To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a “core” sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of ‘super’ off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.

CRISPR/Cas has been coming to prosperity since its discovery and application.It becomes a standard solution for gene editing in the past few years.A guide RNA is used to lead the endonuclease,such as Cas9 and Cpf1,to specific sites and break the double strand.However,there is also possibility that the system will cut a non-specific position,which is called "off-target effect".The off-target cleavage may cause trouble to gene function research or clinic treatment.In order to reveal the target specificity of Cpf1,this study explored the single-nucleotide mismatches by a dual-luciferase system.Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting.Moreover,rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1,which was same as CRISPR/Cas9.The phenomenon might be attributed to the homology of the two enzymes.In summary,our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9,as this is an intrinsic characteristic of the system.

Recently, molecule-based targeted therapy has achieved remarkable success in clinical, and opened up a new era particulraly in the field of cancer therapy.Monoclonal antibodies and small molecule inhibitors are two pillars of this field.Additionally, a single-strand nucleic acid molecule called "chemical antibody" has also attracted increasing attention.These novel targeting agents are called "nucleic acid aptamers (aptamers)".With high specificity and affinity for a variety of target molecules, nucleic acid aptamers represent a new direction in the gene targeting therapy.In this review, we summarizes the recent development of aptamers and their druggability.

Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.