Precise genome editing without exogenous donor DNA via retron editing system in human cells

Dear Editor,CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair(HDR),albeit with relative low efficiency due to the inefficient delivery of exogenous DNA(Cox et al.,2015;Gao,2021).Retrons are bacterial phage-defense related operons composed of a specialized reverse transcriptase(RT)and a relevant non-coding RNA(ncRNA)which can be partially reverse tran-scribed by RT initiating at a conserved guanosine(G)residue to produce a multicopy single-stranded DNA(msDNA)(Yee et al.,1984;Millman et al.,2020).After being reverse transcribed,the msDNA is usually covalently teth-ered to the ncRNA through the 2',5'-phosphodiesterbond between the priming G in ncRNA and 5'end of msDNA(Dhundale et al.,1987).The reverse transcription process,of which the specialized RT recognizes the unique secondary structure of retron ncRNA,is highly specific(Hsu et al.,1989).Additionally,desired msDNA can be generated in vivo by replacing the dispensable region of retron ncRNA with desired sequences(Mirochnitchenko et al.,1994;Simon et al.,2019).Therefore,retrons are promising biological sources for in vivo generation of DNA donors for HDR-me-diated precise genome editing.

An engineered xCas12i with high activity,high specificity,and broad PAM range

Dear Editor,The clustered regularly interspaced short palindromic repeats-Cas(CRISPR-Cas)systems,including type II Cas9 and type V Cas12 systems,which serve in the adaptive immunity of prokaryotes against viruses,have been developed into genome-editing tools(Anzalone et al.,2020;Doudna,2020).Compared with type II systems,the type V systems including V-A to V-K showed more functional diversity(Yan et al.,2019).Amongst them,Cas12i has a relatively smaller size(1,033-1,093 aa),compared to SpCas9 and Cas12a,and has a 5'-TTN protospacer adjacent motif(PAM)preference(Yan et al.,2019).

LincRNA-1614 coordinates Sox2/PRC2-mediated repression of developmental genes in pluripotency maintenance

Large-intergenic noncoding RNAs （lincRNAs） cooperate with core transcription factors to coordinate the pluripotency network of embryonic stem cells. The mechanisms by which lincRNAs affect chromatin structure and gene transcription remain mostly unknown. Here, we identified that a UncRNA （linc1614）, occupied by pluripotency factors at its promoter, was indispensable for both maintenance and acquisition of pluripotency. Linc1614 sewed as a specific partner of core factor Sox2 in maintaining pluripotency, primarily by mediating the function of Sox2 in the repression of developmental genes. Moreover, Ezh2, an essential subunit of polycomb repressive complex 2 （PRC2）, physically interacted with linc1614 and contributed to lincRNA-mediated transcriptional silencing. Thus, we propose that the interplay of linc1614 with Sox2 implicates this lincRNA as a recruitment platform that mediates transcriptional silencing by guiding the PRC2 complex to the loci of developmental genes.

Morphology evolution and breakdown mechanism of cross-linked polyethylene(XLPE)-silicone rubber(SiR)interface induced by silicone grease diffusion

Silicone grease(SG)is used for lubrication during cable accessory installation and can penetrate into the silicone rubber(SiR),leading to properties deterioration of the SiR.In this study,the effects of SG on the breakdown characteristics of the cross-linked poly-ethylene(XLPE)-SiR interface are investigated.First,the variation of the XLPE-SiR interface breakdown voltage with SG coating time is experimentally explored.The interface breakdown voltage significantly increases after SG is applied,remains stable for coating times of up to approximately 144 h and then rapidly decreases;the minimum interface breakdown voltage is lower than that without the SG coating.Next,the effects of SG on the chemical composition and surface topography of the SiR are examined by infrared spectroscopy and optical profilometry,respectively.The SG penetration does not change the functional groups of the SiR but significantly increases its surface roughness.Finally,the interface electric-field distribution after coating with SG is analysed by finite-element simulation,revealing that the residual SG at the interface distorts the interface electric field after a long coating time.The increase in the SiR surface roughness caused by SG diffusion and the interface electric-field distortion caused by the residual SG together lead to the decrease of the interface breakdown voltage.