Optimization And Improvement Of CRISPR Gene Editing Related New Technologies

The CRISPR system has become an indispensable tool in biological research.Now the programmability of the Cas9 enzyme has been more diversified,playing an important role in various fields of medical research,biotechnology and agriculture.CRISPR-Cas9 is no longer just a gene editing tool,and its application areas include gene regulation,epigenetic editing,chromatin engineering,and imaging,which is far beyond the genetic editing capabilities of WT Cas9 itself.The development and application of new technologies about CRISPR will be a hot issue in future research.To explore whether these new technologies are safe and effective or not for future inheritance disease treatment,we explored some new technologies related to CRISPR/Cas9 system to edit the relevant genes in mouse cells,embryos and human embryos.Firstly,To enhance HDR(homoIogy-directedrepair),enabling the insertion of precise genetic modifications,we compared two strategies during surrogate reporter assays in mouse N2 A cells: the suppression of DNA ligase IV,a key molecule in NHEJ,using the CasRx system,and co-expression of Kaposi's sarcoma-associated herpesvirus ORF52 proteins.The resultsdemonstrated that both methods are effective in increasing homology.It is important for achieving precise genome editing in treatments of clinical disease,which require targeted base insertions or targeted point mutations.Secondly,we knocked out multiple genes in mice using BE3 mediated new editing technology which named Crispr-stop.Our results showed that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants,bypassing laborious mouse breeding.Finally,we compared with the traditional CRISPR-medited technology,and found that both BE3 and ABE7.10 systems performed better in gene editing efficiency.Indeed,the BE3 system was used for base editing in human embryos in this study,which confirmed that the BE3 system can achieve efficient and accurate base editing in human trinuclear(3PN)fertilized eggs.It proved that the base editor can be used to correct the possibility of genetic defects in human embryos in future.The main results of the rearch are as follows:1.Homology-directed repair in mouse cells increased by CasRx-mediated knockdown or co-expressing Kaposi's sarcoma-associated herpesvirus ORF52.Precise genome editing with directed base insertion or targeted point mutations can be achieved by CRISPR/Cas9-mediated homology-directed repair(HDR)and is of great significance in clinical disease therapy.However,HDR efficiency,compared with nonhomologous end-joining(NHEJ),is inherently low.To enhance HDR,enabling the insertion of precise genetic modifications,we compared two strategies during surrogate reporter assays in mouse N2Acells: the suppression of DNA ligase IV,a key molecule in NHEJ,using the CasRx system,and co-expression of Kaposi's sarcoma-associated herpesvirus ORF52 proteins.We found that suppression of DNA ligase IV promotes HDR efficiency by 1.4-fold.When co-expressed with the Cas9 system,ORF52 improved HDR efficiency by up to 2.1-fold.In addition,we used ORF52co-expression to modify the ACTB and Tubb3 genes of mouse N2 A and E14 cells,which further increased HDR efficiency by approximately 2~4-fold.In conclusion,our data suggest that ORF52 co-expression is effective for enhancing CRISPR/Cas9-mediated HDR,which may be useful for future studies involving precise genome editing.2.Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.In vivo genetic mutation has become a powerful tool for dissecting gene function;however,multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations,at best difficult to interpret,and,at worst,misleading.Hence,it is necessary to establish an efficient way to disrupt multiple genes simultaneously.CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon;this is referred to as CRISPR-stop.Its application in generating zygotic mutations has not been well explored yet.Here,we first performed a proof-of-principle test by disrupting Atoh1,a gene crucial for auditory hair cell generation.Next,we individually mutated vGlut3(Slc17a8),otoferlin(Otof)andprestin(Slc26a5),three genes needed for normal hearing function.Finally,we successfully disrupted vGlut3,Otof and prestin simultaneously.Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants,bypassing laborious mouse breeding.We believe that CRISPR-stop is a powerful method that will pave the way for highthroughput screening of mouse developmental and functional genes,matching the efficiency of methods available for model organisms such as Drosophila.3.Compareing the base editing efficiency between base editing system and the traditional crispr-medited technology and base editing research through using BE3 in human embryos.Mutant mice with single nucleotide substitutions were generated using ABE7.10 to demonstrate the potential for base editing in mouse embryos.Firstly,we used ABE7.10(TadA-TadA * 7.10-nCas9)to induce point mutations in the Tyr gene.Three mutation sites were designed on the Tyr gene.Base editing was performed in mouse fertilized eggs by microinjection of ABE7.10 mRNA and sgRNA.High base editing rates were observed for all three sites,75%,87.5%,and 75% respectively.Compared with the traditional CRISPR-assisted ssODN-mediated homologous targeted repair,ABE7.10 system showed high base editing efficiency and low Indels(insert or miss)rate,and ABE7.10 system has more advantages in targeted point mutation.Indeed,we made a comparison betweent the BE3 system and the CRISPR-Cas9 system for gene konckout in mouse embryos.Four target sites were designed for the mouse Tyr gene.Bsaedon the results,when a single sgRNA was used to target mouse Tyr gene,a certain ratio of chimeric mice appeared in the two methods.When multiple sgRNAs are used,the chimeric rate is reduced or absent.Although the BE3 and CRISPR-cas9 are highly efficient in gene knockout,the knockout mice produced by BE3 system with a lower chimerism rate and a higher homozygous rate.Finally,gene editing studies in human trinuclear(3PN)fertilized eggs using BE3 with nCas9 and SaKKH-BE3 with SaKKH-nCas9 showed in three genes(HBB,FANCF and DNMT3B).Higher base editing efficiency and no significant off-target effects were observed in genetically engineered human embryos,demonstrating the possibility that base editors can be used to correct genetic defects in human embryos in future.