Optimization Of Guide RNA Cluster Assembly Based On Guide RNA Scaffold Sequence Permutation

CRISPR/Cas9 system is a powerful tecnique for gene editing.In this system,guide RNA(gRNA)associates with Cas9 protein to form an effector complex,which binds to the targeting DNA and cleaves it.The cleavage efficiency is affected by the scaffold of gRNA,besides the influence of PAM and spacer.GRNA scaffold is highly conservative,which can form secondary structure,generating stem loops.In each stem loop,there are some bases that interact with Cas9 protein directly.Changing sequence of the scaffold might affect the interaction between gRNA and Cas9 and the stability of gRNA secondary structure.It's easy to implement CRISPR/Cas9 for multiplex genome editing,which used tRNAs to concatenate gRNAs and cloned gRNA cluster with different spacers through goldengate assembly method.However,the result of this work showed that it was difficult to get gRNA cluster by regular fusion PCR cloning tecnique.To solve the problem,we analysed the gRNA scaffold and permutate some bases,trying to optimize the cloning of gRNA cluster.The main findings are as follows:1.We mutated a few of bases(1-4)in gRNA scaffold's tetra loop or stem loop1 to stem loop3 or the linker between stem loop1 and stem loop2,respectively.These mutated sgRNAs were shown be able to mediate DNA cleavage in vitro.And according to the aboved-mentioned base mutations,we further mutated the gRNA scaffold at multiple sites and designed some sgRNAs with lower homologous scaffolds.We showed that these sgRNAs were able to mediate DNA editing in vitro and in vivo.These results offer a new way to opitimize the cloning of gRNA cluster.2.We cloned gRNA cluster rapidly by using different tRNAs to concatenate sgRNAs with different scaffolds.What's more,we constructed a multigene-targeting vector with the gRNA cluster.And we proved the feasibility of the gRNA cluster cloning method according to the result of agrobacterium transient expression experiment.