Efficient Immortalization Of Bovine Cells Using CRISPR/Cas9-mediated Precise Integration Of BTERT

Currently,new varieties of transgenic cattle are being bred with a greater tendency to edit endogenous functional genes,which provides a significant increase in safety and pro-duction value compared to previous approaches through the integration of exogenous genes.It is therefore necessary to explore and study key genes associated with disease occurrence and production performance in cattle.Immortalized cell lines that can be cultured in vitro for a long time are important tools in molecular cell biology.However,due to the chronic lack of bovine immortalized cell lines,only human or mouse cell lines can be used as model cells for the study of bovine functional genes,which restricts the development of research and the transformation value in transgenic cattle breeding.Therefore,it is of great importance to es-tablish a simple and efficient immortalization strategy for bovine primary cells.The traditional immortalization strategies for bovine cells are usually to introduce hu-man telomerase reverse transcriptase（hTERT）gene into primary cells via transient transfec-tion or virus-mediated delivery system,which have defects in efficiency and reliability.To solve the problem,this study proposed to improve the traditional immortalization strategy in terms of both the immortalization factors introduced and the genetic engineering methods adopted.We demonstrated for the first time that bTERT is a higher quality immortalization factor for bovine primary cells,established an efficient immortalization strategy by perform-ing the CRISPR/Cas9-mediated site-specific insertion of bTERT at the bovine ROSA26（b ROSA26）locus of bovine genome,and then generated stable immortalized bovine fetal fibroblasts（BFFs）.In addition,we attempted to immortalize primary bovine bone marrow-derived macrophages（bBMMs）using the above strategy,but found that the nuclear translo-cation and enzymatic activity ofTERT was somewhat inhibited in bBMMs,preventingTERT-mediated immortalization of bBMMs.The insertion of bTERT mimicking phosphory-lation mutant into the ROSA26 locus of bBMMs genome mediated by CRISPR/Cas9 system could overcome the above limitations to a certain extent and prolong the culture time of bBMMs in vitro.The main contents are as follows:1.We identified bTERT as a high-quality immortalization factor for the first time.First-ly,we demonstrated that the expression level of bTERT was tightly regulated in various tis-sues of the organism,which resembled the expression pattern of hTERT.Secondly,the re-sults of RNA immunoprecipitation assay showed that bTERT could interact with endogenous telomerase RNA subunits in human,bovine,mouse and goat cells,indicating that bTERT participates in telomerase assembly in cells of multiple species.Subsequently,we truncated and interchanged the carboxyl terminal domains（CTDs）of bovine,human and mouseTERT,and found that the full length of bTERT and its CTD were more advantageous in promoting telomerase activity and extending telomere length.Finally,overexpression of bTERT in hTERT^+/-Hela cellsrevealed that proliferation activity and telomere length were restored compared to hTERT^+/-Hela cells.These results suggest that bTERT could play a stronger function than hTERT as an immortalization factor in bovine primary cells.2.A highly efficient immortalization strategy for bovine primary cells was established through the CRISPR/Cas9-mediated insertion of bTERT at the b ROSA26 locus.ROSA26 lo-cus was selected as targeting locus on bovine genome,and the Cas9 target plasmid and the corresponding donor vector bTERT-HMEJ were constructed.After co-transfection with BFFs,the positive colonies with precise integration of bTERT were identified by puromycin selection,junction PCR and Sanger sequencing.The immortalized cell lines were generated after further in vitro culture and passage.We conducted a comprehensive and systematic comparison of the above CRISPR/Cas9-mediated immortalization method with immortaliza-tion methods using transient transfection and virus-mediated delivery system.The results proved that the immortalization strategy relying on CRISPR/Cas9 gene editing system estab-lished in this study is more efficient and of better quality,as evidenced by the higher number of immortalized colonies,more generations in culture,stronger cell viability and more stable telomere length,which had obvious advantages over traditional methods.3.The immortalized BFFs generated by the above immortalization strategy（bT-iBFFs）have stable cellular properties and can be used as model cells in scientific research.The re-sults of Western blot,real-time quantitative PCR and other experiments demonstrated that,the exogenously integrated bTERT expressed normally in bT-iBFFs,and the cell type of bT-iBFFs did not change.In addition,the length of telomere did not shorten obviously with pas-sages in bT-iBFFs,and the 80th passage of bT-iBFFs did not get aging and still retained a high proliferation rate.The results of karyotype analysis and soft agar assay showed that of bT-iBFFs had normal chromosomes and did not undergo malignant transformation.Further-more,bT-iBFFs were capable of receiving secondary transfection and gene editing.4.Using the above immortalization strategy mediated by CRISPR/Cas9 system,at-tempts were made to construct immortalized bBMMs.In this study,the positive bBMM col-onies with accurate integration of bTERT using the above strategy were obtained,but immor-talization was not fully achieved.Through Western blot,co-Immunoprecipitation assay and other experiments,we found endogenous post-translational modification factors in primary bBMMs that hinder the nuclear transport and enzyme activity of bTERT.Firstly,the low ac-tivity of AKT kinase in bBMMs inhibited the phosphorylation of the substrate bTERT and hindered the nuclear translocation of bTERT.Secondly,the high level of endogenous PINX1in primary bBMMs impeded the function of bTERT in the nucleus through interacting with bTERT.In addition,the high level of expression of the“Shelterin”complex and“CST”complex in bBMMs inhibited the function of bTERT.Using mutant strategy and sequence alignment analysis,we found that serine residue 231 of bTERT is a key phosphorylation site modulated by AKT kinase,which could regulate the nuclear transport of bTERT.Therefore,through constructing the phosphorylation-mimicking mutant bTERT-S231E and utilizing CRISPR/Cas9-mediated the mutant gene integrated into the genome of bBMMs,we success-fully extended the culture time of primary bBMMs.In summary,this study established an immortlization strategy for bovine cells with easy-manipulation and high-efficiency performing a CRISPR/Cas9-mediated precise integra-tion of bTERT at the b ROSA26 locus,and realized the immortalization of BFFs.In addition,we found that the limitation of the above strategy in achieving immortalization of primary bBMMs was related to blocked nuclear translocation and inhibited activity of bTERT.We proposed that there are endogenous factors in primary cells could hinderTERT-mediated immortalization for the first time,and then extended the culture time of bBMMs through op-timized strategy.Therefore,when subsequently usingTERT as an immortalization factor to establish immortalized cell lines,the expression and regulatory pattern ofTERT in different cell types need to be fully considered,and customized protocols to eliminate endogenous factors that inhibit the function ofTERT.Meanwhile,the immortalization strategy in this study provides an effective means for the construction of bovine model cell lines,which is important for promoting the study of endogenous functional genes in cattle and their applica-tion in transgenic cattle breeding.