CRISPR Off-target Effect: Transformative Role in Biology and Genetic Research

Off-target effects include accidental point mutations, deletions, insertion inversions, and translocations. Due to its simplicity, scalability, and affordability, designing nuclease systems such as CRISPR-cas9 is becoming increasingly popular.

For the application of Cas9, methods must be considered to minimize the degree of off-target cleavage and detect the presence of off-target cleavage. Although many techniques have been developed to minimize the risk of off-target cleavage, in some cases off-target mutations remain the main focus of CRISPR/Cas9-mediated genome editing. These mutations should be carefully monitored, especially when using CRISPR/Cas9 for treatment. However, off-target analysis of the CRISPR/Cas9 system has been very challenging, especially when performed directly in the cell.

In order to better understand and predict how the off-target effect of Cas9 occurs, considerable efforts have been made. The development of high-throughput sequencing technology has greatly facilitated the detection and prediction of off-target effects. Some of them are specifically used to detect off-target effects, such as GUIDE-seq, Digenome seq, Circleseq or DISCOVER-seq. In addition to whole-genome sequencing technologies, these technologies also determined that Cas9 off-target effects are relatively rare and detectable. Meanwhile, a survey of the effects of Cas9 from thousands of guide RNAs has defined and refined how to predict the efficiency and off-target effects of guide RNA nucleotide sequences on specific organisms.

These technologies, combined with artificial intelligence such as machine learning, can now accurately understand the frequency and possibility of Cas9 deviating from the target frequency which allows researchers to predict the occurrence of efficiency and off-target effects with high precision.

There are two factors to consider while reducing off-target effects. First, controlling the delivery time of Cas9 and reducing the availability of Cas9 is essential to avoid this situation. For example, compared to plasmid RNA, the half-life of Cas9 delivered in the form of ribonucleoprotein (RNP) in combination with Cas9 is shorter. Therefore, Cas9 is rapidly degraded in the cell, thereby reducing the possibility of producing ribonucleic acid. Another important aspect to reduce off-target effects is the choice of reagents. Certain Cas9 variants (such as eSPCas9 or HFCas9) can provide high fidelity and low-to-zero frequency off-target effects. More importantly, guiding RNA design is essential to reduce off-target effects.

The off-target effect of Cas9 is minimal and should be considered more or less for a given specific application. For gene therapy or food production, off-target effects are crucial. The combination of good guide RNA design and highly specific Cas9 and cutting-edge sequencing technologies will ensure the safety of the method and ultimately put safety above efficiency. The balance is to determine the acceptable degree of off-target effects of naturally occurring mutations in the genome. However, for biological studies of genetically modified organisms, the efficiency of this method will take precedence over safety, because researchers can eliminate off-target effects of future generations.

The comprehensive improvement of gene editing technology and the introduction of new technologies will enable us to completely eliminate off-target effects and ensure that safe methods are used when using gene editing technology for treatment or food production. For a given application, the definition of the acceptable level of off-target effects is critical to determine the suitability of using CRISPR gene editing technology.https://www.creative-biogene.com/crispr-cas9/crispr-off-target-effects-analysis.html