The Construction Of Kdm2b Conditional Knockout Mouse Model And Kdm2b Knockout Mouse By CRISPR/Cas9-Mediated Genome Engineering

In the past years,genome editing efficient and precise genetic modifications,which manipulate virtuallyany gene in adiverse range of cell types and organisms.The ease of producing targeting RNAs over the generation of unique sequence-directed nucleases to guide site-specific modifications makes the CRISPR/Cas9 system an appealingly accessible method for genome editing.A single targeting RNA can guide Cas9 to a specific genomic sequence where it induces double-strand breaks that,stimulate error-prone nonhomologous end joining(NHEJ)orhomology-directedrepair(HDR)at specific genomic locations.NHEJ-mediated repair of a nuclease-induced DSB leads to the introduction of small insertions or deletions at the targeted site,resulting in knockout of gene function via frameshift mutations.HDR can lead to the introduction of single or multiple foreign DNA to correct or replace existing genes.Thus,this study use the Kdm2b as the targeted gene to construct Kdm2b knockout mouse by CRISPR/Cas9-mediated genome engineering.Moreover,we identified the correct knockin ofloxp sequencein the Kdm2b target locus in the NIH3T3 cells using the CRISPR/Cas9 technology.This system could used to create kdm2b conditional knockout mice.In this study,We designed 2 sgRNAs targeting exon7 and 2 sgRNAs targeting exon9,accordingly,constructed the sgRNAs expressing plasmid.All the sgRNAs pladmids were transfected into NIH3T3 cells with lipofection respectly.Then,we analysised the efficiency of SgRNA targeting and off-target.As a result,2 sgRNAs(sgRNA5-2 and sgRNA3-2)which can effiently edited the Kdm2b gene and have a low level of off-target were screened.The Cas9,sgRNA5-2 and sgRNA3-2 mRNA were microinjected into mouse zygotes and Kdm2b knockout mice were successfully generated.To futher generate the Kdm2b conditonal kncokout mouse,we co-transfected Cas9,sgRNAs and double-stranded DNA donor which designed the correspond:ing LoxP site sequence with homolgy sequences on each sidesurrounding each sgRNA-mediated DSB into the NIH3T3 cells.Then we analysis the cell DNA by T7E1 enzyme digestion and DNA sequence.These results demonstrate that LoxP could be correctly inserted into targeting locus.To enhance the efficiency of gene targeting,we further try to use the large sigle-stranded DNA(ssDNA)donor instead of double-stranded or short sigle-stranded DNA donor.ssDNA doner were produced by lambda exonuclease and comfirmed by DNA sequencing.The ssDNA doner was co-transfected with sgRNA and Cas9 into NIH3T3 cells to investigate whether it could be knocked into the targeting locus.Compared to the dsDNA,the ssDNA doner have more recombination efficiency.In conclusion,we successfully screened one pair of sgRNAsand donor to construct the Kdm2b conditional knockout mouse,and developed a approach to generate ssDNA which could beused as the homology directed repair doner.We created mutant mice by microinjection of the cytoplasm zygotes with Cas9 and the pair sgRNAs.Our reslut establish the fundation for the construction of Kdm2b conditional knockout mice and other gene-modified mice via Cas9/RNA-mediated gene targeting.