Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants

The clustered regularly interspaced short palindromic repeat (CRISPR) system has been widely adapted to genome editing to either introduce or correct genetic mutations (Wang et al., 2016). However, due to competition with the dominant non-homologous end-joining (NHEJ) pathway, precise genome modi ﬁ cations through Cas9-stimulated homologous recombination (HR) is inef ﬁ cient. Through fusion of cytidine deaminases, APOBEC1 (apolipoprotein B editing complex 1) or AID (activation-induced deaminase), with Cas9 variants, several groups have developed the cytidine base editor (BE) systems (Komor et al., 2016; Li et al., 2018; Nishida et al., 2016). The BE system achieves programmable conversion of C (cid:129) G base pairs to T (cid:129) A without double-stranded DNA cleavage (Zhou et al., 2017). More recently, adenine base editors (ABEs), which ef ﬁ ciently convert A (cid:129) T base pairs to G (cid:129) C in genomic DNA, have been developed via fusion of an engineered tRNA adenosine deaminase (ecTadA from Escherichia coli ) with nCas9 (Gaudelli et al., 2017). The ABE system has quickly been adapted to generate disease models and correction of genetic disease in mice (Ryu et al., 2017; Liu et al., 2018). However, whether the editing ef ﬁ ciency and the targeting scope of ABE could be improved is largely unexplored. In this study, we describe the ef ﬁ cient generation of base-edi-ted mice and rats modeling human diseases through ABEs with highest ef ﬁ ciency up to 100%. We also demonstrate an increase of ABE activity through injection of chemically modi ﬁ ed tracrRNA and crRNA in mouse zygotes, and the expansion of editing scope by fusion of an ecTadA mutant to


Cell culture and transfection.
HEK293T (ATCC CRL-3216) cells lines were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with10% FBS (Gibico), 100 U ml -1 penicillin, and 100 mg ml -1 streptomycin at 37 °C with 5% CO2 incubation. Cells were seeded into 24-well plates (Corning) one day before transfection at a density of 200,000 cells per well, and transfected at 70-80% confluency using polyethylenimine (Polysciences) following the manufacturer's recommended protocol. For each well of a 24-well plate, a total of 500 ng DNA was used. 250 ng of SaKKH-ABE and 250 ng of sgRNA expression plasmids were transfected using 1.5 µl of polyethylenimine per well according to the manufacturer's protocol. The cells in a 24-well plate were seeded into a 12-well plate 3 days after transfection. Fluorescence-activated cell sorting ( FACS) was performed 5 days after transfection following BD's recommended protocol.

Animals.
C57/BL6 strain mice and Sprague-Dawley strain rats purchased from Shanghai Laboratory Animal Center were housed in standard cages in a specific pathogen-free facility on a 12 h light/dark cycle with ad libitum access to food and water. All animal experiments conformed to the regulations drafted by the Association for Assessment and Accreditation of Laboratory Animal Care in Shanghai and were approved by the East China Normal University Center for Animal Research. To obtain homozygous mice carrying mutation on the TGA stop codon of Fah, the crossed founders were kept on 10 mg/L NTBC water.

Targeted deep sequencing of genomic DNA samples
HEK293T or mouse tail tip genomic DNA was isolated using the blood/cell/tissue DNA Isolation Kit (TianGen) according to the manufacturer's instructions. PCR products for targeted deep sequencing were prepared as described in HI-TOM Kits (Novogene).
Mixed samples were sequenced on the Illumina HiSeq platform as previously described (www.hi-tom.net/hi-tom/documentation.html). All primers used for Hi-Tom deep sequencing are listed in table S6.

qPCR
Mice were sacrificed by carbon dioxide asphyxiation. Total RNA was extracted from mouse liver using RNAiso Plus (Takara) and reverse transcribed using HiScript II Q RT SuperMix (Vazyme). qPCRs were run on QuantStudio 3 (Applied Biosystems) using Hieff™ qPCR SYBR® Green Master Mix (YEASEN). Data were normalized to β-Actin. Primers used for qPCR are listed in table S4.

Western blot, histology and immunohistochemistry.
For western blot, mouse liver protein lysates were prepared in RIPA buffer with proteinase and phosphatase inhibitors. Proteins were separated on SDS/PAGE and transferred to nitrocellulose membranes for immunostaining with 1:500 anti-Fah antibody (AbboMax). For immunohistochemistry, mouse livers were fixed with 4% paraformaldehyde (PFA), embedded in paraffin, sectioned at 5 μm and detected with 1:2000 anti-Fah antibody (AbboMax). For measuring glycogen content, rat muscles tissues were fixed with 4% PFA, frozen in Tissue Freezing Medium (Leica), sectioned at 5 μm and stained using Glycogen Periodic Acid Schiff (PAS/Hematoxylin) Stain Kit (Solarbio).

Serum biochemical analysis.
Blood was collected using retro-orbital puncture and centrifuged at 12,000 r.p.m. at 4 for 5 min. The serum was frozen at −80°C before being sent to ADICON Clinical Laboratory to determine AST, ALT, TBIL and ALB levels.

GAA activity assay
Substrate was 6 mM 4-methylumbelliferone-alpha-glucopyranoside Sigma , prepared in 10% 2-methoxyethanol/0.2M phosphate citrate (PC) buffer (0.1M citric acid, 0.2 M Na2HPO4 in DI water pH 4.3). Protein concentrations were determined by BCA Protein Assay Kit (Pierce). The acid α-glucosidase assay reaction mixture in each tube consisted of 50μL muscle tissue homogenates, 10μL 100μM acarbose, and 50μL 6mM 4-MUG. Two blank tubes with 50μL water instead of homogenates were incubated along with the samples. All tubes were incubated for 1 hour at 37°C. The reaction was stopped by addition of 1.25 mL of Glycine Carbonate buffer (glycine, 0.17 M; Na 2 CO 3 , 0.51M; pH 10.3). Muscle tissue homogenate (50μL) was added to each blank tube after the reaction was stopped. Fluorescence intensity was measured with a VICTOR2 spectrofluorometer (Finland, Wallac). Acid β-galactosidase at pH4.0 was used as a control. Enzyme activity is expressed as nmol/hour/mg protein.

Statistics
Data are presented as means ± DS. Means of two groups were compared using Student's t-test (unpaired, 2-tailed), with P < 0.05 considered to be statistically significant.

Data availability.
Plasmids encoding ABE7.10 and SaKKH-ABE are available upon request from