Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery

Gaj, Thomas; Staahl, Brett T.; Rodrigues, Gonçalo M.  C.; Limsirichai, Prajit; Ekman, Freja K.; Doudna, Jennifer A.; Schaffer, David V.

doi:10.1093/nar/gkx154

Title: Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery

Journal Article · Thu Mar 02 00:00:00 EST 2017 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkx154· OSTI ID:1625562

Gaj, Thomas ^[1]; Staahl, Brett T. ^[2]; Rodrigues, Gonçalo M. C. ^[3]; Limsirichai, Prajit ^[4]; Ekman, Freja K. ^[5]; Doudna, Jennifer A. ^[6]; Schaffer, David V. ^[7]

Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology
Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering; Universidade de Lisboa, Lisbon, (Portugal). Instituto Superior Técnico. Dept. of Bioengineering and Inst. for Bioengineering and Biosciences
Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). MBIB Division
Univ. of California, Berkeley, Berkeley, CA (United States). Dept. of Bioengineering; Univ. of California, Berkeley, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering; Univ. of California, Berkeley, Berkeley, CA (United States). Helen Wills Neuroscience Inst.

Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); National Research Service Award (NRSA); F. Hoffman-La Roche Postdoctoral Fellowship; Fundação para a Ciência e a Tecnologia (FCT) de Portugal; National Institutes of Health (NIH)

Grant/Contract Number:: AC02-05CH11231; F32GM113446; RPF311; SFRH/BD/89374/2012; R01EY022975

OSTI ID:: 1625562

Journal Information:: Nucleic Acids Research, Vol. 45, Issue 11; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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