Prime Editing in vivo: Correcting the Leptin Receptor of db/db Mice
Kyung Eun Lee1,†,
Yanping Xu1,†, Bingchuan Geng1,
Jongsoo Kim1, Natalie Kellon1,
Michele He1, Zhentao Zhang1, Deqiang
Li2, Doug A. Gouchoe3, Hua
Zhu1,*
1 Department of Surgery, Davis Heart and Lung Research
Institute, the Ohio State University Wexner
Medical Center, Columbus, OH 43210, USA;
kyungeun.lee@osumc.edu
(K.E.L.);
yanping.xu@osumc.edu
(Y.X.);
bingchuan.geng@osumc.edu
(B.G.);
jongsoo.kim@osumc.edu
(J.K.);
natalie.kellon@osumc.edu
(N.K.);
he.1908@buckeyemail.osu.edu
(M.H.);
zhentao.zhang@osumc.edu
(Z.Z.); hua.zhu@osumc.edu
(H.Z.)2 The Center for Cardiovascular Research at Nationwide
Children’s Hospital, Columbus, OH 43205;
deqiang.li@nationwidechildrens.org
(D.L.)3 COPPER Laboratory, Department of Surgery, The Ohio
State University Wexner Medical Center, Columbus OH 43210, USA;
doug.gouchoe@osumc.edu
(D.G.)* Correspondence:
hua.zhu@osumc.edu (H.Z.)† Shared first authorship.
Abstract: Genetic diseases can be caused by monogenic diseases,
which result from a single gene mutation in the DNA sequence. Many
innovative approaches have been developed to cure monogenic genetic
diseases, namely by genome editing. A specific type of genomic editing,
prime editing, has the potential advantage to edit the human genome
without requiring double-strand breaks or donor DNA templates for
editing. Additionally, prime editing does not require a precisely
positioned protospacer adjacent motif (PAM) sequence, which offers
flexible target and more precise genomic editing. Here we detail a novel
construction of a prime editing extended guide RNA (pegRNA) to target
mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The
pegRNA was then injected into the flexor digitorum brevis (FDB) muscle
of db/db mice to demonstrate in vivo efficacy, which resulted in
pegRNA mediated base transversion at endogenous base transversion.
Genomic DNA sequencing confirmed that prime editing could correct the
mutation of leptin receptor gene in db/db mice. Furthermore, prime
editing treated skeletal muscle exhibited enhanced leptin receptor
signals. Thus, the current study showed in vivo efficacy of prime
editing to correct mutant protein and rescue the physiology associated
with functional protein.
Keywords: Prime Editing; Leptin Receptor; db/db mouse
1. Introduction
Single genetic mutations in the DNA sequence can sometimes result in
monogenic diseases. Most pathogenic alleles arise from specific
insertions, deletions, or base substitutions [1].
The CRISPR-Cas9 system is currently one of the most well-known gene
editing tools that scientists use, which requires the need of
double-strand breaks (DSB) or donor DNA (dDNA) template[2]. However, this may not be the most ideal way
to engage in gene editing. A modified version of the CRISPR-Cas9, Cas9
nickase, can offer higher editing efficiency without DSBs and fewer
indel byproducts [3,4]. However, Cas9 nickase
still has several limitations. It requires a very well-positioned target
base, in which a protospacer adjacent motif (PAM) sequence should be in
the approximately 15 bases from the target site. Additionally, it is
limited in the point mutations it can edit, namely a C:G base pair into
a T:A base pair and an A:T base pair into a G:C base pair[5]. This is one of the main drawbacks of Cas9
nickase, as there is a litany of different classes of mutations,
signaling the need for expanded approaches for single mutation editing.
Recently, Andrew V. Anzalone et al. established a powerful
genome-editing tool known as prime editing (PE), which includes a
Cas9n-H840A and C-terminal reverse transcriptase (M-MLV RTase)[6]. The RNA component of PE, prime editing
extended guide RNA or pegRNA, could specify target locuses and
accurately transmit genomic information from the pegRNA to complete the
desired edit [6]. PE also offers major advantages
since it does not require a PAM sequence, which ultimately offers more
targeting flexibility and greater editing precision. After its
invention, this technology has been applied in many in vitro studies[7]. However, while it has been used in vivo[8-10], its application in animal is limited due
to the large size of PE constructs.
B6.BKS(D)-Leprdb/J (db/db) mice offer an ideal in vivo model in
order to study the efficacy of PE. The db/db mouse shows the phenotype
of obesity caused by G to T point mutation in intron 18 of the leptin
receptor gene, which leads to alternative splicing resulting in the lack
of C-terminal of leptin receptor. Ultimately, this results in the
inability to initiate intracellular signal transduction, causing severe
obesity in db/db mice [11]. In this manuscript, we
describe our design of a guide RNA (gRNA) to target leptin receptor
intron 18, around the position of G to T point mutation. The gene
editing efficacy was confirmed by Sanger sequencing. Then, a pegRNA was
constructed based on the gRNA sequence, which we observed to mediate
successful base transversion in vitro . The pegRNA and PE
constructs were delivered to the skeletal muscle of db/db mice via
established electroporation. Genomic sequencing confirmed the constructs
could effectively and precisely correct leptin receptor gene mutation in
db/db mice. Furthermore, leptin receptor signaling was significantly
enhanced in prime editing treated skeletal muscle, indicating the
corrected leptin receptor protein is functional. Taken together, our
study provided solid evidence for in vivo application of prime
editing to precisely correct disease-causing mutations.
2. Materials and Methods
2.1. Design of gRNA to Target Leptin Receptor
The guide RNAs (gRNAs) were designed using the online predictor
CCTop-CRISPR/Cas9 target (https://cctop.cos.uni-heidelberg.de)[12,13] to target mouse leptin receptor intron 18,
and three gRNAs were selected for the testing. The designs are as
follows: gRNA1: 5’-CACC CCA TCA AAA TGT AAA CAT C-3’; 5’- AAAC GAT GTT
TAC ATT TTG ATG G-3’, gRNA2: 5’- CACC TCA AAA TGT AAA CAT CTT C-3’; 5’
AAAC GAA GAT GTT TAC ATT TTG A-3’ gRNA3: 5’- CACC GTC ATT CAA ACC ATA
GTT T-3’; 5’-AAAC AAA CTA TGG TTT GAA TGA-3’. 1 µl of each 100 µM oligos
were put in annealing buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl), and
were heated and cooled down by a thermocycler for complementary
annealing. Double-strand oligos were ligated into px459 V2.0-eSpCas9
(1.1) plasmid, which was a gift from Yuichiro Miyaoka (Addgene plasmid
# 108292; http://n2t.net/addgene:108292; RRID: Addgene_108292)[14]. The gRNA clone was confirmed by Sanger
sequencing.
2.2. Determining the Efficacy of gRNA in vitro by Using C2C12 Cells
First to demonstrate in vitro efficacy, we demonstrated proper
transfection into C2C12 cells with gRNAs. To determine the concentration
of puromycin to destroy more than 90 % of C2C12 cells, C2C12 cells were
seeded on the 6-well plate and treated with puromycin at the
concentration of 0, 1, 2.5, 5, 7.5, 10 µg/ml. It was determined that 10
µg/ml was the ideal concentration to destroy C2C12 cells and permit
proper transfection. Next, C2C12 cells (1 X 105) were
again seeded in a 6-well plate. On day 2, 2 µg of either: constructed
gRNA CRSIPR vector or green fluorescent protein (GFP) or vector only
construct was transfected into the C2C12 cells by mixing 4 µl of Viafect
transfection (Promega, # E4981) with them. On day 3, cells were treated
with 10 µg/ml of puromycin for 24-hours followed by changing to growth
medium containing high glucose DMEM (Gibco, # 11965092) supplemented
with 10% Fetal Bovine Serum (FBS) (VWR, # 97068-091) and 1%
penicillin–streptomycin (Sigma, #P0781). On day 6, cells were
collected for the genomic DNA isolation. Cas9 sequence PCR primers were
designed to amplify a 277 bp product evenly flanking the CRISPR/Cas9
site. The primers were as follows: forward primer, 5’- CAG AGA ACG GAC
ACT CTT TG -3’reverse primer -3’, reverse primer, 5’- GCA GAG TCC ATG
AAT ATC AAC -3’. Genomic DNA was extracted from a 6-well plate using the
proteinase K (Sigma, #3115879001). PCRs were performed using Q5®
High-Fidelity DNA Polymerase (NEB, #M0491). 100 ng of C2C12 genomic DNA
was used as a template for PCR. Each 20 µl of PCR reaction consisted of
4 µl of 5 x buffer (NEB), 2 µl of 2.5 mM dNTPs, 0.5 µl of Q5 Taq DNA
polymerase (NEB), 1 µl of forward primer and 1 µl of reverse primer at
an initial concentration of 10 µM, with respective amounts of DNA
templates and H2O to reach a reaction volume of 20 µl. The PCR
conditions were set at 95 ℃, 5 min; 30 cycles of 95 ℃ for 3 min, 55 ℃
for 30 sec and 72 ℃ for 1 min; and a final extension at 72 ℃ for 5 min.
PCR products were then extracted using 1.5 % agarose gel followed by
isolation of DNA using a Qiagen kit (Qiagen, #12943). The purified DNA
then underwent Sanger Sequencing to confirm.
2.3. Cloning of pegRNAs to Target the Mutant Sequence of Leptin Receptor
in db/db Mice
Methods for cloning pegRNAs have been described
previsouly[6]. Briefly, three kinds of oligos were
prepared as follows. The 1st component oligo set is
for the pegRNA spacer annealed oligos containing gRNA3 targeting the
leptin receptor mutant sequence of db/db mice: 5’- caccg
GGTCATTCAAACCATAGTTT gttttaga- 3’, 5’- tagctctaaaac AAACTATGGTTTGAATGACC
c -3’. The 2nd component oligo set is for the pegRNA
3’ extension annealed oligos: two kinds of pegRNAs were designed. One is
pegRNA8 containing 8 nucleotides for primer binding site (PBS). The
other is pegRNA10 which has 10 nucleotides for primer binding site
(PBS). pegRNA8: 5’ -gtgc GGAAACAAACCTAAACTATGGTT -3’,
5’-aaaaAACCATAGTTTAGGTTTGTTTCC -3’, pegRNA10: 5’ -gtgc
GGAAACAAACCTAAACTATGGTTTG -3’, 5’-aaaaCAAACCATAGTTTAGGTTTGTTTCC -3’. The
3rd component oligo set is for the pegRNA scaffold
annealed oligos: 5’-
GCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCG -3’,
5’- GCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTC
-3’. For cloning of the pegRNA, 1 µl of each forward and reverse gRNA
oligos at an initial concentration of 100 µM were mixed in the annealing
buffer (10 mM Tris-HCl pH 8.5 and 50 mM NaCl). The oligos were annealed
in a thermocycler, heated at 95 ℃ for 3 minutes, then cooled gradually
(0.1℃/s) to 22℃. Then, the 1st and 2nd component oligos were diluted
with annealed oligonucleotides 1:4 by adding 75 µl of H2O. To
phosphorylate the 3rd component oligos 6.25 µl of 4 µM oligonucleotide
duplex, 2.5 µl of 10x T4 DNA ligase buffer (NEB), 0.5 µl of T4 PNK
(NEB), 15.75 µl of H2O were mixed and incubated at 37 ℃ for 60 min
followed by 65 ℃ for 20 min for the heat inactivation. 2 µg of
pU6-pegRNA-GG-Vector plasmid, which was a gift from David Liu) (Addgene
plasmid # 132777; http://n2t.net/addgene:132777; RRID: Addgene_132777)[6] was digested in a mixture of 1.0 µl of BsaI,
5.0 µl of 10x fast digestion buffer, and H2O to bring the volume to 50
µl and the mixture was subsequently incubated at 37℃ for 3 hours. Cut
plasmids were then isolated by phenol extraction. The dephosphorylation
reaction of the vector consisted of 1.5 µl of 10x Antartic phosphatse
buffer, 1 µl Antartic phosphatase, and 12.5 µl of cut vectors was
incubated at 37℃ for 1 hour followed by 70 ℃ for 10 minutes for heat
inactivation. Next, the pegRNA assembly was performed using 1 µl of 10x
ligation buffer, 1 µl of 10x PEG, 0.5 µl of T4 DNA ligase, 0.25 µl of
BsaI, 1 µl at 30 ng of digested pU6-pegRNA-GG plasmid vector, 1 µl at 1
µM of 1st component oilgos, 1 µl at 1 µM of 2nd component oligos, 1 µl
at 1 µM of 3rd component oligos, which was incubated in a thermocycler,
cycle between 5 min at 16℃ and 5 min at 37 ℃ for 8 cycles followed by 15
min at 37 ℃, then 15 min at 80 ℃, then hold at 12 ℃. Transformation of
competent E. coli cells (Promega, Bacterial Strain JM109, # P9751) with
the ligation reaction was performed by the conventional calcium method.
Three clones were picked for sequencing. The sequencing primer used was
the human U6 promoter forward primer: 5’-GAC TAT CAT ATG CTT ACC GT-3’.
2.4. Flexor Digitorum Brevis Electroporation of pegRNA into db/db Mice
All in vivo experiments used B6.BKS(D)-Leprdb/J mice (db/db mice) (The
Jackson Laboratory, Bar Harbor, ME) that were approximately 8 weeks old
and weighed about 40g. All animals were housed under standard conditions
and were provided access to water and standard diet. Animal experiments
were carried out with the approval of the Institutional Animal Care and
Use Committee (IACUC protocol #2016A00000017). All experiments were
performed in strict accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals[15].
Exact details for flexor digitorum brevis (FDB) electroporation have
been previously described [16]. Briefly, the
constructed pegRNA plasmid and PE2-GFP plasmid (pCMV-PE2-P2A-GFP was a
gift from David Liu (Addgene plasmid # 132776;
http://n2t.net/addgene:132776; RRID: Addgene_132776)[6] were purified from E. coli using the QIAGEN
endotoxin free maxiprep kit (Qiagen, #12362) following the
manufacturer’s instructions. The plasmids were dissolved in sterile H2O
at 2 µg/µl, and 10 µg of constructed pegRNA and 30 µg of PE2-GFP were
prepared by mixing in a final volume of 20 µl. The db/db mice were
anesthetized and placed in the supine position. 10 µl of 1x
hyaluronidase solution (8 unit) (Sigma, #H4272) was then injected into
the footpad of mouse and allowed to absorb for 1 hour while the mouse
was sedated. After this, the mixed plasmids were injected into the FDB
followed by electroporating the muscle (20 pulses, 20 msec in
duration/each, at 1Hz, at 100V/cm).
After 5 days, the mouse was then again anesthetized and the FDB muscle
was isolated, and GFP positive single myofibers were collected under
epifluorescence microscopy. Additionally, the ipsilateral extensor
digitorum longus (EDL) muscle was harvested as a negative control. PCR
was then performed by using of Q5 (NEB) Taq DNA polymerase. The primer
set used as: forward primer, 5’- CAG AGA ACG GAC ACT CTT TG -3’reverse
primer -3’, reverse primer, 5’- GCA GAG TCC ATG AAT ATC AAC -3’.
2.5. Protein Extraction and Western Blot Analysis
All muscle samples were washed with PBS and homogenized with RIPA lysis
buffer (EMD Millipore, 20-188) containing protease inhibitors (Sigma,
#P8340) and phosphatase inhibitors (Sigma, #P0044). The denatured
proteins (20ug/well) were separated by 10% SDS/polyacrylamide gels and
electrotransferred onto PVDF membranes (Merck Millipore, Berlin,
Germany). Membranes were incubated in 5% w/v nonfat dry milk for 1 hour
at room temperature, further probed with primary antibody and incubate
at 4 °C with gentle shaking overnight. Then, they were washed with
Tris-buffered saline with 0.1% Tween 20 detergent (TBST) and probed
with secondary antibody. Immunodetection was performed by Bio-Rad
imaging system using SuperSignal West Pico PLUS Chemiluminescent
Substrate (Thermo Fisher Scientific, #34580). Primary antibodies used
were as follows: STAT3 (Cell Signaling, #9139), phospho-STAT3 (Tyr705)
(Cell Signaling, #9145), both were diluted in 5% w/v BSA (Cell
Signaling, #9998) at a ratio of 1:1000; GAPDH (ABclonal, #AC002) was
diluted in 2.5% w/v nonfat dry milk at a ratio of 1:1000.
2.6 Statistical Analysis
Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad
Software Inc., La Jolla, CA, USA). The data are presented as
mean ± standard deviation, depicted by error bars for each group.
Student’s t-test was employed for two-group comparisons. One-way ANOVA
was utilized for comparisons involving more than two groups. The
significance level was set at P < 0.05.
3. Results
3.1. The Screening of gRNA for Mouse Leptin Receptor
We tested three gRNAs to determine their efficiency to bind to the mouse
leptin receptor (intron 18) in C2C12 cells. First, we determined the
concentration of puromycin to kill more than 90 % of C2C12 cells and
found the concentration of 10 µg/ml of puromycin to be suitable.
Following transfection of each gRNA into C2C12 cells, subsequent
treatment with 10 µg/ml of puromycin for 24 hours, genomic DNA was
isolated from cells. PCR was performed to check the peak of gRNA target
sequence (Figure1), and Sanger sequencing revealed the noise peaks
around PAM sequence from each gRNA of interest. gRNA1 and gRNA2 were
deemed to be insufficient matches. Ultimately, gRNA3 targeted the leptin
receptor most efficiently, and therefore was chosen as the backbone of
the pegRNA. According to previous studies [6,17],
editing efficiency was dependent on primer binding site (PBS) length. We
constructed pegRNAs using gRNA3 combined with reverse transcription (RT)
template 15 nucleotides (nt) adding PBS with length of 8 nt (pegRNA8),
10 nt (pegRNA10) for verification.