Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase.

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase.

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

[Expression of connective tissue growth factor in sciatic nerve after chronic compression injury and effect of rhodiola sachalinensis on its expression].

OBJECTIVE: To investigate the expression of connective tissue growth factor (CTGF) in the chronic sciatic nerve compression injury and to explore the effect of rhodiola sachalinensis on the expression of CTGF. METHODS: Forty-five adult male Sprague Dawley rats were randomly divided into groups A, B, and C: In group A (sham-operated group), only the sciatic nerve was exposed; in group B (compression group), sciatic nerve entrapment operation was performed on the right hind leg according to Mackinnon method to establish the chronic sciatic nerve compression model; and in group C (compression and rhodiola sachalinensis group), the sciatic nerve entrapment operation was performed on the right hind leg and rhodiola sachalinensis (2 g/mL) was given by gavage at a dose of 0.5 mL/100 g for 2 weeks. The nerve function index (SFI) was observed and neural electrophysiology was performed; histology, transmission electron microscope, real-time fluorescent quantitative PCR, and Western blot were performed to observe the morphological changes of the compressed nerve tissue and to determine the mRNA and protein levels of CTGF, collagen type I, and collagen type III at 2, 6, and 10 weeks after operation. RESULTS: At 6 and 10 weeks after operation, SFI of groups A and C were significantly better than that of group B (P < 0.05), but there was no significant difference between groups A and C (P > 0.05). The nerve function test showed that the nerve motor conduction velocity (MCV) and the amplitude of compound muscle action potential (CMAP) of group B were significantly lower than those of groups A and C, and distal motor latency (DML) was significantly prolonged in group B (P < 0.05), but there was no significant difference between groups A and C (P > 0.05). Histology and transmission electron microscope observations showed that myelinated nerve fibers degenerated and collagen fiber hyperplasia after sciatic nerve chronic injury in group B, and rhodiola sachalinensis could promote the repair of nerve fibers in group C. At 2 weeks postoperatively, the number of myelinated nerve fibers in groups B and C were significantly less than that of group A (P < 0.05), and the myelin sheath thickness of groups B and C were significantly larger than that of group A (P < 0.05). At 6 and 10 weeks postoperatively, the number of myelinated nerve fibers in groups B and C were significantly more than that of group A (P < 0.05); the myelin sheath thickness of group B was significantly less than that of groups A and C (P < 0.05). The effective area of nerve fiber had no significant difference among groups at each time point (P > 0.05). Real-time fluorescent quantitative PCR and Western blot results showed that the mRNA and protein expressions of CTGF, collagen type I, and collagen type III in group B were significantly higher than those in groups A and C at each time point (P < 0.05), but there was no significant difference between groups A and C (P > 0.05). CONCLUSION: Sciatic nerve fibrosis can be caused by chronic nerve compression. The increased expression of CTGF suggests that CTGF plays an important role in the process of neural injury and fibrosis. Rhodiola sachalinensis can significantly reduce the level of CTGF and plays an important role in nerve functional recovery.