Designer nuclease systems such as CRISPR-cas9 are becoming increasingly popular research tools as a result of their simplicity, scalability and affordability
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), mega nucleases, 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.
Drug Designing: Open Access