Assessing the off-target effects of gene editing

Ways of predicting and reducing off-target effects​. By Garrett Rettig, Research Scientist, Integrated DNA Technologies (IDT)

Since CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing was discovered seven years ago, it has been generating a lot of excitement in academic circles and pharma and biotech businesses, as well as driving mainstream conversations. CRISPR gene editing makes use of enzymes, particularly nucleases, that have been programmed to target specific sequences in the genome and then introduce cuts into the DNA strands to edit existing DNA via deletions, sequence changes or insertions. This opens the door to a whole host of opportunities in agricultural science, medicine and microbiology.

While research is still in the early stages, the focus to-date has been on finding treatments and cures for diseases caused by a single gene mutation, including sickle cell anemia (SCA), Severe Combined Immunodeficiency (SCID), commonly known as ‘bubble boy disease’, and beta thalassemia. In fact, the first use of an investigational ex vivo CRISPR-based therapy to treat both SCA and beta thalassemia is already underway in at least two patients, with many more trials likely to begin soon.

With the first human trials in progress and the first gene-edited babies continuing to spark discussion, it is more important than ever to understand the genetic background being manipulated using CRISPR and predict and reduce the risk of off-target effects (OTEs) that may occur. 

Off-target effects of gene editing

While CRISPR gene editing is being considered for numerous applications in animals and plants alike, one cause for concern remains the potential occurrence of OTEs. These concerns are not new. For viral vector-based gene editing, researchers were worried about the potential for a mutagenic vector to integrate into the genome; for zinc finger nucleases, there was concern that the method could cause OTEs according to cell-based experiments.[1] 

In the case of CRISPR, the technology comprises two components: a nuclease (e.g., Cas9), which acts like a pair of scissors and is responsible for cleavage of double-stranded DNA, and a single guide RNA (sgRNA), which forms a complex with the nuclease and guides it to the target site. However, the RNA-guided nuclease may cleave sites in the genome other than the intended one(s). This can occur since multiple sites in the genome can have the same or similar sequences to that being targeted. Additionally, the enzyme might cleave the target site but then go on to cleave other unintended sites thereafter. Another concern is that since no two genomes are identical, OTEs may differ from one individual to the next, making them hard to accurately predict.

There are number of ways researchers are trying to better predict OTEs and determine their effect, one of which is to improve the specificity of the enzymes used in the process.

Improving the CRISPR enzymes 

As the most commonly used enzyme in CRISPR gene editing, Cas9 can be applied to target specific stretches of genetic code and edit DNA at precise locations. However, use of wild type (WT) Cas9 has been shown to have more than 50% of the edits being made off-target. While most of these events occur in areas of the genome thought to have no function, there is always a risk that some OTEs may have unintended, adverse consequences. Many researchers have tried modifying the guide RNA and Cas9 proteins to improve target specificity, but these alterations often also reduce editing performance in terms of nuclease activity. 

IDT recently engineered an effective new Cas9 nuclease by devising an unbiased bacterial screen to isolate a high-fidelity Cas9 that has greater targeting specificity and similar nuclease activity to the WT Cas9. The company's HiFi Cas9 is a highly active and specific high-fidelity Cas9 enzyme, delivered as an RNP complex, which provides optimal targeting specificity. It is these characteristics that make it an ideal option for research and clinical applications. 

A new tool that may provide a solution

In addition to increasing enzyme specificity, researchers have been using web-based algorithms to predict which regions of the genome might be the site of OTEs. While each CRISPR has some known OTEs, predicted by these web-based algorithms, there can be many additional unknown sites.

In response to these concerns, earlier this year, IDT launched a new product that can be used in preclinical CRISPR OTE risk assessments called rhAmpSeq. This new technology enables a researcher to rapidly characterise hundreds to thousands of individual off-target and on-target sites from a single DNA sample using modern next generation sequencing (NGS) methods. 

Where do we go from here?

Current scientific advances have shown that CRISPR gene editing is versatile, precise and increasingly safe and cost effective. There is much excitement about the potential to develop therapies for previously untreatable diseases, from cancers to muscular dystrophy, as well as better understand disease mechanisms and develop personalised treatment options. However, there is still much progress to be made to understand OTEs and their consequences if we are to see the full potential of CRISPR gene editing realised and being used in patient treatments. By developing innovative solutions, IDT hopes to both improve the utility of CRISPR-Cas9 as well as address the need to minimise OTEs, in order to help make CRISPR-based treatments and cures a reality for many patients around the world.

 [1] Keep off-target effects in focus volume. Nature Medicine. 2018(24): 1081.

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