Delivery Methods to Maximize Genome Editing with CRISPR-Cas9

Diseases caused by genetic mutations cause suffering for millions of people worldwide. Rare genetic diseases can impact as much as 5.9% of the global population at any point in time. Many of these diseases, such as cystic fibrosis and sickle cell disease, are caused by single mutations. In response, researchers have searched for ways to correct these mutations. These efforts have yielded several gene editing methods, such as prime editing (insert link to Excedr article on prime editing).

The discovery of the CRISPR-Cas9 system accelerated these efforts. Expressed by bacteria, the Cas9 system protects microbes from foreign DNA by cutting the DNA  upon detection. Since its discovery, researchers have used the machinery to develop Cas9-mediated gene editing. Although the system has paved the way for therapeutic gene editing, its components must be packaged for it to act on target cells. To that end, researchers have developed several delivery systems for diverse cell lines and cell types. Each system adopts a unique approach to deliver the CRISPR-Cas nucleic acids and proteins into cells. Knowing which system to use will maximize the chances of a successful gene edit.

In this article, we will delve into several aspects of delivering CRISPR-Cas9 systems into cells. We will discuss the methods that researchers have developed, how they are being used for clinical practice, and how we can lease you the equipment you need to produce effective delivery systems.

What Is CRISPR-Cas9 Gene Editing?

Before we can discuss how to deliver CRISPR-Cas9 systems, we need to discuss what is being packaged. The CRISPR-Cas system is an adaptive immune system found in bacteria and archaea. This machinery protects its hosts from genetic elements introduced by phages and other microorganisms from the environment. The system consists of four components that coordinate the breakdown of foreign DNA. They are:

• Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs): CRISPRs are DNA sequences that form a series of repeated palindromic sequences within a region of bacterial DNA. These sequences come from plasmids and bacteriophages, viruses that exclusively infect bacteria. It is from these sequences that CRISPR-Cas identifies foreign DNA to destroy in cells.
• The Cas endonuclease: Cas is a DNA nuclease that cleaves the DNA sequences after being targeted. The enzyme, with the help of a gRNA sequence, identifies target DNA sequences to be broken down and cuts them apart.
• CRISPR-associated RNA (crRNA): crRNA comprises the first of two components of the guide RNA (gRNA) sequence. As its name suggests, gRNA guides the Cas nuclease to the area where the DNA will be cut. The crRNA is the mRNA transcript produced from the CRISPR array. This sequence is what makes the guiding possible.
• Trans-activating CRISPR (TracrRNA): TracrRNA is the other component of the gRNA sequence. The RNA sequence is one of the most abundant small RNAs in cells. The molecule acts as a scaffold for the Cas nuclease to bind and cleave the target DNA sequences.

When each component comes together, the Cas9 enzyme forms a ribonucleoprotein complex (RNP complex) that forms double-stranded breaks (DSBs) at specific DNA regions. When researchers adopted the system for cas9 gene editing, they generated an editing tool that comprises the following components:

• Single guide RNA (sgRNA): sgRNA is an RNA oligonucleotide sequence that combines the CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA) elements of a CRISPR-Cas system into a single sequence.
• Cas9 protein: The cas9 protein is a member of the Cas9 family of enzymes. The sgRNA guides the cas9 protein to target genomic regions, cut the genomic region of interest, and replace it with the edited DNA sequence. In gene therapy, this process is known as Cas9-mediated insertional mutagenesis.
• Repair template: The DNA molecule contains the new DNA sequence to be integrated after CRISPR-Cas9-mediated cleavage.

Put together, the RNP complex mediates gene editing through homology-directed repair (HDR). In homology-directed repair, DSBs yield gene knockouts by deleting the target gene. HDR of the repair DNA template then produces a knock-in with the DNA to be integrated into the cell. When done properly, CRISPR-mediated gene editing is a rapid process that can take as quickly as seconds to complete.

The Basic Components of CRISPR-Cas Delivery Systems

A functional gene editing system only operates well when it can be transported into the nuclei of primary cells, where genetic information is stored. As such, researchers have sought different approaches to develop robust gene delivery methods. Any successful delivery of CRISPR components and CRISPR genes requires two components: the cargo and the vehicle. The cargo comprises the components of the CRISPR system, whether encoded in genes or provided as proteins. On the other hand, the vehicle encapsulates the cargo for their in vivo delivery.

The CRISPR-Cas machinery can be supplied as cargo in one of three ways:

• DNA plasmid: DNA plasmids are circular DNA molecules smaller than whole genomes. Researchers engineer plasmid DNA to encode the components of the CRISPR-Cas system. Using a plasmid ensures that gene expression of the Cas9 RNP can occur once the plasmids are introduced into the cells. However, the cells must express and produce the cas9 RNP themselves, resulting in higher editing times and increasing the risk of off-target effects.
• mRNA delivery: In mRNA delivery, the transcripts encoding the Cas9 RNP are provided directly to the cells. Like DNA plasmid delivery, every CRISPR component is encoded within the transcript. Unlike plasmids, however, mRNA transcripts have short half-lives. This minimizes Cas9 protein expression and limits the duration of gene editing. On the other hand, mRNA is easy to degrade, and keeping them intact during delivery is a challenge.
• Cas9 enzyme and gRNA together: When the enzyme and gRNA are combined, the complete RNP complex is formed, enabling RNP delivery directly into cells. As such, this is the most widely used form of cargo for delivering the CRISPR-Cas machinery into target cells.

Delivery Methods for CRISPR-Cas Systems

Researchers have several ways to deliver CRISPR-Cas cargo into target cells. These methods can be divided into three distinct categories:

• Physical delivery methods: With physical approaches, researchers introduce the CRISPR-Cas9 cargo into cells using force. To make the cells more amenable to accepting cargo, an opening is created at the cell membrane. Scientists use one of three approaches to open them:
• Microinjection: Microinjection is the most common physical method for delivering CRISPR-Cas components into cells. Microinjection is most often done on the embryonic stem cells of animal models. There, a needle punctures the cell membrane and injects the cargo into target cells. The approach enables cargo of any size and type to be injected into cells. However, microinjection raises the risk of cell death when cells are injected twice in quick succession or even hours apart.
• Electroporation: In electroporation, researchers pulse high-voltage electric current at the cell membranes. The current transiently produces nanosized-diameter pores at the cell membrane. The pores allow the CRISPR-Cas9 cargo to enter cells while enabling the cells to reseal the pores once the cargo is introduced. Its biggest advantage lies in its flexibility; electroporation can be used on many cell types and at various cell cycle stages. Nonetheless, care must be taken to minimize the toxicity of each electric pulse.
• Hydrodynamic delivery: This approach quickly pushes a large-volume solution containing the gene-editing cargo into the host’s bloodstream. This approach is most useful for delivering gene editing content into the liver and other organs connected directly to the bloodstream. However, the approach can also cause organ trauma, which leads to increased blood pressure, liver dilation, and other physiological side effects.‍
• Viral vectors of delivery: This delivery method uses a virus to carry and inject the CRISPR-Cas cargo into target cells. This process is called transduction. Its primary advantage comes from being able to introduce the CRISPR-Cas9 genes into cells with minimal off-target effects and subsequent toxicity.‍
• Adeno-associated virus (AAV): AAVs are single-stranded DNA viruses whose DNA can be modified to contain solely CRISPR-Cas DNA sequences of interest. When using AAVs, researchers prepare a DNA plasmid encoding the components of the cas9 ribonucleoprotein. AAVs can infect many kinds of cells without causing adverse reactions, making them ideal candidates for gene therapy. However, AAVs cannot carry genetic content more than ~4.5-5.0 kilobases (kb) large. For context, the plasmid containing the RNP would take up 4.2 kb of space. In response, researchers searched for alternatives, such as encoding the sgRNA and SpCas9 plasmids into two different AAVs.‍
• Lentiviral vectors: Lentiviruses are a viable alternative to AAVs. Importantly, lentiviruses can carry bulky and complex transgenes while maintaining long-term expression for dividing and non-dividing cells. These viruses, such as HIV-1 particles, are adept at infecting cells. Although HIV-1 causes AIDS, the lentivirus used for CRISPR-Cas editing can hold multiple plasmids that encode every component needed to express the CRISPR-Cas machinery inside target cells.‍
• Non-viral delivery methods: Researchers have also developed polymeric materials to deliver CRISPR-Cas components into target cells. They typically comprise biomolecules such as lipids that conduct the encapsulation. Once the cargo enters the cells, the complexes dissociate so that the cells’ machinery can express the RNP complex.‍
• Lipid nanoparticles: Lipid nanoparticles stabilize nucleic acids inside their packaging and help them get transported into cells. The lipids are cationic, carrying a positive charge that stabilizes its cargo for delivery into cells. Lipofectamine is the first commercialized lipid nanoparticle for molecular delivery. For CRISPR-based applications, the lipofectamine CRISPRMAX has shown substantial promise for delivering the entire Cas9 RNP into cells.‍
• Lipoplexes/polyplexes: Lipoplexes and polyplexes represent any other nanocomplex that packages and delivers cargo into target cells. Zwitterionic amino lipids (ZALs) are one such delivery lipoplex. They combine components of lipids and amino acid peptides to bind and deliver nucleic acids while reducing the risk of degradation before the cargo is released inside cells.‍
• Nanoparticle delivery: Nanoparticles are tiny materials no larger than 100 nanometers in size. Nanoparticles can form polymers viable for delivering CRISPR-Cas9 cargo in vivo. For instance, gold nanoparticles are well known for their high delivery efficiency. When combined, their cationic characteristics can deliver Cas9 RNP and DNA into target cells to correct DNA mutations.

CRISPR-Cas9 technologies can play a key role in pushing therapeutic genome therapies forward. Nonetheless, researchers must still grapple with the immunogenicity of the CRISPR-Cas cargo. Addressing these and other factors will continue to ensure safety by minimizing off-target effects at the gene and protein levels.

Clinical Applications: Implications for Gene Therapy

As researchers develop ways to ensure the delivery of CRISPR cargo into cells, genome editing has seen more use in developing novel therapeutics.  Some of the clinical applications that robust gene delivery methods have helped develop include:

• CAR T cell engineering: Researchers have sought to enhance immune activity against cancers without hurting our body’s cells. CAR T cell engineering represents one such approach. CAR T cells are T cells with the added ability to identify cancer cells for killing. These specialized T cells are edited with a CAR transgene, a foreign gene that encodes a tumor-specific antigen receptor. In CAR T cell engineering, researchers typically use viral delivery approaches such as AAVs to deliver the cas9 system cargo into T cells.
• Vaccines: Researchers have also used the CRISPR-Cas9 machinery as a vaccine. For instance, scientists used a lentivirus vector to carry CRISPR-Cas9 cargo and remove genes encoding the PD-1 receptor. The deletions prevented tumor cells from downgrading immune responses against them by binding to PD-1.
• Treating genetic diseases: Recent efforts have begun to see clinical trials for CRISPR-based gene therapies against diseases. Particularly exciting within this field is the FDA examining a CRISPR-based gene therapy for treating sickle cell disease. Developed by Vertex Pharmaceuticals and CRISPR Therapeutics, the treatment employs electroporation to introduce gRNA and the Cas9 protein into human hematopoietic stem cells and progenitor cells (HSPCs).

Key Technologies for Successful CRISPR-Cas9 Delivery

Researchers have multiple technologies available for delivering gene editing cargo. Maximizing the editing efficiency of this system, however, requires scientists to conduct a series of quality-control experiments. These experiments ensure that the cargo remains intact upon delivery and that the cells express the Cas9 machinery.

Aware of the growth in the gene editing sector, Excedr has established a leasing program to accelerate molecular genetics research. Although we do not hold an equipment inventory, we can acquire the instruments you need from your manufacturer of choice. Through our leasing program, we can supply you with the equipment you need to assess whether you successfully delivered the CRISPR-Cas machinery into your cells:

• Cell counters: Cell counters help researchers determine the number of viable and non-viable cells in a cell culture. Knowing the total viable cell counts in vitro is essential for a successful delivery. For one, various factors relating to cell counts affect the efficiency of mammalian cell transfections, including cell seeding density, incubation time, and cargo dosage. Knowing how many live cells you have in your preparations is essential for successful transfection. Hemocytometers, through the Trypan blue stain, provide an easy, automated process for counting viable cells in CRISPR-Cas delivery experiments. Check out some of the brands we have leased from here.
• Flow cytometers: Flow cytometers measure multiple cellular characteristics in a solution. Aside from size, eukaryotic cells can be stained with fluorescent dyes. When excited by a point light source at specific wavelengths, the dyes are excited, emitting light through the photoelectric effect. This phenomenon is regularly harnessed in confocal microscopy, another technology that characterizes and visualizes cell populations. Check out the brands of flow cytometers we lease from here.
• PCR systems: Successfully integrating DNA into the genome requires amplifying genes encoding the CRISPR-Cas machinery or the gRNA being sent into the target cells. Amplification with PCR features primers and other reagents associated with DNA replication to amplify gene fragments of interest. Along with its derivative, qPCR, PCR is a vital tool for determining the gene editing efficiency in edited cells. Check out the brands of PCR and qPCR thermocyclers we lease from here.

Lease Essential Editing Tools with Excedr

The in vivo delivery of the CRISPR-Cas9 RNP complex into cells requires a robust vehicle for transporting the genome editing cargo into cells. Knowing that the cargo was delivered and that the target genes were successfully edited involves a slew of technological equipment to conduct quality-control experiments. From PCR thermocyclers to cell counters, researchers must find affordable ways to own and operate the equipment.

Excedr’s leasing program can help advance your laboratory’s gene editing efforts by procuring the equipment your lab needs for developing gene therapies. From reducing upfront costs to extending your cash runway, speak with our team today to learn exactly how we can help.