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

Tomoko Nakanishi,Aya Maekawa,Izumu Saito,Kumiko Sato,Mai Mori,Hirotaka Tabata,Mariko Suzuki

doi:10.1038/s41598-021-83259-0

Abstract

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

Highlights

Genome editing using the CRISPR/Cas[9] system is widely used in various basic studies and in applied fields in vitro and in vivo[1,2,3]
We developed a method for construction of a DNA fragment consisting of four multiplex-guide RNAs (gRNAs) units in one step (“Tetraplex-guide Tandem”) (Fig. 1a, bottom, called head-block) by using only PCR without plasmid cloning
Because the aim of this study is the construction of AdVs expressing multiplex-gRNA units, we introduced the fragment into a cassette cosmid pAxc4wit[223] for construction of replication-deficient adenovirus vector

Summary

Introduction

Genome editing using the CRISPR/Cas[9] system is widely used in various basic studies and in applied fields in vitro and in vivo[1,2,3]. Simultaneous expression of multiplex gRNAs is valuable for knockout of multiple genes. If the simultaneous expression of four multiplex gRNAs is available, specific and efficient knockout of the target genes would become possible. If adenovirus vectors, lentivirus vectors and AAV vectors expressing highly-multiplex gRNAs are available, they certainly contribute to more specific and efficient genome-editing therapy. Eight red diamonds represent BsaI and the vertical arrows show cleavage positions to excise the cassette fragments. In the second and third rows, the specific sticky four nts produced by BsaI are shown as colored horizontal waves on both sides of the cassette fragments (the colors are identical to the boxed four nts in b, the detailed junction sequences are shown in Supplementary Fig. S1a). The structures of the resultant cassette fragments of mid-A and mid-E are identical to those of head-A and head-E, respectively, as for BsaI-producing sticky ends, and the same cassette fragments of B, C, and D for head-block can be used for mid-block construction

Objectives

Methods

Results

Conclusion