Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.
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.
Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.
Thomas
T
Gaj
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Brett T
BT
Staahl
Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.|Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisbon, Portugal.
Gonçalo M C
GMC
Rodrigues
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Prajit
P
Limsirichai
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
Freja K
FK
Ekman
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.|Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.|Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.|MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Jennifer A
JA
Doudna
Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.|Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA.|Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
David V
DV
Schaffer
D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D002454:Cell Differentiation; D000229:Dependovirus / Q000235:genetics*; D004720:Endonucleases / Q000737:chemistry*; D005347:Fibroblasts / Q000502:physiology; D055879:Gene Knock-In Techniques; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases