What are off-target effects of CRISPR and why are they concerning?

Written by Gaetan Burgio, Australian National University

Gaetan Burgio of the Australian National University (Canberra, Australia) discusses the off-target effects of CRISPR, how they occur, how to avoid them and their impacts on the uses of the technique.

CRISPR gene editing technology has been transformative in investigating important biological questions or studying the function of genes. Importantly the technology shows a lot of promise in improving crop or food production, and in detecting or treating diseases such as cancer, infections or severe hereditary disorders. To date, 23 active clinical trials at various stages of progression using CRISPR gene editing technology are registered in ClinicalTrials.gov. Most of the diseases investigated are cancer related, such as a recently published clinical trial on a patient with HIV and leukemia or severe hereditary conditions such as sickle cell anemia, highly prevalent in the African American population. The use of CRISPR gene editing technology could potentially lead to transformative changes, resulting in cure or diagnosis of diseases, and improved food or fuel production. Ensuring safety for gene editing applications, such as treating medical conditions, is paramount for a successful treatment. Ultimately, it will improve lives. Therefore, it is important to determine whether off-target effects are a concern for gene editing applications, the acceptable level for gene therapy or food production, and how to alleviate them. This short overview of the question provides an overall picture of this question and provides guidance on how to detect and minimize off-target effects.

How does the CRISPR-Cas9 enzyme generate off-target effects?

Cas9 is a bacterial enzyme that uses a guide RNA scaffold to direct the DNA cutting. The enzyme scans and unwinds the DNA to create an R loop, changing its conformation to expose the nucleases domains and cleaving the DNA to generate a double-stranded break of the DNA. The specificity of the guide RNA is critical to enable Cas9 to cut the right target and to avoid cutting another piece of DNA (off target). Therefore, the combination specificity of the enzyme/guide RNA is crucial to avoid occurrence of off-target effects. A series of studies has demonstrated that Cas9 is sensitive to mismatches of the guide RNA, which in short means Cas9 is a highly specific enzyme. Additional recent studies further refined Cas9 specificity and have shed light on how off-target effects occur. Briefly, Cas9 binds and hybridizes to off-target sites, but fails to generate a break in the DNA. In short, this means Cas9 is highly sensitive to the nucleotide composition at the target site, which means that off target effects are therefore predictable.

How can we predict off-target effects?

Considerable efforts have been undertaken to better understand and predict how Cas9 off-target effects occur. Development of high throughput sequencing technologies has largely contributed to improvements in the detection and prediction of off-target effects. Some of those are specifically dedicated to detecting off-target effects such as GUIDE-seq, Digenome seq, Circleseq or DISCOVER-seq. In addition to whole-genome sequencing techniques, those techniques have established that Cas9 off-target effects are relatively rare and detectable. In parallel, surveys on the effect of Cas9 from over tens of thousands of guide RNAs have defined and refined how to predict efficiency and off-target effects from a guide RNA nucleotide sequence on a specific organism. These techniques, combined with artificial intelligence such as machine learning, now give an accurate landscape of the frequency and the likelihood of Cas9 off-target cutting. Today, this enables us to predict efficiency and occurrences of off-target effects with high accuracy.

How can we minimize or overcome off-target effects?

Two factors are important to consider in reducing off-target effects. First, controlling the timing of Cas9 delivery and reducing Cas9 availability is critical to avoid them. For example, delivery of Cas9 in a form of ribonucleoprotein (RNP) in a complex with the guide RNA has a short half-life compared to plasmid delivery and consequently, Cas9 degrades rapidly in the cell, which in turn reduces the likelihood of generating an off-target effect. Another important aspect in the reduction of off-target effects is the choice of reagent. Some Cas9 variants such as eSPCas9 or HFCas9 offer high fidelity and low-to-null frequency of off-target effects. More importantly, the guide RNA design is critical in reducing off-target effects. A good design using online available guide RNA design tools such as CRISPOR, CCTOP or GtScan predicts quite well the frequency of off-target effects for a given guide RNA sequence.

Are off-target effects really such a concern?

Accumulation of a large amount of evidence suggests that Cas9 off-target effects are minimal and should be more or less considered for a given specific application. For gene therapy or food production, off-target effects are critical and good guide RNA design combined with the use of highly specific Cas9 and cutting-edge sequencing technologies will ensure the safety of the approach and ultimately will prioritize safety over efficiency. The balance is to determine how acceptable CRISPR off-target effects are from mutations that naturally occur in the genome. However, for studying biology by the generation of genetically modified organisms, the efficiency of the approach will be prioritized over safety as the researcher has the possibility to eliminate off-target effects in subsequent generations. Overall refinement of the gene-editing technology and the introduction of novel techniques will enable us to fully eliminate off-target effects and ensure a safe approach in the use of gene-editing technology for therapy or food production. I would, therefore, argue that given the evolution of CRISPR technology, off-target effects are not such a concern. For a given application definition of the acceptability level for off-target effects will be key to determine the suitability of using CRISPR gene-editing technology.