The CRISPR-Cas9 system has completely subverted the fields of gene function research, gene therapy, and biotechnology development with its unprecedented convenience and efficiency. It is known as gene scissors, and its core charm lies in its ability to achieve accurate targeted editing. However, this powerful targeting ability is accompanied by an inherent and crucial challenge, that is, the off-target effect.

Off-target effect means that the CRISPR system cuts at unexpected sites in the genome, which may lead to unpredictable gene mutation, thus posing a fundamental threat to the reliability of basic research, the safety of agricultural applications, and the success or failure of clinical treatment. This paper aims to deeply discuss the core paradox of on-target and off-target effects in CRISPR technology, and systematically expound its definition, molecular mechanism, influence, and coping strategies, in order to provide a comprehensive and profound understanding framework for researchers and promote the development of this technology in a safer and more reliable direction.

What is the Goal: Defining On-Target Editing

The ultimate ideal of CRISPR-Cas9 technology is to realize accurate and efficient on-target editing. The so-called on-target editing refers to the process that Cas9 nuclease recognizes and binds to a preset genome target site (target site) under the precise guidance of single-stranded guide RNA (sgRNA), and produces a double-strand break (DSB) of DNA at this site. This process is the cornerstone of all subsequent gene editing operations. A successful on-target editing event depends on a highly specific molecular "lock and key" mechanism:

Recognition of PAM Sequence: The Key for CRISPR-cas9

PAM (Protospacer Adjacent Motif) is a short sequence located beside the target DNA sequence (for commonly used Streptococcus pyogenes Cas9, namely SpCas9, its PAM sequence is 5'-NGG-3'). The Cas9 protein first scans the whole genome, looking for the PAM sequence. The existence of PAM is the absolute prerequisite for Cas9 to interact with DNA and start the subsequent cleavage procedure. Without the correct PAM, even if the sgRNA sequence is completely complementary to a certain DNA, Cas9 can't cut it there. Therefore, PAM recognition is the first step and the most basic threshold for editing on the target.

Complementary Pairing of sgRNA and Target DNA: Accurate Location

Once Cas9 is anchored to DNA through PAM, the guiding sequence of sgRNA will be base-complementarily paired with the DNA sequence upstream of PAM. When sgRNA and target DNA sequence are nearly perfectly complementary, Cas9 protein will undergo conformational changes, activating its HNH and RuvC nuclease domains. HNH domain cleaves the DNA strand complementary to sgRNA, while RuvC domain cleaves the non-complementary strand, resulting in a blunt-ended double-stranded break at the target site.

Realization of DNA Repair and Editing Results: from Breakage to Change

Cells themselves have a powerful DNA damage repair mechanism. CRISPR technology uses these repair mechanisms to achieve the ultimate goal of gene editing. After the target site produces DSB, it will mainly trigger two repair pathways:

• Non-Homologous End Joining (NHEJ): This is an error-prone repair mechanism. Cells will directly reconnect the ends of broken DNA, but this process often introduces small fragments of insertions or deletions. If an indel occurs in the coding region of the gene, it is very likely to lead to a frameshift mutation, thus inactivating the target gene. This is the most important way to achieve gene knockout.
• Homology-directed repair (HDR): Under the condition of providing an exogenous DNA repair template, cells can use this template to repair DSB accurately by homologous recombination. This enables researchers to achieve accurate gene typing and point mutation correction.

Therefore, an efficient on-target editing means that a high proportion of DSB occurs at the expected site, successfully induces the expected cell repair path, and finally produces the expected genotype change. Measuring the editing efficiency on the target is the first step of any CRISPR experiment, which is usually evaluated by techniques such as T7E1 restriction enzyme detection, TA clone sequencing, or NGS.

Precise genome-editing with site specific nucleases (Zheng et al., 2024)

The Unintended Consequence: What Are CRISPR Off-Target Effects

Like any powerful tool, the CRISPR-Cas9 system is not perfect. The most obvious and worrying side effect is the off-target effect. Off-target effect refers to the cleavage of the CRISPR-Cas9 complex at unexpected sites similar to the target sequence in the genome, which leads to DNA double-strand breaks and subsequent mutations at these sites.

The off-target effect is the inevitable result of the internal working mechanism of CRISPR technology. The core problem is that the recognition of DNA sequence by the Cas9-sgRNA complex allows a certain degree of mismatch tolerance. In other words, even if there is a mismatch, insertion, or deletion of several bases between the guide sequence of sgRNA and the DNA sequence, as long as the PAM sequence is correct and there is enough complementarity in the key region (especially the seed region near PAM), Cas9 may still identify it as a target and cut it. Miss distance effect can be divided into several types according to its location and characteristics:

Miss Distance Based on Sequence Similarity

• Complete complementary miss-target: Some genomic sites may be exactly complementary to the sgRNA guiding sequence and have the correct PAM. Although this situation is rare, once it exists, the probability of a miss is extremely high.
• Mismatch tolerance miss: This is the most common type of miss. There is a mismatch of 1 to 5 or more bases between the DNA site and sgRNA sequence, but it is still recognized and cleaved by Cas9. The risk of mispairing is closely related to the number, location, and type of mismatch (such as a G-T mismatch, which is relatively stable).

Off-target Based on Genomic Position

• Expected miss: A genomic site that is highly similar to the target sequence and can be predicted by bioinformatics tools (such as Cas-OFFinder, CRISPOR).
• Unexpected miss: It occurs at a site with low similarity to the target sequence and is difficult to predict by conventional algorithms. This may be related to the complex factors, such as the three-dimensional structure of chromatin and the accessibility of local DNA, which is the most challenging part of off-target detection.

Other Types of Unexpected Editing

In addition to the traditional double-strand breaks, the CRISPR system may also cause other unexpected effects, such as:

• Chromosome translocation: When there are two or more DSBs (both on target and off target) in the same cell, the broken ends may be wrongly connected, leading to a wide range of chromosome structural variation.
• Deletion and rearrangement of large fragments: In or around the target site, sometimes large-scale deletion or rearrangement of DNA sequences will occur far beyond expectations.
• Transgenic integration of Cas9: When Cas9 is delivered by plasmid DNA, its coding sequence may be randomly integrated into the host genome, which brings long-term expression and safety risks.

The existence of off-target effects means that after CRISPR editing, the genome of the cell may have uncontrollable changes at one or more unknown sites, which constitutes the most important safety hazard in the application of this technology.

Differences between the CRISPR-Cas9 and base editors methodologies (Preta et al., 2023)

Why Does Cas9 Make Mistakes: Key Mechanisms

In order to effectively avoid and reduce the miss effect, we must deeply understand the root causes of it. The mistake of Cas9 is not a random event, but is determined by its molecular structure and cell environment.

Mismatch Tolerance: The Core Fuzzy Matching Mechanism

This is the core mechanism that leads to off-target effects. The binding of the Cas9-sgRNA complex to DNA target is a dynamic and multi-step process. Studies have shown that Cas9 does not require all 20 bases of sgRNA and DNA to be perfectly matched. It can tolerate a certain number of base mismatches, especially at the 5' end of sgRNA, far from the PAM sequence.

The structural basis of this tolerance is that the combination of Cas9 and DNA will form an R-loop structure, in which the stability of sgRNA-DNA heteroduplex determines whether cleavage occurs. Even if there is a mismatch, cleavage will occur as long as the energy state of the whole complex is stable enough to trigger the conformational change of Cas9.

Key Role of Seed Sequence: The Core Area of Accuracy

Not all positional mismatches will be treated equally. In the sgRNA guiding sequence, about 10-12 nucleotide regions immediately upstream of PAM are called "seed sequences". This area is very important for the identification and cutting of Cas9. The bases in the seed sequence must be highly complementary to the target DNA, and any mismatch will greatly weaken the cleavage activity.

On the contrary, at the 5' end of sgRNA, the tolerance of mismatch is much higher. This explains why the differences between many off-target sites and target sequences are mainly concentrated at the 5' end, while the seed sequences remain highly consistent.

Variant Identification of Sequences: Looseness of the First Level

Although Cas9 has strict requirements on PAM sequences (for example, SpCas9 requires NGG), research shows that Cas9 can recognize and tolerate some non-classical PAM variants, such as NAG and NGA, in some cases, although its efficiency is far lower than that of standard NGG PAM. This loose recognition of PAM greatly expands the library of potential off-target sites, so that some sites that only meet the requirements of loose PAM and partial sequence similarity may also be cut.

Influence of Cell Environment: Genome is Not A Homogeneous Substrate

The genome does not exist in a uniform linear state in the nucleus, but is tightly wrapped in chromatin. The open and closed state of chromatin (accessibility) significantly affects the cutting efficiency of Cas9.

• Open chromatin region: regions with active transcription, such as gene promoters and enhancers. The chromatin structure is loose, DNA is easy to contact, and Cas9 is easier to combine and cut. Therefore, even if the sequence similarity of an off-target site is not the highest, if it is located in a highly open chromatin region, the risk of editing it may be much higher than that of a site with higher sequence similarity but located in heterochromatin (closed state).
• Epigenetic modification: DNA methylation and histone modification may also indirectly affect the binding efficiency of Cas9, which increases the complexity of off-target prediction.

Expression Level and Action Time: Toxicity of Dose and Time

Experiments show that the expression level of Cas9 and sgRNA in cells and their duration are positively correlated with off-target effect. When a strong promoter (such as CMV) is used to continuously express Cas9 and sgRNA through the plasmid, a high concentration of CRISPR components will be maintained in the cell for a long time, which greatly increases the probability that Cas9-sgRNA complexes can find and cleave off-target sites with low similarity. High concentration and long exposure time amplify the fuzzy matching characteristics of the system.

To sum up, the off-target effect is the inevitable product of the interaction between the intrinsic molecular characteristics of the CRISPR-Cas9 system and the complex cellular microenvironment. It is not a bug that can be completely eliminated, but a system feature that must be strictly managed and monitored.

Protection of mismatch target sequences from Cas9 digestion (Handelmann et al., 2023)

The Impact: Why Off-Target Effects Matter for Research and Therapy

The potential consequences of off-target effects are far-reaching and multi-level, which directly challenge the reliability, safety, and ethics of CRISPR technology application.

Influence on Research: Data Misinterpretation and Conclusion Distortion

In functional genomics research, the goal is to clarify the functions of specific genes. If an undetected off-target mutation occurs in a gene knockout experiment, and this mutation just leads to the observed phenotype, then researchers may mistakenly attribute the phenotype to the knockout of the target gene, thus drawing a completely wrong conclusion. This misinterpretation will pollute scientific literature, waste huge scientific research resources, and hinder the correct understanding of life phenomena. Therefore, when publishing the research results based on CRISPR, it is gradually becoming a rigid requirement for academic journals to provide rigorous miss-effect analysis data.

Fatal Threat to Gene Therapy and Clinical Application: Safety Risk

This is the most concerning field of off-target effects. When CRISPR technology is applied to the human body for disease treatment, the risk caused by the off-target effect is fatal.

• Cancer risk: This is the most worrying risk. If off-target cleavage occurs in or within the regulatory region of a proto-oncogene, it may accidentally activate its expression, leading to cancer. For example, the famous p53 gene is an important tumor suppressor gene, and its inactivation is the common pathway of many cancers. An undiscovered off-target mutation that leads to the loss of p53 function may sow the seeds of cancer.
• Risk of loss of function: Off-target effect may destroy important housekeeping genes, metabolic genes, or immune-related genes, leading to cell dysfunction or even death, affecting the therapeutic effect or triggering new diseases.
• Immunogenicity and Long-term Risk: Continued expression of Cas9 protein may trigger an immune response. In addition, when editing in germ cells or early embryos, off-target mutations may be integrated into the genome and passed on to future generations, which will bring irreversible and far-reaching ethical and social problems.

Challenges to the Safety and Public Acceptance

In the agricultural field, using CRISPR technology to cultivate crops and livestock with disease resistance, high yield, and enhanced nutrition has great potential. However, if the edited organism carries an unknown off-target mutation, it may affect its nutritional composition, introduce new allergens, or have an unpredictable impact on the environment. This is not only about food safety and ecological safety, but also directly affects the public's trust and acceptance of this new technology. Strict off-target assessment is the premise of obtaining regulatory approval and consumer recognition.

Bottleneck of Technology and Business Development

For biotech companies that are committed to developing CRISPR therapies and products, the off-target effect is the core obstacle to whether their products can pass the approval of drug regulatory agencies. In clinical trials, sufficient data must be provided to prove that the off-target risk of therapeutic products is at an acceptable low level. Any safety incident related to off-target may lead to the failure of the whole R&D project, causing huge economic losses and hitting the confidence of the whole industry.

Components of AI predictive pipelines for CRISPR (Abbasi et al., 2025)

Conclusion

The paradox of on-target and off-target presented by the CRISPR-Cas9 system profoundly reflects the double-edged sword characteristics of modern biotechnology. It not only gives us the magical ability to accurately modify the blueprint of life, but also reminds us of the great responsibility that comes with this ability. Off-target effect is not the end point of CRISPR technology, but the core driving force to promote its continuous evolution and improvement.

Faced with this challenge, the scientific community has not been sitting still, but has developed a set of multi-level and systematic coping strategies:

• More accurate tools: The invention of high-fidelity Cas9 variants (such as eSpCas9, SpCas9-HF1, HiFi Cas9) greatly reduced their mismatch tolerance through the protein project.
• Smarter design: An improved sgRNA design algorithm can more accurately predict and avoid potential off-target sites.
• More controllable delivery: Using in vitro transcribed mRNA or directly delivering preassembled ribonucleoprotein (RNP) complex, the CRISPR module is provided instantly, which significantly shortens its action time, thus reducing the off-target risk.
• More stringent detection: The development of unbiased off-target detection technologies at the whole genome level, such as WGS, enables us to draw an edited genome panorama in unprecedented depth and breadth.

Therefore, the application of CRISPR in the future is bound to be an accurate control system of the whole process from "design-delivery-editing-verification". According to the specific application scenario (whether it is basic research, agricultural breeding, or clinical treatment), researchers must establish the analysis and management standards of off-target effects that match their risk levels.

In a word, understanding and solving the off-target effect of CRISPR is the only way for us to release its great potential safely and effectively. We are learning how to polish this powerful "gene scissors" more sharply and accurately, so as to maximize our goals and minimize the unexpected consequences in the grand journey of rewriting the life code. This journey of dancing with precision has just begun.

Uncertainty in CRISPR editing holds you back. CD Genomics provides precise, reliable detection of on-target modifications and genome-wide off-target effects. Utilizing validated, unbiased methods, we deliver the critical data you need to confirm edit specificity, assess potential risks, and ensure the integrity of your results.

FAQ

1. What defines CRISPR on-target editing?

It's when Cas9, guided by sgRNA, binds the preset target via PAM recognition and sgRNA-DNA complementarity, creates DSBs, and triggers NHEJ/HDR to achieve intended genetic changes.

2. What's the most common type of CRISPR off-target effect?

Mismatch tolerance off-target—Cas9 cleaves DNA sites with 1-5 + base mismatches to sgRNA, as long as PAM is correct and seed region complementarity is sufficient.

3. Why does chromatin state affect CRISPR off-target risk?

Open chromatin (e.g., promoters) has loose structure, making DNA more accessible to Cas9. Off-target sites here have higher cleavage risk than those in closed heterochromatin.

4. How do off-target effects distort research results?

Undetected off-target mutations may cause observed phenotypes, leading researchers to mistakenly attribute them to target gene edits, resulting in wrong conclusions.

5. What's a key strategy to reduce CRISPR off-targets?

Using high-fidelity Cas9 variants (e.g., eSpCas9, SpCas9-HiFi) engineered to lower mismatch tolerance, plus optimized sgRNA design and RNP delivery.