Gene-editing They performed gene-editing experiments using jetOPTIMUS following Polyplus recommendations. They dissolved 80 ng of editor plasmid and 40 ng of sgRNA plasmid in 12.5 μl of jetOPTIMUS buffer. They mixed the solution with 0.12 μl of jetOPTIMUS and incubated it at room temperature for 10 minutes. They used the resulting mixture to transfect HEK293T cells in 96-well plates. Lentivirus production They also produced lentiviruses. They seeded HEK293T cells into 6 cm tissue culture dishes at a density of 1.2 x 10^6 cells per dish and transfected them 18 hours post-seeding at a confluency of roughly 70-80%. They mixed 2.5 μg of lenti sgRNA-target library plasmids, 1.84 μg of a lentiviral packaging plasmid, and 1.2 μg of a lentiviral envelop plasmid with 572 μL of jetOPTIMUS buffer and 5.25 μl jetOPTIMUS, incubated the mixture at room temperature for 10 minutes, and added it dropwise to the culture. They harvested the lentiviruses 48 hours post-transfection and stored them at -80 °C.

Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. Existing ABEs function most effectively when the target A is in a TA context. Here we evolve the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to generate TadA8r, which extends potent deoxyadenosine deamination to RA (R = A or G) and is faster in processing GA than TadA8.20 and TadA8e, the two most active TadA variants reported so far. ABE8r, comprising TadA8r and a Streptococcus pyogenes Cas9 nickase, expands the editing window at the protospacer adjacent motif-distal end and outperforms ABE7.10, ABE8.20 and ABE8e in correcting disease-associated G:C-to-A:T transitions in the human genome, with a controlled off-target profile. We show ABE8r-mediated editing of clinically relevant sites that are poorly accessed by existing editors, including sites in PCSK9, whose disruption reduces low-density lipoprotein cholesterol, and ABCA4-p.Gly1961Glu, the most frequent mutation in Stargardt disease.