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The CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and offtarget cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less offtarget mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable 2 diseases caused by genetic mutation.

Objective Various surface modification techniques that can further improve the function and usability of stainless steel as a medical device have been reported. In the present study, the physical and biological properties of nanoporous stainless steel as well as its usefulness for drug delivery were assessed. Methods The specimen was prepared with a circular disk shape (15 mm in diameter and 1 mm in thickness). The disk was subjected to electropolishing at a constant voltage of 20 V and 10 A for 10 min in an acidic environment (50% H2SO4). Everolimus (EVL) was used as a testing drug for drug-loading capacity of the material surface and release kinetics. The physiobiological properties of the material were assessed using platelet adhesion, and smooth muscle cell (SMC) adhesion, migration, and proliferation assays. Results The surface roughness of the postpolishing group was greater than that of the nonpolishing group. Platelet adhesion and SMC adhesion and migration were inhibited in the postpolishing group compared to those in the prepolishing group. In the postpolishing group, the total amount of EVL on the surface (i.e., drug storage rate) was higher and the drug release rate was lower, with half the amount of the EVL released within 4 days compared with only 1 day for that of the prepolishing group. Conclusion Taken together, this stainless steel with a nanoporous surface could be used as a medical device for controlling cellular responses and carrying drugs.

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