With the growing influence and applicability of genome editing technologies, TALENs and more recently CRISPRs, offer genetic capabilities to an ever growing list of organisms. In turn, as more and more researchers become interested in using these technologies, a common question is often “How do I begin?”
After deciding the gene or genes of interest to target, both TALEN and CRISPR technologies have specific target site requirements that must be first taken into consideration.
TALEN technologies rely on the production of a TALE repeat domain fused to a Fok1 nuclease domain. To produce a double strand break, target sites must be recognized by two TALENs on opposite DNA strands ~14-20 bp apart in order to promote dimerization and DNA cleavage.
In contrast, the CRISPR/Cas9 system is dependent on a single-guide RNA (sgRNA) to direct target site specificity and Cas9 cleavage. Specificity is defined by a 20 bp target sequence, adjacent to a PAM sequence (NGG) that is recognized by the Cas9 enzyme to initiate cleavage.
Further confounding TALEN or sgRNA design is the need to prevent off-target effects. TALENs and especially sgRNAs (due to their short recognition sequence) require the user to evaluate target specificity to eliminate potential side-effects produced by non-specific genome editing.
To address some of these pitfalls in designing TALENs or CRISPR sgRNAs, Montague et al. (2014) developed a new web based tool, CHOPCHOP, to facilitate with these important first steps in experimental design.
CHOPCHOP is a user friendly web-based tool that identifies both TALEN and CRISPR target sequences using an interactive graphical interface. Accepting a variety of input sequences, CHOPCHOP evaluates candidate sequences for genomic location (coding regions, orientation) and target specificity. In addition, CHOPCHOP integrates primer design to amplify the region of interest for downstream analysis and provides restriction site information that may expedite screening.
Currently available for several model organisms including Drosophila melanogaster and Anopheles gambiae, CHOPCHOP is a significant advancement in the genetic toolbox to ease users into the use of TALEN and CRISPR technologies. With an intuitive and customizable user interface, CHOPCHOP offers many researchers an excellent resource to begin their foray into genome editing technologies.
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