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Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

MicroRNAs (miRNA) are small noncoding RNAs that are critical regulators of gene function. In the recent years, miRNAs have been increasingly noted for their capacity to regulate key malignant properties of tumor cells. MicroRNA-128 (miR-128) is a brain-enriched miRNA that is normally involved in the development of the nervous system and in the maintenance of neural physiological functions. In tumorcells, miR-128 expression is dysregulated through a variety of genetic and epigenetic events. Dysregulation: of miR-128 has profound effects on tumorigenesis and maintenance of tumor cells through alterations in cellular proliferation, differentiation, metabolism, and apoptosis. This article will review the latest advances in our understanding of miR-128, specifically in the context of clinical and fundamental cancer biology. Further characterization of miR-128 will likely identify its new roles in cancer biology. The use of miR-128 as a diagnostic and/or therapeutic tool may result in improvements in diagnosis, prognosis, and treatment of numerous cancers.