| With the continuous development of sequencing technology,various life blueprints that composed of base sequences have been gradually revealed.Based on huge genomic sequencing data,researchers have been able to master genetic maps and subsequent advances have been carried out in various fields,including specific gene function studies,detection of genetic diseases related base mutations and genomic localization,as well as improvement of production traits of livestock and poultry.These studies mainly rely on genome editing technologies that have been applied to achieve site-specific insertions,sequence deletions,and single-base mutations.Genomic editing relies on artificial site-specific nucleases that firstly create double-strand breaks(DSBs)in the genome,then these DSBs are repaired by two main repair pathways.The first repair pathway called non-homologous end-joining(NHEJ)that can perform rapid and complete double chain repair but leaves behind base insertion or deletion.In the second repair pathway,insertions,deletions or replacements of DNA sequences are obtained by homology-directed repair(HDR)in the presence of donor DNA with homologous sequences at both ends of the cleavage.In the practical application of disease treatment and improvement of livestock traits,it is required to specifically introduce deletions,insertions or substitutions to the target site without additional unintended modifications for the achievement of precise genome editing.However,CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing.The existing precise genome editing technologies can be divided into one-step and two-step methods based on the number of genomic targeting events.The one-step method uses a circular plasmid,double-stranded DNA or single-stranded DNA as a template to introduce point mutations(specific base deletion or insertion)directly into the genome by HDR.In the two-step method,a screening sequence for positive clone selection is introduced into the genome by HDR,simultaneously with the point mutation at the first round of genome targeting.In the second step,the screening sequence is deleted through Cre-Loxp,piggyBac,and secondary HDR,resulting in precise genome editing.The result of the one-step method can only be confirmed by the genome sequencing of the picked single colonies,which is a time-consuming and labor-intensive process.Selection cassettes are generally introduced in the form of a drug or fluorescent screening system to help in obtaining nuclease-transfected or nuclease-targeted positive clones.This method is generally used for embryonic stem cells,induced pluripotent stem cells or pronuclear injection.In the existing two-step techniques,the combination of CRISPR/Cas9 with Cre-LoxP demonstrates a higher efficiency but leaves behind a 34-base pair of tag sequences due to its inherent property.Another method utilizes piggyBac transposon for removing the selection cassette,but its disadvantage is the difficulty in controlling its random reintegration after releasing.The secondary HDR approach faces false-positive results because of the silence of screening genes after genomic integration.Here,we report a novel two-step precise gene-editing method by leveraging the single strand annealing(SSA)-mediated repair mechanism into the CRISPR/Cas9-mediated genomic editing system.An integrating cassette was developed with positive and negative selection markers,which was flanked by direct repeat sequences as SSA arms,and was introduced into genome sequence through the homologous arm with desired mutations.After the targeted integration of the cassette mediated by CRISPR/Cas9-induced HDR,cell clones were first selected through the positive selection.In the second-round targeting,the selection cassette was removed by SSA-mediated DNA DSBs repair without any scar left behind.The main results of this study are summarized as follows:1.The novel seamless genome editing technique was tested on CCR5,APP,and?-syn loci,and finally demonstrated up to 45.83%,68%and 50%of precise genome editing efficiency,respectively.2.Afterward,we optimized the system efficiency,including the formation of homologous recombination donors in the first round of targeting and the homologous arm length of SSA in the second round of targeting.We found that the bilateral linearization of a donor has higher integration efficiency and the efficiency of single strand annealing is positively correlated with the length of the homologous arms.3.According to the optimization results,the donor vector was modified and tested in the IGF2 and RELA loci of PK15 cells,and obtained 30%and 53.33%precise genome editing efficiency,respectively.This study provides a new efficient approach for precise genome editing and gene correction.4.Furthermore,since the result of biallelic editing was not observed in the exploitation of SSA-mediated precise genome editing system.The TK-Puro~R-eGFP/TK-Zeo~R-mRFP biallelic genome editing system was established based on the positive and negative screening cassettes that are used in the precise genome editing system.The biallelic genome editing system achieved 52.94%and 54.90%biallelic editing efficiency in the APP and PSEN1 loci of 293T cells,respectively.Later,the length of the screening cassette was optimized,and two sets of more efficiency biallelic genome editing systems,Puro~R-eGFP-TK/Zeo~R-mRFP-TK and Puro~R-TK/Zeo~R-TK,were constructed and reached 82%and 70%biallelic genome editing efficiency at APP locus,respectively.In this study,we provided a SSA-mediated precise genome editing system,which has operational-simplicity,high-efficiency,and universal-adaptability.This system also provides a new scheme for gene function researches,genetic disease treatment,and livestock production improvement.In addition,the three sets of efficient biallelic editing systems provide a new idea for biallelic manipulation and lay a foundation for subsequent biallelic precise editing. |
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