Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency

Abstract

Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.

Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

“…Optimizations of linker composition and length [3,53], as well as domain-inlaid systems [29,[54][55][56][57][58], may confer greater control over the editing window. Finally, future optimization of the single-AAV vector construct, such as changing the transgene cassette orientations, may potentially increase expression levels of the BE effector, the sgRNA, or both [18,85]. Our studies also suggest factors that must be considered for optimal guide expression.…”

“…To test if different arrangements of sgRNA and effector cassettes in the Nme2-ABE8e all-in-one AAV construct can further increase in vivo editing efficiency, we moved the U6-sgRNA cassette to the 3’ end of the AAV genome and reversed its orientation, similar to the optimal arrangement reported by Fry et al . [85] ( Figure 6a ). Using the same Rosa26 guide described above ( Figure 5d ), we packaged the rearranged construct in AAV9 capsids and performed tail-vein injections in 8-week-old mice.…”

“…We did not detect any abovebackground A•T-to-G•C editing at these sites (Supplementary Figure 5). Optimizing effector and sgRNA arrangement improves editing efficiency by AAV delivery Previous studies have shown that effector and sgRNA placement and orientation within the AAV genome can affect transgene expression levels and editing efficiencies [18,26,85]. To test if different arrangements of sgRNA and effector cassettes in the Nme2-ABE8e all-in-one AAV construct can further increase in vivo editing efficiency, we moved the U6-sgRNA cassette to the 3' end of the AAV genome and reversed its orientation, similar to the optimal arrangement reported by Fry et al [85] (Figure 6a).…”

CRISPR/Cas9 for hepatitis B virus infection treatment

“…Another way to overcome the pre‐existing immunity of AAV capsids is to give transgenic capsids and decoy (empty) capsids. 44 , 45 …”

Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector

“…Optimizing effector and sgRNA arrangement improves editing efficiency by AAV delivery Previous studies have shown that effector and sgRNA placement and orientation within the AAV genome can affect transgene expression levels and editing efficiencies [18,26,85]. To test if different arrangements of sgRNA and effector cassettes in the Nme2-ABE8e all-in-one AAV construct can further increase in vivo editing efficiency, we moved the U6-sgRNA cassette to the 3' end of the AAV genome and reversed its orientation, similar to the optimal arrangement reported by Fry et al [85] (Figure 6a). Using the same Rosa26 guide described above (Figure 5d), we packaged the rearranged construct in AAV9 capsids and performed tail-vein injections in 8-week-old mice.…”