SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing

Abstract

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5′- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications.

A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA

“…However, Pol III promoters provide limited options for transcriptional regulation. In contrast, Pol II promoters allow for more versatile experimental designs for RNA expression 38 , but result in substantial post-transcriptional modifications that interfere with sgRNA functionality 39,40 . Recently, a novel technique for sgRNA multiplexing under a single promoter was described using self-cleaving ribozymes 41 .…”

Daisy-chain gene drives for the alteration of local populations

“…One way to reliably prevent such events is to eliminate regions of homology between the elements. Promoter homology can be removed by using different U6, H1, or tRNA promoters to express the required guide RNAs (27–29); if there are insufficient promoters, then each can drive expression of multiple guide RNAs by using tRNA (30, 31) or miRNA processing (32–34). However, each element must still encode multiple guide RNAs >80 base pairs in length to prevent the creation of drive-resistant alleles, precluding safe and stable daisy-drive designs.…”

Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing

“…This presents a considerable barrier when genome editing is to be restricted to a selected population of cells in the tissue. The expressions of Cas9 endonuclease and sgRNA are typically driven by RNA polymerase II and III promoters respectively [124]. Viral RNA polymerase II promoters such as cytomegalovirus (CMV) and simian virus 40 (SV40) and eukaryotic promoters such human elongation factor 1 alpha (EF1α) and chicken β-actin (CBA) RNA polymerase II promoters are commonly used and are constitutively active [125,126].…”

“…Viral RNA polymerase II promoters are preferred over eukaryotic promoters because viral promoters induce a higher level of transcription [127]. Expression of the sgRNA is usually regulated by an RNA polymerase III promoter such as the human U6 promoter which transcribes genes that encode for small RNA sequences in eukaryotes [124,128]. This is because sgRNA expressed under the RNA polymerase II promoter is non-functional when unique sequences like the 5′-cap and 3′-polyadenylation tail are included in the transcript [124].…”