Large Genomic DNA Fragment Editing Via CRISPR/CAS

Triditional genomic editing technologies, including gene targeting and chemical mutagenesis such as N-ethyl-N-nitrosourea, are costly, time and labour consuming because of the low self initiating homologous recombination (HR) rate and fussy screening process. However the arising of some artificial endonucleases including ZFN and TALEN brought the genomic editing techonology into a bright new era. By fusing the DNA binding domain of Zinc Finger or TAL to the FokI endonuclease catalytic domain, researchers can induce double strand DNA breatks (DSBs) in virtually any DNA sequences to stimulate two kinds of repair pathways. With the help of ZFN and TALEN, site-specific gene editing has been accomplished in many classical animal models including the rat, which was a break through back then. Despite many beneficials, there are still limitations in ZFN and TALEN techonologies. For instance, the specificity of ZFN is greatly influenced by the binding context of a target. On the other hand, the DNA binding domain of a TALEN contains numerous amino acid repeats, which brings difficulties to the vector construction. The emergence of the CRISPR/Cas system, circumvent the flaws of ZFN and TALEN. This new system consists of a Cas9 protein that serves as the endonuclease and a single strand guide RNA (sgRNA), which directs the Cas9 protein to the target DNA sequence by Watson-Crick base-paring rules. To induce the DSBs, there has to be a PAM sequence adjacent to 3’end of the target sequences.Owing to its flexibility and high efficiency, CRISPR/Cas has been applied to genomic editing in pig, monckey, zebrafish, drasophila, and etc. Our group is one the pioneers that reported knockout mouse and rat by the CRISPR/Cas. Microinjecting the mRNA or DNA encoding Cas9 along with the sgRNAs into one-cell embryos is the common method to induce DSBs that will further cause error-prone NHEJ repair process. However, the alternative method of direct injection of Cas9 protein:sgRNA complex into the one-cell embryos to has seldom been reported. In this paper we achieved in making point mutations, via direct injection of Cas9 protein:sgRNA complex. Interestingly, in our point mutation experiment, we found this method resulted in a lower off target rate. Long non-conding genes, including lncRNAs and eRNAs have been found to play more important roles in regulating the gene nextwork. Lacking the open reading frame, the long non-coding genes can’t be knocked out or inactived by just inducing the indels. Here, we tried to delete the 53kb lncRNA GM14005 and the 95kb Fprl-3 gene cluster by a "two sgRNA cut" stretagy derived from the CRISPR/Cas. Lineage tracing is a useful tool for the study of stem cell or oncogenesis. However strategies that facilitate lineage tracing with the help of CRISPR/Cas has never been reported. Here, we knocked the IRES-Cre-ERT2 cassette into the exon9 of mouse Nfatcl gene. After crossing with Rosa26-lsl-LacZ-/- mouse to obtain the Nfatcl-CreER+/-,Rosa26-lsl-LacZ+/- mouse, we successfully traced the progenies of the Nfatcl expressing stem cells in mouse hair follicles under the induction of Tamoxifen. Conditional knockout is a pivotal tool when the whole body knockout is lethal. By knocking in the oppositely oriented SA-GFP-polyA cassette into the intronl of Lgr5 locus we generated the Lgr5 conditional knockout rat, which is also the first conditional knock model in rat. By combining the CRISRP/Cas system with the Cre mediated DNA inversion and excision, we achieved mosaic analysis in the rat small intestine cells. This mosaic analysis model can be used to trace and viualize KO cells in vitro, which is very helpful in the study of gene functions in a spatio-temproal manner. In summary we find that by direct injection of Cas9 protein: sgRNA complex into one cell embryos large DNA fragment editing can be achieved in vivo with high efficiency and germ line transmission rate.