The CRISPR Toolbox

Since CRISPR (clustered regularly interspaced short palindromic repeats) gene editing was discovered in 2012, its ability to make precise and permanent changes in the DNA of both animals and plants has been generating excitement. The focus of medical science research to-date has been on diseases caused by a single gene mutation, such as sickle cell anemia (SCA) and beta thalassemia, and the improvement of anti-tumor immunotherapy. The first use of an investigational ex vivo CRISPR-based therapy to treat SCA and beta thalassemia is already underway in at least two patients in clinical trials. 

CRISPR gene editing makes use of enzymes, particularly nucleases, that have been programmed to target specific sequences in the genome and then introduce edits. However, DNA cleavage and editing may occur at additional off-target sites in the genome that have similar but different DNA sequence from that of the intended site.

Although Cas9 is the most commonly used CRISPR nuclease, others do exist, and applications are being developed. In fact, over the last few years, more than 10 different CRISPR/Cas proteins have been engineered for gene editing and there are many more known and being discovered across bacterial species. These other enzymes can work alongside Cas9 or be used to serve different functions – and there will likely be no shortage in the variety to satisfy the targeted nuclease application. 

Though Cas9 remains the best-characterized and most widely used nuclease for gene editing, Cas12a has recently emerged as an alternative (1). There are several unique features of Cas12a that distinguish it from Cas9 – most notably the fact that it works across a broad range of temperatures, including lower temperatures. Additionally, it targets AT-rich regions of the genome, which makes it suitable for editing plants, which are AT-rich. Until recently though, Cas12a was not very efficient at cutting DNA. However, our team has isolated a new and improved variant, Cas12a Ultra, which provides specificity and efficiency as good as that of Cas9, as well as different targeting characteristics.

However, with all gene editing, whether it be in plants or in animals, off-target effects remain an important consideration. Despite its advantages in potency, DNA cleavage and editing may occur at unintended sites throughout the genome that have a similar DNA sequence and differ by typically one to three bases. Often, these events occur in areas of the genome thought to have no function, nevertheless there is always a risk of unintended, adverse consequences. Generally, the longer Cas9 persists in cells, the more opportunity there is for unintended side effects, like off-target effects. My work has focused on assessing the newly engineered Cas9 and Cas12a enzymes that have improved targeting specificity and thus reduced off-target effects. 

As CRISPR technology advances, it is important to balance perspectives on both the potential benefits and risks. It is paramount that we have not only the tools to edit more precisely and effectively, but also tools to check for and eventually address off-target effects.