Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing.

The increasing emergence of Cas9 variants has attracted broad interest, as these variants were designed to expand CRISPR applications. New Cas9 variants typically feature higher editing efficiency, improved editing specificity, or alternative PAM sequences. To select Cas9 variants and gRNAs for high-fidelity and efficient genome editing, it is crucial to systematically quantify the editing performances of gRNAs and develop prediction models based on high-quality datasets. Using synthetic gRNA-target paired libraries and next-generation sequencing, we compared the activity and specificity of gRNAs of four SpCas9 variants. The nucleotide composition in the PAM-distal region had more influence on the editing efficiency of HiFi Cas9 and LZ3 Cas9. We further developed machine learning models to predict the gRNA efficiency and specificity for the four Cas9 variants. To aid users from broad research areas, the machine learning models for the predictions of gRNA editing efficiency within human genome sites are available on our website.

Gene Silencing via PDA/ERK2-siRNA-Mediated Electrospun Fibers for Peritendinous Antiadhesion.

Sustained delivery of small interfering RNA (siRNA) is a challenge in gene silencing for managing gene-related disorders. Although nanoparticle-mediated electrospun fibers enable sustainable gene silencing, low efficiency, loss of biological activity, toxicity issues, and complex electrospinning techniques are all bottlenecks of these systems. Preventing peritendinous adhesion is crucial for their successful use, which involves blocking cellular signaling via physical barriers. Here, a multifunctional, yet structurally simple, cationic 2,6-pyridinedicarboxaldehyde-polyethylenimine (PDA)-mediated extracellular signal-regulated kinase (ERK)2-siRNA polymeric delivery system is reported, in the form of peritendinous antiadhesion electrospun poly-l-lactic acid/hyaluronan membranes (P/H), with the ability to perform sustained release of bioactive siRNA for long-term prevention of adhesions and ERK2 silencing. After 4 days of culture, the cell area and proliferation rate of chicken embryonic fibroblasts on siRNA+PDA+P/H membrane are significantly less than those on P/H and siRNA+P/H membranes. The in vivo results of average optical density of collagen type III (Col III) and gene expression of ERK2 and its downstream SMAD3 in the siRNA+PDA+P/H group are less than those of P/H and siRNA+P/H groups. Consequently, siRNA+PDA+P/H electrospun membrane can protect the bioactivity of ERK2-siRNA and release it in a sustained manner. Moreover, adhesion formation is inhibited by reducing fibroblast proliferation and Col III deposition, and downregulating ERK2 and its downstream SMAD3.