Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure

Abstract

Background: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.Methods: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection. Results:We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites. Conclusions:We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors. KEYWORDS cosmid, double nicking, lambda packaging, multiplex guide RNA, non-viral vector, nucleic acid therapy ---This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

“…In contrast to transformation, lambda packaging is size-selective and transfers only large cosmids typically between approximately 36 and 52 kilobases (kb) in size. Consequently, unwanted deleted cosmids generated by homologous recombination are selected out 18 .…”

Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

“…Therefore, the limiting step is not AdV construction and handling, but rather the construction of plasmids containing a >30 kb AdV genome with eight gRNA units with lack of deletion. We recently developed a method, Tetraplex-guide Tandem [ 29 , 33 ], and completely stable cosmids containing AdV genome bearing eight multiplex gRNA units were obtained in a single step utilizing lambda in vitro packaging. The AdVs that we used are commercially available Adex vectors [ 34 , 35 ], which possess the full Ad5 packaging domain [ 36 ], while other AdVs, for example, those of Bett et al [ 37 ] and Mizuguchi et al [ 38 ], do not.…”

Short term but highly efficient Cas9 expression mediated by excisional system using adenovirus vector and Cre

“…Fragments of the gRNA expression unit were inserted into the E4 cloning site of pAxSRNCre-4c located in the E4 region 162 nucleotides downstream from the right end of the adenovirus-5 (Ad5) genome, and the resulting cosmids were designated gRNA G-1/Cre AdV, gRNA G-2/Cre AdV, and gRNA G-3/Cre AdV. The fragments of multiplex gRNA expression cassettes were constructed using polymerase chain reaction (PCR) and restriction enzymes that produce non-palindromic termini, and were introduced into the E4 cloning site of pAxSRNCre-4c 56 . All plasmids were confirmed by DNA sequencing.…”

“…) using polymerase chain reaction (PCR) and restriction enzymes that produce non-palindromic termini, and were introduced into the E4 cloning site of pAxSRNCre-4c56 . All plasmids were confirmed by DNA sequencing.All plasmids and adenovirus vectors can be provided by Y. Kanegae.…”