Introduction to CRISPR gene editing and off-target effects

CRISPR is diligently investigated as a gene editing tool in various research and clinical applications. It is based on the CRISPR-Cas9 system, which has been repurposed from an adaptive immune response mechanism in bacteria. The system comprises a single-guide RNA (sgRNA) targeting a specific DNA sequence and the Cas9 nuclease that cuts the DNA at the target site. Thus, CRISPR-Cas9 gene knockouts, insertions, or corrections pave the way for studying the genetic causes of diseases and administering gene editing to treat cancer, cardiovascular diseases, autoimmune disorders, and rare genetic deficiencies. While the potential of CRISPR is promising, a key concern poses severe limitations in clinical applications: off-target effects. More specifically, unintended CRISPR editing can result in mutations and genomic instability that may put patients' lives at risk. That is why improving the on-target precision of CRISPR-Cas9 constructs is crucial to ensure their safety and accuracy.

Mechanisms behind off-target activity in CRISPR-Cas9

Role of Cas9 and sgRNA in target recognition

The CRISPR system owes its targeting capability to a specially designed sgRNA containing a 20-nucleotide sequence complementary to the target DNA. When introduced into the host cell, the sgRNA binds the target sequence by complementary base pairing and activates Cas9, which cuts both DNA strands to create a double-stranded break.

A key mediator in this mechanism is a short DNA sequence, the Protospacer Adjacent Motif (PAM), immediately downstream of the target DNA. The Cas enzyme can only bind and cut the target DNA if it recognizes the PAM sequence. Thus, the requirement for PAM sites improves the specificity of the CRISPR gene editing.

Causes of off-target cleavage by CRISPR-Cas nucleases

However, the CRISPR-Cas9 system can occasionally attach to and cut a different site than intended. This can happen for one of the following reasons:

1. Mismatches between sgRNA and DNA: Cas9 can tolerate mismatches between the sgRNA and the target DNA to an extent. Research suggests that the mismatches in the target seed region, containing the 8-12 sequences closest to the PAM, can cause erroneous cleavage when tolerated by Cas9. ¹
2. Sequence similarity and homology in the genome: Genomic regions sharing similar sequences are prone to off-target editing. Therefore, sgRNAs for repetitive or conserved sequences will likely induce unintended cuts.
3. PAM-dependent and PAM-independent off-target events: The PAM sequence is specific to the Cas used in the CRISPR application. For the conventional Streptococcus pyogenes Cas9, the PAM sequence is 5'-NGG-3'. On the other hand, cleavage may occur even in the absence of PAM sites.
4. Cas9 structure and binding dynamics: The target sequence's guanine-cytidine (GC) content can increase the risk of off-target editing. While sufficient GC content is necessary for stabilizing the CRISPR-Cas9 structure, excessive GC content (e.g., poly-G sequences) can cause Cas9 misfolding, potentially triggering cleavage at the wrong site. ²

Consequences of off-target effects

Impact of off-target mutations

In CRISPR-Cas systems, the double-stranded break after DNA cleavage is fixed by the host's DNA repair mechanism, which creates random mutations at the site. In successful CRISPR-mediated gene therapy, these mutations result in gene knockout and loss of function, preventing the gene from exerting disease-promoting activity. On the other hand, when the mechanism acts on a random location in the genome, it may disrupt essential genes and interfere with regulatory biological pathways. Accumulation of off-target mutations compromises genomic integrity.

Implications for therapeutic applications

Off-target mutations and their accumulation have negative implications in therapeutic applications. Unprecedented mutations in the genome can disrupt gene regulatory and signal transduction networks, producing adverse immunogenicity or oncogenesis. For example, unintended mutations increase the risk of carcinogenesis by inadvertently activating oncogenes and inhibiting tumor suppressor genes.³ Furthermore, off-target editing can have more deleterious effects on patients who have diseases characterized by multiple gene defects.

While unintended CRISPR editing is a critical bottleneck in somatic gene therapy, a new set of issues arises for embryonic and germline editing. Off-target germline mutations may cause heritable changes that disrupt cellular functions in future generations. This risk lies at the heart of ethical and legal concerns around CRISPR-Cas9 editing, such as the lack of consent from future generations.

Regulatory agencies, such as the FDA and EMA, account for these implications and implement stringent criteria for evaluating CRISPR-based therapies. Therefore, companies must present them with a comprehensive report that addresses precision and off-target risks.

Factors influencing CRISPR off-target activity

sgRNA design and optimization

sgRNA is vital in directing the Cas enzyme to the target DNA. Precise Cas cleavage requires an effective design of the sgRNA, accounting for the sgRNA sequence, length, PAM proximity, GC content and chromatin accessibility.

Sequence and structural considerations

The most obvious criterion is the custom sequence's complementarity to the target DNA. Errors during this step underlie mismatch-related discrepancies, such as disruptions in sgRNA base pairing and correct Cas cleavage.

The GC content of a sequence determines whether it is an ideal target. The optimal GC content has been reported as 40-60%.² While lower GC reduces efficiency by generating an unstable sgRNA-Cas construct, excessive GC content can make the sgRNA too rigid and increase the chances of misfolding and erroneous Cas cleavage.^1,4

The target site's physical accessibility can also determine CRISPR-Cas precision. Accessibility mainly depends on the region's chromatin structure and epigenetic modifications. For instance, histone modifications may block access to the DNA by tightening the chromatin around it.⁵

Role of Cas9 variants

A powerful strategy to minimize off-target editing is to engineer Cas9 variants with improved target specificity. These variants include high-fidelity Cas9 enzymes, which are more effective at discriminating against mismatches than the traditional Cas9.⁶

Detection and prediction of off-target effects

Experimental off-target detection methods

Risks of unintended mutations can be mitigated by assessing off-target effects during the research and development phase.

Whole Genome Sequencing (WGS) is often the initial detection method. The entire genome is screened before and after editing, and researchers can compare the two versions to identify aberrant edits. However, WGS may miss low-frequency off-target events.

Genome-wide off-target detection methods amplify off-target cuts, making them more sensitive than WGS. Examples include:

1. Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-seq)
2. Digested Genome Sequencing (Digenome-seq)
3. Selective enrichment and Identification of Tagged genomic DNA Ends by Sequencing (SITE-Seq)
4. Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq)

Computational prediction strategies

Computational tools accelerate off-target analysis by predicting off-target sites before CRISPR experiments begin. Early detection can help researchers optimize sgRNA and Cas design.

Bioinformatics tools scan the sgRNA sequence against a reference genome to identify similar sequences that the sgRNA may mistakenly target.⁷

GuideScan offers insights into genome accessibility and chromatin data to verify the biological significance of sites.⁸ Thus, researchers can ascertain whether a computationally predicted mismatch sequence can be accessed by the CRISPR system, revealing the actual risk.

Deep learning tools can improve prediction accuracy by inferring on-target and off-target scores from many sgRNA features.⁹

Challenges in off-target identification

Despite computational and experimental advancements, off-target detection faces several limitations. While genome-wide assays may skip rare off-target events, other methods may falsely detect off-target edits that did not occur. Even when the in vitro methods accurately detect off-target effects in cell lines, the same edits may not occur in vivo. On the other hand, not all off-target cleavage events are functionally significant. Therefore, RNA-seq and phenotypic sequencing must be employed to distinguish between benign and disruptive off-target edits.

Strategies to reduce off-target effects

Several strategies are investigated to balance on-target precision and minimum off-target effects.

1. Rational design of sgRNAs through chemical and structural modifications ^10,11
2. Shortening the sgRNA sequence by 1-2 nucleotides, which increases specificity and reduces mismatch tolerance
3. Regulating Cas9 and sgRNA expression duration to reduce the time that the Cas9 remains active inside the cell. Regulating Cas9 activity after cleavage at the target site prevents further unwanted edits.

References

1. Manghwar H, Li B, Ding X, Hussain A, Lindsey K, Zhang X, et al. CRISPR/Cas systems in genome editing: methodologies and tools for sgRNA design, off‐target evaluation, and strategies to mitigate off‐target effects. Adv Sci 2020;7(6):1902312.
2. Liu X, Homma A, Sayadi J, Yang S, Ohashi J, Takumi T. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system. Sci Rep 2016;6(1):19675.
3. Chehelgerdi M, Chehelgerdi M, Khorramian-Ghahfarokhi M, Shafieizadeh M, Mahmoudi E, Eskandari F, et al. Comprehensive review of CRISPR-based gene editing: mechanisms, challenges, and applications in cancer therapy. Mol Cancer 2024;23(1):9.
4. Jung WJ, Park S-J, Cha S, Kim K. Factors affecting the cleavage efficiency of the CRISPR-Cas9 system. Anim Cells Syst 2024;28(1):75-83.
5. Chen E, Lin-Shiao E, Trinidad M, Saffari Doost M, Colognori D, Doudna JA. Decorating chromatin for enhanced genome editing using CRISPR-Cas9. Proc Natl Acad Sci 2022;119(49):e2204259119.
6. Zhang W, Yin J, Zhang-Ding Z, Xin C, Liu M, Wang Y, et al. In-depth assessment of the PAM compatibility and editing activities of Cas9 variants. Nucleic Acids Res 2021;49(15):8785-8795.
7. Naeem M, Alkhnbashi OS. Current bioinformatics tools to optimize CRISPR/Cas9 experiments to reduce off-target effects. Int J Mol Sci 2023;24(7):6261.
8. Schmidt H, Zhang M, Chakarov D, Bansal V, Mourelatos H, Sánchez-Rivera FJ, et al. Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2. Genome Biol 2025;26(1):1-25.
9. Ding S, Zheng J, Jia C. DeepMEns: an ensemble model for predicting sgRNA on-target activity based on multiple features. Brief Funct Genomics 2025;24:elae043.
10. Riesenberg S, Helmbrecht N, Kanis P, Maricic T, Pääbo S. Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage. Nature Comm 2022;13(1):489.
11. Liu X, Xiong W, Qi Q, Zhang Y, Ji H, Cui S, et al. Rational guide RNA engineering for small-molecule control of CRISPR/Cas9 and gene editing. Nucleic Acids Res 2022;50(8):4769-4783.

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