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.
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References
1.
MaederML, StefanidakisM, WilsonCJ, et al.Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med, 2019; 25:229–233. DOI: 10.1038/s41591-018-0327-9.
2.
XueK, JollyJK, BarnardAR, et al.Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia. Nat Med, 2018; 24:1507–1512. DOI: 10.1038/s41591-018-0185-5.
3.
Cehajic-KapetanovicJ, XueK, Martinez-Fernandez de la CamaraC, et al.Initial results from a first-in-human gene therapy trial on X-linked retinitis pigmentosa caused by mutations in RPGR. Nat Med, 2020; 26:354–359. DOI: 10.1038/s41591-020-0763-1.
4.
RussellS, BennettJ, WellmanJA, et al.Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, Phase 3 trial. Lancet, 2017; 390:849–860. DOI: 10.1016/S0140-6736(17)31868-8.
5.
RanFA, CongL, YanWX, et al.In vivo genome editing using Staphylococcus aureus Cas9. Nature, 2015; 520:186–191. DOI: 10.1038/nature14299.
6.
LiA, LeeCM, HurleyAE, et al.A self-deleting AAV-CRISPR system for in vivo genome editing. Mol Ther Methods Clin Dev, 2019; 12:111–122. DOI: 10.1016/j.omtm.2018.11.009.
7.
YangY, WangL, BellP, et al.A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol, 2016; 34:334–338. DOI: 10.1038/nbt.3469.
8.
JarrettKE, LeeCM, YehY-H, et al.Somatic genome editing with CRISPR/Cas9 generates and corrects a metabolic disease. Sci Rep, 2017; 7:44624. DOI: 10.1038/srep44624.
9.
YuW, MookherjeeS, ChaitankarV, et al.Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun, 2017; 8:14716. DOI: 10.1038/ncomms14716.
10.
KimE, KooT, ParkSW, et al.In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun, 2017; 8:1–12. DOI: 10.1038/ncomms14500.
11.
HungSSC, ChrysostomouV, LiF, et al.AAV-mediated CRISPR/Cas gene editing of retinal cells in vivo. Invest Opthalmol Vis Sci, 2016; 57:3470–3476. DOI: 10.1167/iovs.16-19316.
12.
TsaiYT, WuWH, LeeTT, et al.Clustered regularly interspaced short palindromic repeats–based genome surgery for the treatment of autosomal dominant retinitis pigmentosa. Ophthalmology, 2018; 125:1421–1430. DOI: 10.1016/j.ophtha.2018.04.001.
13.
MurlidharanG, SakamotoK, RaoL, et al.CNS-restricted transduction and CRISPR/Cas9-mediated gene deletion with an engineered AAV vector. Mol Ther Nucleic Acids, 2016; 5:e338. DOI: 10.1038/mtna.2016.49.
14.
GuoY, VanDusenNJ, ZhangL, et al.Analysis of cardiac myocyte maturation using CASAAV, a platform for rapid dissection of cardiac myocyte gene function in vivo. Circ Res, 2017; 120:1874–1888. DOI: 10.1161/CIRCRESAHA.116.310283.
15.
IshizuT, HigoS, MasumuraY, et al.Targeted genome replacement via homology-directed repair in non-dividing cardiomyocytes. Sci Rep, 2017; 7:9363. DOI: 10.1038/s41598-017-09716-x.
16.
TabebordbarM, ZhuK, ChengJKW, et al.In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science, 2016; 351:407–411. DOI: 10.1126/science.aad5177.
17.
NelsonCE, HakimCH, OusteroutDG, et al.In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science, 2016; 351:403–407. DOI: 10.1126/science.aad5143.
18.
LongC, AmoasiiL, MireaultAA, et al.Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science, 2016; 351:400–403. DOI: 10.1126/science.aad5725.
19.
CurtinJA, DaneAP, SwansonA, et al.Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Ther, 2008; 15:384–390. DOI: 10.1038/sj.gt.3303105.
20.
ParkSK, HwangBJ, KeeY. Promoter cross-talk affects the inducible expression of intronic shRNAs from the tetracycline response element. Genes Genomics, 2019; 41:483–490. DOI: 10.1007/s13258-019-00784-z.
21.
Semple-RowlandSL, CogginWE, GeeseyM, et al.Expression characteristics of dual-promoter lentiviral vectors targeting retinal photoreceptors and Müller cells. Mol Vis, 2010; 16:916–934.
22.
NieL, Thakur MDas, WangY, et al.Regulation of U6 promoter activity by transcriptional interference in viral vector-based RNAi. Genomics Proteomics Bioinformatics, 2010; 8:170–179. DOI: 10.1016/S1672-0229(10)60019-8.
23.
LambethLS, Van HaterenNJ, WilsonSA, et al.A direct comparison of strategies for combinatorial RNA interference. BMC Mol Biol, 2010; 11:77. DOI: 10.1186/1471-2199-11-77.
24.
LevyJM, YehW-H, PendseN, et al.Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat Biomed Eng, 2020; 4:97–110. DOI: 10.1038/s41551-019-0501-5.
25.
FriedlandAE, BaralR, SinghalP, et al.Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol, 2015; 16:257. DOI: 10.1186/s13059-015-0817-8.
26.
KleinstiverBP, PrewMS, TsaiSQ, et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 2015; 523:481–485. DOI: 10.1038/nature14592.
27.
McClementsME, BarnardAR, SinghMS, et al.An AAV dual vector strategy ameliorates the Stargardt phenotype in adult Abca4−/− mice. Hum Gene Ther, 2019; 30:590–600. DOI: 10.1089/hum.2018.156.
28.
LipinskiDM, BarnardAR, SinghMS, et al.CNTF gene therapy confers lifelong neuroprotection in a mouse model of human retinitis pigmentosa. Mol Ther, 2015; 23:1308–1319. DOI: 10.1038/mt.2015.68.
29.
FischerMD, McClementsME, Martinez-Fernandez de la CamaraC, et al.Codon-optimized RPGR improves stability and efficacy of AAV8 gene therapy in two mouse models of X-linked retinitis pigmentosa. Mol Ther, 2017; 25:1854–1865. DOI: 10.1016/j.ymthe.2017.05.005.
30.
BrinkmanEK, ChenT, AmendolaM, et al.Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res, 2014; 42:e168. DOI: 10.1093/nar/gku936.
31.
LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 2001; 25:402–408. DOI: 10.1006/meth.2001.1262.
32.
YeungE, DyAJ, MartinKB, et al.Biophysical constraints arising from compositional context in synthetic gene networks. Cell Syst, 2017; 5:11–24.e12. DOI: 10.1016/j.cels.2017.06.001.
Penaud-BudlooM, Le GuinerC, NowrouziA, et al.Adeno-associated virus vector genomes persist as episomal chromatin in primate muscle. J Virol, 2008; 82:7875–7885. DOI: 10.1128/JVI.00649-08.
37.
NakaiH, YantSR, StormTA, et al.Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol, 2001; 75:6969–6976. DOI: 10.1128/jvi.75.15.6969-6976.2001.
38.
YanZ, SunX, FengZ, et al.Optimization of recombinant adeno-associated virus-mediated expression for large transgenes, using a synthetic promoter and tandem array enhancers. Hum Gene Ther, 2015; 26:334–346. DOI: 10.1089/hum.2015.001.
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