Establishment, Optimization And Application Of Eukaryotic CRISPR/Cas9 Systems Derived From Streptococcus Thermophilus

Targeted genomic modification plays an important role in genomic functional study, gene therapy and transgenic breeding study. The development of zinc finger nucleases(ZFNs) and transcription activator-like effector nucleases(TALENs) has promised targeted genomic modification as effective technology tools. However, it is a still great challenge for selecting or assembling efficient ZFNs and TALENs, which has limited their wide application in gene function study and generation of transgenic organisms. Thus, an economic and efficient, but simple and quick programmable nuclease technology remains to be developed for mammalian genome editing. The Streptococcus pyogenes type II CRISPR/Cas immune system was firstly reported in 2012 to be used for in vitro DNA targeting, which has soon gained great attention. Early in 2011, the Streptococcus thermophilus type II CRISPR/Cas system CRISPR3 has been reported to function in Escherichia coli for plasmid DNA interference. Under this background, we would like to develop a novel nuclease technology based on the Streptococcus thermophilus CRISPR3 locus to facilitate the future precise gene editing and animal genome editing study.In this study, we have successfully cloned the Cas9 gene from Streptococcus thermophilus strain; established successively the yeast St CRISPR/Cas9 expression system, the yeast activity validation system based on Gal4 reporter, the target integrated yeast reporter strain system, the mammalian St CRISPR/Cas9 expression system, the mammalian activity validation system based on Ds Red-e GFP reporter and the target integrated HEK293 T reporter cell line system; and performed functional assays for the St CRISPR/Cas9 activity on levels of yeast reporter vector, integrated yeast genome, mammalian reporter vector, integrated mammalian genome and mammalian endogenous genes. The main results of our study are shown as follows:1. By the development and application of the yeast St CRISPR/Cas9 expression system and the Gal4 reporter-mediated yeast activity validation system, the nuclease function of St CRISPR/Cas9 was preliminarily confirmed within yeast cells. By investigating different domains of sg RNA, we saw the importance of tracr RNA 3’-terminal and Bulge motif. The St CRISPR/Cas9 system was further proved to suffer the position-dependent off-target effects. Besides, the r G::d T reorganization between sg RNA and target DNA may cause serious off-target events. Finally, the optimized sg RNA.Opti design and codon-humanized h St Cas9 both improved the St CRISPR/Cas9 to some extent, and provided the highest activity in yeast assays as 14.32%.2. To construct the target integrated yeast reporter strains, we first confirmed that the SSA arm length with 20 bp is sufficient to manage the Gal4 reporter gene repair. Yeast transformation assays with reporter strains did indicated that the St CRISPR/Cas9 system functions on yeast genome DNA. The St CRISPR/Cas9 activity on yeast genome was doubled after the optimization with sg RNA.Opti/h St Cas9, and the highest was 6.64%.3. By the development and application of the mammalian St CRISPR/Cas9 expression system and the Ds Red-e GFP reporter-mediated mammalian activity validation system, the nuclease function of St CRISPR/Cas9 was further confirmed within human HEK293 T cells. We have tried the bacterial b St Cas9 gene and the codon-humanized h St Cas9 gene, as well as 8 different guiding RNA expression strategies. Results demonstrated that, St CRISPR/Cas9 firstly suffered low efficiency in HEK293 T cells for the inefficient b St Cas9 expression; however, the leader sequences and the cr RNA::tracr RNA expression strategies may help to some extent. The h St Cas9 gene improved St CRISPR/Cas9 activity dramatically in HEK293 T cells and the highest was 41.93%.4. By the construction and application of the target integrated HEK293 T reporter cell lines, we next confirmed that the St CRISPR/Cas9 system can function on integrated targets in HEK293 T genome. The undetectable of off-target events in 293 T.SP1.P5M4 and 293 T.SP1.PAM5 cells targeted correspondingly by SP1.sg RNA.WT/h St Cas9 and SP1.sg RNA.Opti/h St Cas9 further demonstrated the specificity of our St CRISPR/Cas9 system.5. By application of our St CRISPR/Cas9 system, we successfully conducted genome targeting on HEK293 T endogenous targets, and the indel frequencies for CCR5a、CCR5b and AAVS1 were 20.84%, 26.68% and 15.46%, respectively.