Establishment And Application For CRISPR/Cas9-Mediated Site-Specific DNA Integration

a powerful and versatile tool,CRISPR/Cas9 gene editing technology has a wide variety of applications including basic biological research,development of biotechnology products,and treatment of diseases,for example,gene cutting,gene integration,and base editing,as well as for human diseases.Currently,CRISPRmediated targeted transgene integration is generally based on homologous recombination(HDR)and non-homologous end-joining(NHEJ).HDR-based integration is precise without any insertions and deletions(indels)at the junction sites,but restrained in dividing cells with low efficiency.NHEJ-based approach is an efficient integration method without homology arm.CRISPR/Cas9-mediated homology-independent targeted integration(HITI),a NHEJ-based approach,introduces robust targeted knock-in by in vivo cleaving DNA donor without HA in both dividing and non-dividing cells.Once the donor DNA is inserted in the desired direction,the Cas9 target site is disrupted and further Cas9 cutting is prevented.When the donor DNA is inserted in the reverse direction,the Cas9 target site will remain intact and the inserted transgene will be cut out by a second round of Cas9 cutting.Thus,the direction of HITI-mediated transgene insertion is predetermined.We developed hESCs for inducible and multiplex orthogonal gene knockout and activation,named iKA-CRISPR hESCs.In cells,when complexed with a short guide RNA containing a 14-bp target sequence(14-bp gRNA)or a long 20-bp gRNA,the doxycycline-induced Cas9-p300 protein could activate gene transcription or cleave genomic DNA,respectively.Thus,iKA-CRISPR hESCs provide a convenient platform to control gene expression networks and,therefore,facilitate the applications of hESCs in basic and translational biomedical research.The CRISPR/Cas system is a widely used tool for genome editing.However,robust and targeted insertion of a DNA segment remains challenging.Here,we present a fusion nuclease(Cas9-N57)to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex.The insertion was unidirectional and specific,and DNA fragments up to 12 kb in length were successfully integrated.As a test of the system,Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor(CD19CAR)cassette into the AAVS1 locus in human T cells,and induced intrahepatic cholangiocarcinoma(ICC)in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten.Moreover,variants of the Nuclease-N57 fusion proteins based on eSpCas9,CjCas9,and AsCpfl exhibited similar activity.These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.In summary,the main achievement is as follow:HITI is an efficient gene integration method that can be effectively applied to human embryonic stem cells.IKAhESC is a better platform to explore biological questions related to hESCs.Cas9-N57 can catalyze efficient targeted DNA insertions in a unidirectional,site-specific,and homology-independent manner,both in cell culture and in mouse.Moreover,engineered Cas9,Cpfl,and small Cas9 were successfully used to construct variants of Nuclease-N57,thereby endowing the Nuclease-N57 with distinctive characteristics.