Study On The Off-target Effect Of CRISPR/Cas9 In Bovine Fetal Fibroblasts Using CIRCLE-seq

The CRISPR / Cas9 nuclease of the type II CRISPR(clustered,regularly interspaced,short palindromic repeat;CRISPR)system in the bacterial acquired immune system has been widely used as the third-generation gene editing technology in recent years.CRISPR /Cas9 technology mainly relies on single-guide RNA(sg RNA)and genomic sequence base complementary pairing to achieve precise gene editing,but the recognition module has a certain tolerance for base mismatch,resulting in off-target effects(Off-target effects).The existence of off-target effects severely restricts the application of this technology,especially in the production of transgenic animals will cause problems such as insertion and deletion of genomes(inserts and deletes,indels),the occurrence of cancer and low production efficiency.Therefore,the detection and evaluation of off-target effects of CRISPR / Cas9 technology has also become a research hotspot.In this study,we first used Cas-OFFinder to initially screen the sg RNA sequence with the fewest potential off-target sites in the bovine ROSA 26 locus,and then used the CIRCLE-seq method to detect the off-target activity of different sg RNAs at corresponding sites,with a view to comprehensively assess the off-target effect of CRISPR / Cas9 technology in the breeding process of transgenic cattle.The main research contents are as follows:1.Detection of in vitro cleavage efficiency of potential targeting sites in the bovine ROSA 26 locus.Using Cas-OFFinder to screen loci 11,34 and 45 on the bovine ROSA 26 locus,and design the corresponding sg RNAs.The Cas9-sg RNAs complex was incubated with the target site sequence amplified in vitro,and the cutting efficiency of different target sites was detected by gel electrophoresis imaging and grayscale analysis.The results showed that the cutting efficiencies of the target sites were significantly different.Among them,the cutting efficiency of the 11 th site was the highest,followed by the 34 th site,and the 45 th site had no obvious cutting effect.2.Fragmentation and circularization of bovine genome and CRISPR / Cas9 cleavage reaction in vitro.In order to ensure the effect of genome fragmentation,this study used micrococcal nuclease,contact sonicator and non-contact sonicator to fragment the genome,and compared the interruption efficiency and repeatability of these three approaches.The results show that the non-contact ultrasound system has the highest interruption efficiency and repeatability under the condition that the number of ultrasound cycles is fixed at 9times,and the optimal concentration of bovine genome samples(120 ng/?L)can be processed into 300 ? 500 bp DNA Fragment.Subsequently,a linker with a palindrome isadded to the fragmented DNA through a ligation reaction,and it is circularized through a series of enzymatic reactions.Finally,the circularized genomic fragments were co-incubated with sg RNAs,and PCR amplification and Sanger sequencing were used to identify the circularization and cleavage effects of the target site,and finally the samples that could be used for subsequent self-built libraries were obtained.3.CIRCLE-seq self-built library and gene targeting verification of sequencing results.By setting the molar ratio gradient of Adapter and Input DNA,explore the best self-built library reaction system.The results show that for Input DNA of 100 ng ? 500 ng,the best library building effect can be achieved when the molar ratio of Adapter: Input DNA is 100:1.By using the combination of Qubit fluorescence quantification and q PCR quantification,the progress of sequencing adapter connection reaction and library amplification reaction can be monitored in time.Based on the CIRCLE-seq results,through the screening and statistics of gene targeting positive clones,it was verified that the actual targeting efficiency of locus 11 is the highest.In summary,this study optimized the CIRCLE-seq technology reaction system.This method was first applied to the detection of off-target effects on the ROSA 26 locus of cattle,which has improved the breeding process of disease-resistant transgenic cattle breeds.Gene targeting efficiency is of great significance.