini-era" in genome editing, enabling more efficient in vivo delivery.

While pDNA, mRNA, and RNP formats each have their advantages and limitations, recent publications by prominent researchers such as Feng Zhang and David Liu suggest that RNP-based protein delivery may emerge as the next frontier in CRISPR therapeutics, following the success of mRNA technologies.

Delivery Technologies for CRISPR/Cas Systems

Given the diversity of biological formats, various delivery strategies have been developed, broadly categorized into biological, chemical, and physical methods.

Figure 2. Different delivery methods of CRISPR technology^[2]

1. Viral Vectors

Viral vectors, including adeno-associated virus (AAV), lentivirus, and baculovirus, are widely used for CRISPR component delivery due to their high transduction efficiency and biocompatibility. AAV has become the most prevalent vector in in vivo gene therapy and has been approved for clinical use in delivering CRISPR components.

AAV is favored for its ability to cross species barriers and its low immunogenicity, minimizing inflammatory responses. However, its limited packaging capacity (~4.7 kb) poses a challenge for larger CRISPR systems. Strategies to overcome this include using smaller Cas orthologs (e.g., SaCas9) or splitting the system across dual AAV vectors.

Lentiviral vectors, capable of transducing both dividing and non-dividing cells, offer a larger cargo capacity (~10 kb), enabling the packaging of full-length CRISPR systems. However, their tendency for random genomic integration raises concerns regarding insertional mutagenesis and potential oncogenesis.

2. Lipid Nanoparticles (LNP)

Although viral vectors have enabled clinical translation of CRISPR therapies, challenges such as immunogenicity, cargo limitations, and difficulties in repeat dosing persist. Non-viral delivery systems, particularly lipid nanoparticles (LNPs), have emerged as promising alternatives.

LNPs typically consist of four lipid components: ionizable (or cationic) lipids, PEGylated lipids, helper phospholipids, and cholesterol. The key innovation lies in the pH-responsive ionizable lipids. These lipids remain neutral at physiological pH but become positively charged in the acidic environment of endosomes. This charge shift facilitates endosomal escape by disrupting the endosomal membrane, thereby enhancing delivery efficiency and reducing systemic toxicity.

Notably, several LNP-based RNA therapeutics have received FDA approval, underscoring the clinical viability and safety of this platform.

Figure 3. Composition of LNPs^[2]

2. Emerging Delivery Systems

Despite the clinical success of LNPs, alternative nanocarriers are being actively explored. These include polymer-based nanoparticles, engineered protein capsids, and virus-like particles (VLPs), which offer tunable properties for targeted intracellular delivery.

Conclusion

This article has outlined the three primary biological formats (pDNA, mRNA, RNP) and major delivery strategies (viral, chemical, physical) for CRISPR/Cas systems. These foundational concepts are essential for understanding the broad applicability of CRISPR technology. In subsequent articles, we will explore applications of CRISPR/Cas in cell-based gene therapies, agriculture, and other fields. Stay tuned for more insights.

Ordering Information

Application	Product Type	Product Name	Catalog Number
General-purpose	SpCas9 with NLS	Cas9 Nuclease	14701ES
Compact size for delivery	Cas12a	ArCas12a Nuclease	14702ES
	Cas12b	AapCas12b Nuclease	14808ES
sgRNA preparation	sgRNA synthesis	Hifair® Precision sgRNA Synthesis Kit	11355ES
	sgRNA purification	Hieff NGS® RNA Cleaner	12602ES

References

[1] Yi Lin, Ernst Wagner and Ulrich Lächelt, Non-viral delivery of the CRISPR/Cas system: DNA versus RNA versus RNP, Biomater. Sci., 2022, 10, 1166–1192

[2] Taha EA, Lee J, Hotta A. Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges. J Control Release. 2022;342:345-361.

Extended Reading

CRISPR Gene Editing Product Selection Guide

Unraveling the Mechanism of Gene Editing: A Primer on CRISPR Technology