Off-target effects

Extracted Attributes

• Frequency

  Frequent; early CRISPR-Cas9 agents showed >50% of mutations were not on-target

• Mechanism

  Engineered nucleases binding to homologous sequences or tolerating mismatches, leading to off-target double-stranded breaks

• Definition

  Nonspecific and unintended genetic modifications during genome editing

• Manifestations

  Point mutations, deletions, insertions, inversions, translocations, chromosomal rearrangements

• Clinical Impact

  Genotoxic effects, cancer, disruption of vital coding regions, adverse outcomes

• Research Impact

  Confounding variables, misleading and non-reproducible results in biological studies

• Associated Technologies

  CRISPR-Cas9, TALENs, Meganucleases, Zinc Finger Nucleases, Base Editors (CBE, ABE)

• Mismatch Tolerance (Cas9)

  Up to 3 base pairs between sgRNA and genomic DNA

• Mitigation Strategy: Detection

  High-throughput sequencing, RNA-seq (for RNA off-target effects)

• Mitigation Strategy: Nuclease Design

  Developing new Cas9 homologs with rarer PAM sequences (e.g., SaCas9, St1Cas9, St3Cas9), using Cas nickases with two gRNAs

• New Off-target Types (Base/Prime Editors)

  RNA editing, sgRNA-independent DNA editing

• Mitigation Strategy: Computational Prediction

  Tools like COSMID, machine-learning-based tools

Timeline

• Research efforts are focused on improving the specificity of genome editing tools and developing methods to detect and mitigate off-target effects. (Source: Summary, Wikipedia)

  Ongoing

• Development of designer nucleases, computational prediction programs and databases, and high-throughput sequencing to reduce and anticipate mutational occurrence. (Source: Wikipedia)

  Ongoing

• Development of new AI-powered gene editing tools like OpenCrisper-1 by profluent bio to create more effective CRISPR technology, aiming to reduce off-target effects. (Source: Related documents)

  Ongoing

Off-target genome editing

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA ("target"), which they cleave (or "cut"), creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms (non-homologous end joining (NHEJ) and homologous recombination (HR)) and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations. Designer nuclease systems such as CRISPR-cas9 are becoming increasingly popular research tools as a result of their simplicity, scalability and affordability. With this being said, off-target genetic modifications are frequent and can alter the function of otherwise intact genes. Multiple studies using early CRISPR-cas9 agents found that greater than 50% of RNA-guided endonuclease-induced mutations were not occurring on-target. The Cas9 guide RNA (gRNA) recognizes a 20 bp target DNA sequence, which it binds and cleaves to "edit" the DNA sequence. However, target sequence binding can tolerate mismatches up to several base pairs, meaning there are often thousands of possible binding sites which present several experimental and safety concerns. In the research sphere, off-target effects can confound variables in biological studies leading to potentially misleading and non-reproducible results. In the clinical sphere, the major concerns surround the disruption of vital coding regions leading to genotoxic effects such as cancer. Accordingly, the improvement of the specificity of genome editing tools and the detection of off-target effects are rapidly progressing research areas. Such research incorporates designer nuclease development and discovery, computational prediction programs and databases, and high-throughput sequencing to reduce and anticipate mutational occurrence. Many designer nuclease tools are still in their relative infancy and as their molecular properties and in vivo behaviors become better understood they will become increasingly precise and predictable.

Web Search Results

• Off-target effects in CRISPR/Cas9 gene editing - PMC

  Although CRISPR/Cas systems exhibit tremendous potential in translational medicine, off-target effects remain a major challenge (Fu et al., 2013; Hsu et al., 2013; Pacesa et al., 2022). The off-target effects occur when Cas9 acts on untargeted genomic sites and creates cleavages that may lead to adverse outcomes. The off-target sites are often sgRNA-dependent, since Cas9 is known to tolerate up to 3 mismatches between sgRNA and genomic DNA (Fu et al., 2013; Hsu et al., 2013; Wang et al., [...] Although CBE and ABE greatly reduce the classic off-target effects of CRISPR/Cas9 systems, they create new formats of off-target effects such as RNA editing and sgRNA-independent DNA editing (Grünewald et al., 2019; Jin et al., 2019; Zhou et al., 2019; Zuo et al., 2019). These effects are likely introduced by the excessive deaminase activity that are not restricted by the Cas9/sgRNA binding to the corresponding genomic loci. The RNA off-target effects can be detected by RNA-seq (Grünewald et [...] The off-target effect can also be reduced by discovering new Cas9 homologs that use rarer PAM sequences, thereby exhibiting less probability to dock on non-targeted genomic DNA. For example, in contrast to SpCas9, which uses a relatively common 5’-NGG-3’ PAM, the SaCas9 that is derived from _Staphylococcus aureus_ requires a more complicated PAM sequence of 5’-NGGRRT-3’ (Kumar et al., 2018). Similarly, St1Cas9 and St3Cas9 from _Streptococcus thermophilus_ recognize longer PAM sequences, which

• On-target and off-target-based toxicologic effects - PubMed

  Adverse toxicologic effects are categorized as chemical-based, on-target, or off-target effects. Chemical-based toxicity is defined as toxicity that is related to the physicochemical characteristics of a compound and its effects on cellular organelles, membranes, and/or metabolic pathways. On-target refers to exaggerated and adverse pharmacologic effects at the target of interest in the test system. Off-target refers to adverse effects as a result of modulation of other targets; these may be

• Mapping the off-target effects of every FDA-approved drug in ...

  pipelines. After all, off-target effects are among the most common reasons for late-stage trial failures and post-approval black box warnings, and even if the creation of an EvE-like dataset doesn’t fix the problem, I can’t imagine it’d hurt. [...] Yes, that’s a roundabout way of describing ‘off-target effects’ — defined as the action of a drug at a gene product other than the gene product it was intended to affect — but I think it’s a helpful intuition pump. Viewing the drug discovery process as ‘not paying attention to anything that is unrelated to the drug working’ is useful in that it contextualizes the situation we’re in. Drugs are meant to make money, and money is derived from drugs working. To spend time on understanding what else [...] Well, wait a minute. Aren’t we missing something? Off-target effects of a small molecule can be summarized purely by these GPCR/NR measurements, but we’d be failing to capture something else that is of vital importance: whether the drug outright kills the cell. One could imagine this also affecting our receptor experiments! Perhaps a drug is an antagonist and there is no color shift, or perhaps the cell is just dead, and nothing is being expressed at all. Conversely, a drug might look like an

• CRISPR 101: Off-Target Effects - Addgene Blog

  “Off-target effects” a vague catch-all term for unintended consequences in an experiment. For CRISPR work, this often means DNA cleavage at unanticipated sites. While off-target effects are nearly impossible to eliminate, it’s essential to understand them in order to design the most specific assays possible. In this blog, we will discuss when off-targets can be an issue in CRISPR, how to predict and minimize them, and ways to control for and quantify them when they can’t be avoided. [...] unintended sites with sequence similarity to the on-target site. When a CRISPR-Cas system localizes to unintended sites and performs its programmed function there, this is an off-target effect. [...] Off-targets that come from mutations arise from base/prime editing or Cas cleavage. In the case of Cas cleavage, off-target mutations can be mitigated by using two gRNAs in close proximity that guide Cas nickases. When two nicks are generated near each other, this will result in a DNA double strand break (DSB), the event required for frameshift and knock-in mutations. If one nickase acts on an off-target site, it will generate a ssDNA break which can easily be repaired by cells with a near zero

• [PDF] Assessing Genetic Heterogeneity in the Context of Genome Editing ...

  modalities hold great promise for highly specific genetic engineering, it is crucial to critically examine potential off-target effects to refine the techniques and optimize their safety and efficacy. The potential impact of unintended changes, also referred to as off-targets or off-target editing, is a key consideration for the safety of genome editing as a therapeutic strategy. Unintended changes to the genome could be caused by modifying DNA at sites other than those being deliberately [...] This can cause mutations, deletions, translocations, inversions, and other chromosomal rearrangements, which may lead to gain or loss of function. Because of this, off-target effects need to be carefully and exhaustively analyzed. Dr. Bao’s lab created the in silico prediction tool COSMID to identify and rank the potential off-target sites. Bao said it is important to further develop machine-learning-based tools using training and test data sets. In silico tools developed for CRISPR/Cas9 [...] targeted (National Academies of Sciences 2017). Off-target editing can potentially lead to unwanted mutagenesis and chromosomal rearrangements, such as translocations, deletions, and duplications, producing genomic instability. The potential for these off-target events is concerning given the causal function of certain known chromosomal rearrangements in human cancers (Taki and Taniwaki 2006) such as lung cancer (Maddalo et al. 2014) specifically human non-small cell lung cancers (Soda et al.