Targeted genome engineering enables researchers to disrupt, insert, or replace a genomic sequence precisely at a predetermined locus. One well-established technology to edit a mammalian genome is known as gene targeting, which is based on the homologous recombination (HR) mechanism. However, the low HR frequency in mammalian cells (except for mice) prevents its wide application. To address this...

Adaptive nucleic acid-based immune systems of bacteria and archaea provided new tools for genome editing applications and transcription silencing. Despite the rapid success in developing targetable nucleases based on the clustered regularly interspersed short palindromic repeats (CRISPR)-associated (Cas) enzymes, our understanding of how these ribonucleoprotein complexes recognize and process t...

Genome editing with site-specific nucleases, such as zinc-finger nucleases or transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, is becoming the new standard for targeted genome modification in various organisms. Application of these techniques to the man...

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of...

Gene targeting by homologous recombination at chromosomal endogenous loci has traditionally been considered a low-efficiency process. However, the effectiveness of such so-called genome surgery or genome editing has recently been drastically improved through technical developments, chiefly the use of designer nucleases like zinc-finger nucleases (ZFNs), meganucleases, transcription activator-li...

Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator–like effec...

The recent emergence of targeted nucleases has opened up new opportunities for performing genetic modifications with human pluripotent stem cells (hPSCs). These modifications can range from the creation of a routine knock-out to the more challenging single point-mutation. For both the new and established user, deciding on the best approach for the specific modification of interest can be an ard...

Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, us...

In order to investigate the possible role of RNA secondary structure in determining the efficiency and specificity of mRNA splicing, the structures of sequences at three acceptor splice sites in adenovirus were studied. Transcripts spanning intron-exon junctions were synthesized using SP6 RNA polymerase and analyzed using single and double-strand specific nucleases. Distinctive patterns of nucl...

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human ...