Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53–88% and 44–100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit.
Platelet-rich fibrin (PRF) is an autologous platelet concentrate that consists of cytokines, platelets, leukocytes, and circulating stem cells. It has been considered to be effective in bone regeneration and is mainly used for oral and maxillofacial bone. Although currently the use of PRF is thought to support alveolar ridge preservation, there is a lack of evidence regarding the application of PRF in osteogenesis. In this paper, we will provide examples of PRF application, and we will also summarize different measures to improve the properties of PRF for achieving better osteogenesis. The effect of PRF as a bone graft material on osteogenesis based on laboratory investigations, animal tests, and clinical evaluations is first reviewed here. In vitro, PRF was able to stimulate cell proliferation, differentiation, migration, mineralization, and osteogenesis-related gene expression. Preclinical and clinical trials suggested that PRF alone may have a limited effect. To enlighten researchers, modified PRF graft materials are further reviewed, including PRF combined with other bone graft materials, PRF combined with drugs, and a new-type PRF. Finally, we will summarize the common shortcomings in the application of PRF that probably lead to application failure. Future scientists should avoid or solve these problems to achieve better regeneration.
Background: Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase (nCas9), enable efficient C-toT conversion in various organisms. However, current base editors can induce unwanted bystander C-toT conversions when multiple Cs are present in the~5-nucleotide activity window of cytidine deaminase, which negatively affects their precision. Here, we develop a new base editor which significantly reduces unwanted bystander activities. Results: We used an engineered human APOBEC3G (eA3G) C-terminal catalytic domain with preferential cytidine-deaminase activity in motifs with a hierarchy CCC>CCC>CC (where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive CC context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3-58.0% and 54.5-92.2% with excellent CC context-specificity were generated in human cells and rabbit embryos, respectively. In addition, a base editor that can further recognize relaxed NG PAMs is achieved by combining hA3G with an engineered SpCas9-NG variant. The A3G-BEs were used to induce accurate single-base substitutions which led to nonsense mutation with an efficiency of 83-100% and few bystander mutations in Founder (F0) rabbits at Tyr loci. Conclusions: These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.
Dear Editor,Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of β-hexosaminidase A (HexA) 1 . The four-bases (TATC) insertion in exon 11 of the HEXA (HEXA ins TATC) accounts for 80% of Tay-Sachs disease from the Ashkenazi Jewish population 2 . However, no typical clinical phenotypes, such as neurological abnormalities, the restricted pattern of distribution of GM2-ganglioside and membranous cytoplasmic bodies in the brain, were observed in HEXA −/− mouse models, due to the difference in the ganglioside degradation pathways in mice and human 3 . Thus, it is desired to generate an ideal animal model to accurately mimic HEXA ins TATC in TSD patients. CRISPR-Cas9 systemmediated HDR 4 has been used to generate the mutation of HEXA ins TATC, however, low efficiency and high indels impede its application.Recently Anzalone et al. 5 described a "search-andreplace" genome editing technology named prime editing (PE) that mediates 12 possible base-to-base conversions, without requiring DSBs or donor DNA templates in human cells. In addition, a previous study showed that, compared to mice, the late onset of TSD in adult rabbits 6 shared more similarities with human regarding physiology, anatomy, and genetics 7 . Thus, we generated a novel TSD rabbit model using the PE system, and characterized
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