Delivery Methods to Maximize Genome Editing with CRISPR-Cas9

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:

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:

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:

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:

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.