The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and its associated protein (Cas9) system is a genome-editing technology that holds the potential to alleviate multiple genetic disorders.1
However, this gene editing approach has a risk of off-target DNA cleavage that might lead to unwanted or even adverse alterations in the genome.2
Image Credit: Gohang/Shutterstock.com
Understanding CRISPR-Cas9 Off-Target Effects
In bacteria and archaea, the CRISPR-Cas9 system functions as the RNA-based adaptive immune system. This system confers resistance to viruses by introducing short repeats of the viral DNA into the bacterial genome. Therefore, when these bacteria are infected, the transcripts of these repeats guide a nuclease to the complementary DNA of the virus and destroy it.3
Scientists adopted CRISPR-Cas9 as a gene editing technology that uses short guide RNAs (gRNAs) to assist Cas9, a DNA-cleaving enzyme, to reach its genomic target DNAs.4 CRISPR-Cas9 is often called a molecular scissor made of RNA and enzymes.
Even though this technology holds immense potential in medicine, there is a risk of off-target editing, which could lead to unfavorable outcomes. Off-target DNA cleavage could occur when the molecular scissor targets the wrong section of the host genome, resulting in unwanted or unexpected genetic modification.2
Different guide RNA structures and variable configurations also influence the cleaving of on-target and off-target sites.5
Off-target cleavage activity occurs on DNA sequences with only three to five base pair mismatches in the protospacer adjacent motif (PAM) -distal part of the sgRNA-guiding sequence. Generally, mismatches are more easily tolerated by gRNAs at their 5′ end than at their 3′ end.
CRISPR Explained
Risks of CRISPR-Cas9 Off-Target Gene Editing
An off-target gene editing by CRISPR-Cas9 could result in the introduction of new genetic mutations instead of fixing them.6 An off-target mutation in critical genes could disrupt normal cellular activities and potentially cause adverse conditions or diseases.
It could also lead to a wide array of unanticipated serious consequences, including oncogene activation, undesired gene activation or inactivation, cytotoxicity, and immune responses.
Off-target genome editing could significantly reduce the therapeutic potential of CRISPR-Cas9.1 Furthermore, it could create doubts about the existing information about certain genetic activities.
Emerging Technologies in Forensic Science: From CRISPR to AI
Strategies to Reduce Off-Target Gene Editing
Scientists have developed multiple strategies to reduce CRISPR-Cas9 off-target effects, including off-target detection, sgRNA optimization, and Cas9 nuclease modification.6 Some of the important strategies are discussed below:
Detection
Several in silico tools, such as CasOT, Cas-OFFinder, FlashFry, and Crisflash, can predict CRISPR/Cas9 off-target effects.5 Many of these tools are open-source online software and can be easily accessed using internet services. The majority of prediction algorithms are based on sgRNA sequences, increasing the risk of bias towards sgRNA-dependent off-target effects.
Besides computational methods, several cell-free and cell-culture-based experiments are designed to identify off-target DNA cleavage.2
Digenome-seq (digested genome sequencing), CIRCLE-seq (circularization for in vitro reporting of cleavage effects by sequencing), and SITE-seq (selective enrichment and identification of tagged genomic DNA ends by sequencing) enable direct identification of genomic cleavages in cells.
Discover–seq (discovery of in situ Cas off-targets and verification by sequencing) method helps detect off-target effects in tissues and even in living organisms. Detection of CRISPR-Cas9 off-target effects in tissues is extremely crucial to ensure the safety of genetic modifications.
sgRNA Improvement
Various guided RNA modifications, such as sgRNA length, GC content, truncated sgRNA, and chemical modification, have reduced off-target effects.6
GC content in the gRNA sequence between 40% and 60% elevates on-target activity because higher GC content brings stability to DNA: RNA duplex and destabilizes off-target binding.
The length of sgRNA determines its specificity to a particular Cas nuclease. A shorter sgRNA sequence reduces the off-target effect without impacting the gene editing effect.
Chemical modifications, such as the incorporation of 2′-O-methyl-3′-phosphonoacetate at specific sites in the ribose-phosphate backbone of sgRNAs, significantly decrease off-target cleavage activities and improve on-target performance.7
Cas Variants
Besides protein engineering, another method to effectively reduce off-target effects is by employing CRISPR nickase to modify one nuclease domain in only one strand of DNA.6
Unlike normal Cas9, Cas9 nickase breaks down only one DNA strand, thereby reducing the likelihood of further damage to the target DNA. This strategy reduces unwanted mutations in the genome.
The discovery of novel Cas9 homologs with rarer PAM sequences that have a lower affinity to bind to non-targeted genomic DNA can effectively reduce the off-target effect.
For example, in contrast to SpCas9, which uses a relatively common PAM sequence, utilization of the SaCas9 produced from Staphylococcus aureus reduces off-target effects because it requires a more complex PAM sequence to bind.8
Scientists at the University of Texas at Austin redesigned Cas9 and named the new version SuperFi-Cas9, which is 4000 times less likely to cut off-target sites.
Delivery Method Improvement
The activity of gene editing is significantly dependent on the duration and expression levels of the editors in the cell. The methods used to deliver Cas9/sgRNA into the target cells strongly influence its off-target effect.
Generally, Cas9/sgRNA is delivered via viral transduction, ribonucleoprotein (RNP) electroporation, or plasmid transfection method.2 Compared to all delivery methods, RNP electroporation exhibited higher on-target editing efficiency and lower off-target mutations.
To improve genome editing fidelity, Cas9 mRNA and sgRNA can delivered using liposome-based vectors. Lipid nanoparticles (LNPs)-delivered Cas9 mRNA and sgRNA can be quickly degraded in vivo, making it a most popular vector for in vivo gene editing.
The Role of CRISPR in Developing Next-Generation Antibiotics
Future Outlook
The profound potential of gene editing technology in medicine inspires scientists worldwide to improve the efficiency and specificity of CRISPR/Cas9-based editing of the genome.
New strategies are required to enable direct assessment of off-target effects in vivo, particularly in patients under treatment. Advancements in genome editing tools could accelerate the arrival of the gene therapy era.
References
- Li T, et al. CRISPR/Cas9 therapeutics: progress and prospects. Sig Transduct Target Ther. 2-23;8, 36. https://doi.org/10.1038/s41392-023-01309-7
- Guo C, Ma X, Gao F, Guo Y. Off-target effects in CRISPR/Cas9 gene editing. Front Bioeng Biotechnol. 2023;11:1143157. doi: 10.3389/fbioe.2023.1143157
- Zhang XH, et al. Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. Mol Ther Nucleic Acids. 2015;4(11):e264. doi: 10.1038/mtna.2015.37.
- Asmamaw M, Zawdie B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing. Biologics. 2021;15:353-361. doi: 10.2147/BTT.S326422.
- Naeem M, et al. Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing. Cells. 2020;9(7):1608. doi: 10.3390/cells9071608.
- Mengstie MA, et al. Recent Advancements in Reducing the Off-Target Effect of CRISPR-Cas9 Genome Editing. Biologics. 2024;18:21-28. doi: 10.2147/BTT.S429411.
- Ryan DE, et al. Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Res. 2018;46(2):792-803. doi: 10.1093/nar/gkac110.
- Yang ZX, et al. Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing. Genomics Proteomics Bioinformatics. 2023;21(6):1206-1220. doi: 10.1016/j.gpb.2022.12.003.
Further Reading
New Class of CRISPR-Cas Enzymes for Precise Genome Editing