CRISPR-offinder is a CRISPR sgRNA design and off-target searching tool for user-defined protospacer adjacent motif (PAM). This tool could help predict off-target activity and design highly specific sgRNA for use in clinical and agricultural applications of CRISPR system.
CRISPR-Cas9/Cpf1/C2c1 mechanisms recognize DNA targets that are complementary to a short CRISPR sgRNA sequence. The part of the sgRNA sequence that is complementary to the target sequence is known as a protospacer. In order for Cas9/Cpf1/C2c1 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9/Cpf1/C2c1 gene.
Recognition of the PAM by the Cas9/Cpf1/C2c1 nuclease is thought to destabilize the adjacent sequence, allowing interrogation of the sequence by the sgRNA, and resulting in RNA-DNA pairing when a matching sequence is present. Cas9 nucleases with alternative PAMs have also been characterized and successfully used for genome editing. It is important to note that the PAM is not present in the sgRNA sequence but needs to be immediately downstream or upstream of the target site in the genomic DNA.
Cas9/gRNA system, one type of the CRISPR/Cas systems, is a kind of engineered endonuclease (Fig1). It consists of two components: Cas9, a protein with DNA nuclease activity can be used universally in this system; and sgRNA, an ~100-nt single guide-RNA, of which the first ~20 nt in the 5'-end is responsible for recognizing the target site DNA in a DNA-RNA complementary manner. Cas9/gRNA recognizes and cleaves the target DNA and causes a DSB (double-strand break), which provides the opportunity of gene mutagenesis and other types of genome manipulation.
CRISPR/Cpf1 is a DNA-editing technology (Fig2). It works analogously to CRISPR/Cas9 which has revolutionized biological research. Like its predecessor, it is derived from a mechanism that bacteria use to prevent genetic damage from viruses. CRISPR/Cpf1 may be better than CRISPR/Cas9 in that Cpf1 is a smaller and simpler endonuclease (a type of enzyme) than Cas9. That simplifies delivery to the cells whose genes need modifying. Two candidate enzymes from Acidaminococcus and Lachnospiraceae display efficient genome-editing activity in human cells.
CRISPR/C2c1 is a DNA-editing technology (Fig3). It works analogously to CRISPR/Cpf1 which has revolutionized biological research. C2c1 system can mediate DNA interference in a 5' - PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage.
The paired-gRNAs strategy is reported to show higher specificity than the above single-gRNA strategy, it consists of three components: Cas9-nickase, a mutant form of Cas9 protein, which has no nuclease activity but nickase activity, only cleaving one of the DNA strands with the assist of one gRNA; and a pair of gRNAs, target two sites with offset no more than tens of nt in the opposite strands of DNA (Fig4). The two close nicks induced by the gRNA pair can cause a DSB. However, single nick in a potential off-target induced by only one gRNA would be difficult to cause DSB.
Potential off-target site is an unwanted target site in the genome. This is an important consideration when application of CRISPR technology. The specificity of the CRISPR system is determined in large part by how specific the sgRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a sgRNA will have perfect match to the target DNA with no homology elsewhere in the genome. In fact, most of sgRNA targeting sequence will have additional sites throughout the genome where partial homology exists.
The PAM sequence is located on the non-complementary strand. In other words, it is on the strand of DNA that contains the same DNA sequence as the target sgRNA. The PAM sequence should not be included in the design of the sgRNA.