PMID- 32833533
OWN - NLM
STAT- MEDLINE
DCOM- 20210804
LR  - 20240104
IS  - 2573-1602 (Electronic)
IS  - 2573-1599 (Print)
IS  - 2573-1599 (Linking)
VI  - 3
IP  - 4
DP  - 2020 Aug
TI  - Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene 
      Editing Efficiency.
PG  - 276-283
LID - 10.1089/crispr.2020.0021 [doi]
AB  - 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.
FAU - Fry, Lewis E
AU  - Fry LE
AD  - Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical 
      Neurosciences and NIHR Oxford Biomedical Research Centre, University of Oxford, 
      Oxford, United Kingdom; and Oxford University Hospitals NHS Foundation Trust, 
      Oxford, United Kingdom.
AD  - Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, 
      United Kingdom.
FAU - Peddle, Caroline F
AU  - Peddle CF
AD  - Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical 
      Neurosciences and NIHR Oxford Biomedical Research Centre, University of Oxford, 
      Oxford, United Kingdom; and Oxford University Hospitals NHS Foundation Trust, 
      Oxford, United Kingdom.
FAU - Stevanovic, Marta
AU  - Stevanovic M
AD  - Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical 
      Neurosciences and NIHR Oxford Biomedical Research Centre, University of Oxford, 
      Oxford, United Kingdom; and Oxford University Hospitals NHS Foundation Trust, 
      Oxford, United Kingdom.
FAU - Barnard, Alun R
AU  - Barnard AR
AD  - Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical 
      Neurosciences and NIHR Oxford Biomedical Research Centre, University of Oxford, 
      Oxford, United Kingdom; and Oxford University Hospitals NHS Foundation Trust, 
      Oxford, United Kingdom.
AD  - Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, 
      United Kingdom.
FAU - McClements, Michelle E
AU  - McClements ME
AD  - Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical 
      Neurosciences and NIHR Oxford Biomedical Research Centre, University of Oxford, 
      Oxford, United Kingdom; and Oxford University Hospitals NHS Foundation Trust, 
      Oxford, United Kingdom.
FAU - MacLaren, Robert E
AU  - MacLaren RE
AD  - Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical 
      Neurosciences and NIHR Oxford Biomedical Research Centre, University of Oxford, 
      Oxford, United Kingdom; and Oxford University Hospitals NHS Foundation Trust, 
      Oxford, United Kingdom.
AD  - Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, 
      United Kingdom.
LA  - eng
GR  - MC_PC_18059/MRC_/Medical Research Council/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - CRISPR J
JT  - The CRISPR journal
JID - 101738191
RN  - 0 (RNA, Guide, CRISPR-Cas Systems)
RN  - EC 3.1.- (CRISPR-Associated Protein 9)
SB  - IM
MH  - CRISPR-Associated Protein 9/*genetics
MH  - CRISPR-Cas Systems
MH  - Clustered Regularly Interspaced Short Palindromic Repeats
MH  - Dependovirus
MH  - *Gene Editing
MH  - Genetic Vectors
MH  - *Promoter Regions, Genetic
MH  - RNA, Guide, CRISPR-Cas Systems/*genetics
MH  - Staphylococcus aureus/enzymology
PMC - PMC7469699
COIS- The authors have no competing financial interests to declare. The funders had no 
      role in the design of the study; in the collection, analyses, or interpretation 
      of data; in the writing of the manuscript; or in the decision to publish the 
      manuscript.
EDAT- 2020/08/25 06:00
MHDA- 2021/08/05 06:00
PMCR- 2020/08/01
CRDT- 2020/08/25 06:00
PHST- 2020/08/25 06:00 [entrez]
PHST- 2020/08/25 06:00 [pubmed]
PHST- 2021/08/05 06:00 [medline]
PHST- 2020/08/01 00:00 [pmc-release]
AID - 10.1089/crispr.2020.0021 [pii]
AID - 10.1089/crispr.2020.0021 [doi]
PST - ppublish
SO  - CRISPR J. 2020 Aug;3(4):276-283. doi: 10.1089/crispr.2020.0021.