A quick guide to CRISPR sgRNA design tools

Abstract

Targeted genome editing is now possible in nearly any organism and is widely acknowledged as a biotech game-changer. Among available gene editing techniques, the CRISPR-Cas9 system is the current favorite because it has been shown to work in many species, does not necessarily result in the addition of foreign DNA at the target site, and follows a set of simple design rules for target selection. Use of the CRISPR-Cas9 system is facilitated by the availability of an array of CRISPR design tools that vary in design specifications and parameter choices, available genomes, graphical visualization, and downstream analysis functionality. To help researchers choose a tool that best suits their specific research needs, we review the functionality of various CRISPR design tools including our own, the CRISPR Genome Analysis Tool (CGAT; http://cropbioengineering.iastate.edu/cgat ).

Figure 1.

The CRISPR-Cas adaptive immune system. Three processes underlie the system, acquisition, expression, and interference. Foreign DNA is shown entering the cell. During acquisition, target DNA (beige; next to the PAM sequence shown in green) is incorporated into the CRISPR locus. Expression involves transcribing target DNA into noncoding pre-crRNAs to which tracrRNAs attach. During interference the Cas9 endonuclease uses these sequences to target foreign DNA for cleavage.

Figure 2.

CGAT example functionality using OsSWEET11. (A) Paste into the box a sequence (or select a sequence from the database). (B) Specify design parameters including target length, the maximum number of tandemly repeated nucleotides, and minimum/maximum GC content (which has been shown to correlate with sgRNA efficiency; Ren et al. 2014). (C) Select a genome to query for potential off-target recognition and hit the ‘Analyze’ button. (D) Evaluate and prioritize targets using sequence identity as well as (E) off-target sequence identity.