Dear Editor,
CRISPR/Cas9 is a powerful tool for genome editing (Komor et al., 2017). Recently,
it has been employed in several attempts to edit the human embryos (Liang et al.,
2015; Kang et al., 2016; Tang et al., 2017). A major technical concern particularly
relevant in studies involving human embryos is the potential off-target effects (Callaway,
2016; Plaza Reyes and Lanner, 2017). Consequently, development of safer genome editing
strategy in human embryos is highly anticipated (Cyranoski and Reardon, 2015). The
off-target mutation result in part from Cas9-mediated double strand break (DSB) of
DNA. Recently, base editing (BE) without the introduction of DSB has been achieved.
The key design for BE is to use a catalytically inactive Cas9 to recruit the cytidine
deaminase APOBEC to target sequences, leading to conversion of C to T within a window
of approximately five nucleotides (Komor et al., 2016). Therefore, BE is apparently
determined by additional features of the target sequence and offers a potentially
safer approach for genome editing. Here we report the initial technical assessment
of applying BE3, base editor 3 (Komor et al., 2016), in discarded human tripronuclear
embryos.
We targeted two human gene sites, HEK293 site 4 and RNF2 (Komor et al., 2016). BE3
and sgRNAs were prepared in vitro as described (Shen et al., 2014), and microinjected
into the cytoplasm of the tripronuclear zygotes with the concentration of one hundred
nanogram BE3 and fifty nanogram sgRNA per microliter. The zygotes were collected 48
h after microinjection, with the embryos containing different numbers of cells ranging
from 1 to 8 (Table S1). In total, 8 zygotes for each of the two targets (#1–8 for
HEK293 site 4, #9–16 for RNF2) were collected (Fig. 1A). Whole genome of each individual
sample was amplified and used as the template for further analysis.
Figure 1
Highly efficient base editing by BE3 in discarded human tripronuclear embryos. (A)
Summary of the information of the experiments. (B) The representative sequence chromatogram
of site4 from sample #1. The sequence was the targeted sites. The red star indicated
the conversion of C to T. (C) Summary of the deep sequencing of the on-target site
for HEK293 site4. The editing efficiency of every C within the target site was indicated.
The PAM was highlighted in red. (D) The representative sequence chromatogram of RNF2
from sample #11. The sequence was the targeted sites. The red star indicated the conversion
of C to T
To detect the efficiency of base editing, the region around the target sites was amplified
and analyzed initially by the T7EN1 cleavage assay. For HEK293 site 4, we did not
detect any cleavage bands in any of the samples (Fig. S1A). However, sequencing of
the bulk PCR products revealed C to T conversion at the sixteenth base distal from
the PAM in 7 of the samples (#1–6, #8) (Figs. 1B and S1B), which is in accordance
with the original report in human cell lines (Komor et al., 2016). We cloned 3 (#1–3)
of the 8 bulk PCR products and sequenced multiple colonies from each primary product.
For PCR products #2 and #3, each clone sequenced displayed C to T substitution, while
PCR product #1 yielded one wildtype genotype besides the identical mutation genotypes
(Fig. S1C), indicating highly efficient editing.
To more carefully analyze the on-target editing effects, deep sequencing was applied
to samples #2 and #3. In total, more than 3 M clean reads for each sample were generated.
The results showed that only the 16th nucleotide distal from the PAM completely carried
C to T conversion with the efficiency as high as 0.97 for sample #3, and 0.99 for
sample #2. No other nucleotide alteration was detected (Fig. 1C). Besides, no on-target
indel was found (Table S2). These results demonstrated the BE led to highly precise
and efficient genome editing in human embryos.
The same tests were performed for RNF2. T7EN1 cleavage bands were detected in 7 out
of the 8 samples (#9–13, #15–16) (Fig. S2A). Sanger sequencing of PCR products confirmed
C to T conversion in the 7 samples with cleavage (Figs. 1D and S2B). To further analyze
the editing, 3 samples (#10–12) were selected for genotyping by TA cloning and subsequent
sequencing. As reported before (Komor et al., 2016), in most cases, 2 cytosines (at
the 18th and 15th nucleotide distal from the PAM) were simultaneously mutated to T,
and triple C to T conversion (at the 18th, 15th, and 9th nucleotide distal from the
PAM) also occurred (sample #10 and #12) (Fig. S2C). Collectively, these results demonstrated
highly efficient and precise on-target base editing by BE3 in human embryos.
We next tried to mutate the two genes simultaneously in the tripronuclear zygotes.
To avoid possible toxicity, the concentration of each sgRNA was lowered to 25 nanogram
per microliter. Nine embryos (#17–25) were collected and the target sites were analyzed
by sequencing (Fig. 1A). For HEK293 site 4, the expected substitution in the sixteenth
base distal from the PAM was observed in all samples (Fig. S3A), although the wild
type genotype was also detectable in a few samples (Fig. S3B). A sample (#18) was
randomly selected for on-target analysis by deep sequencing. The results showed that
the conversion rate in the 16th C was about 0.68, which was consistent with the results
of bulk PCR products sequencing (Fig. 1C). For RNF2, mutations were detected in all
samples although with lower efficiency than the HEK293 site 4 site in some samples
(Fig. S4). Similar to previous results in human cell lines (Komor et al., 2016), besides
C→T conversion, we observed rare C→G substitution (sample #21) and C→A conversion
(sample #22). This result was verified by re-sequencing of the cloned products. Similar
to the situations of targeting RNF2 alone shown above, triple substitution was found
in sub-clones from sample #23. These results further demonstrated efficient base editing
of BE3 in human embryos.
The off-target mutagenesis is a major concern for all genome editing approaches (Tsai
and Joung, 2016). Since no off-target for the sgRNA against RNF2 was detected by the
GUIDE-seq (Tsai et al., 2015), we therefore focused on the off-target analysis when
editing HEK293 site 4. We selected 7 off-target sites according to the GUIDE-seq results,
and amplified the regions around the selected off-target sites for samples #2, #3,
#5, #8, #18, #20, #22, and #25. All PCR products were sequenced and compared with
the reference sequence. The results showed, among the 56 sites of the 8 samples, two-site
substitution for off-target site #3 and one-site substitution for off-target site
#4 were found on sample #3 (Fig. 2A and 2B). No off-target mutagenesis was detected
in other samples (Fig. S5).
Figure 2
Low off-target mutation detection by BE3 in discarded human tripronuclear embryos.
(A) The representative sequence chromatogram of off-target site #3 detected in sample
#3. The black stars indicated the conversion of C to T. (B) The representative sequence
chromatogram of off-target site #4 detected in sample #3. The black stars indicated
the conversion of C to T. (C) Summary of the deep sequencing of the off-target site
#3. The editing efficiency of every C within the target site was indicated. The PAM
was highlighted in red. The different nucleotides compared with the target site were
highlighted in yellow. The PAM was highlighted in red. (D) Summary of the deep sequencing
of the off-target site #4. The editing efficiency of every C within the target site
was indicated. The PAM was highlighted in red. The different nucleotides compared
with the target site were highlighted in yellow. The PAM was highlighted in red
To further characterize the possible off-target by BE in human embryos, deep sequencing
analysis, which detects targeting effect with the most sensitivity, was performed
on 5 reported off-target sites (#1, #3, #4, #6, #8) (Komor et al., 2016). The sample
#3 with off-target mutagenesis, together with randomly selected sample #2 and #18
were subjected for analysis. As described above, more than 3 M clean reads for each
off-target site of every sample were generated. The C→T conversion in these off-target
sites were firstly calculated. As expected, off-target site 3 containing two-site
alteration and site 4 containing one-site alteration were demonstrated in sample #3
(Fig. 2C and 2D), and further confirmed by sequencing the PCR products. In addition,
a C→T conversion at the 16th nucleotide distal from the PAM of the off-target site
3 was detected in sample #18 with the frequency of 0.18.
Considering the possible indel mediated by BE at off-target sites, then, the indel
frequency was calculated for all the off-target sites. The results showed, the indels
were found in off-target site 3 of all the 3 analyzed samples with the frequency of
0.09 for sample #2, 0.10 for sample #3, and 0.05 for sample #18, respectively (Table
S2). We also detected indels in some other off-target sites with the frequency of
0.01 at off-target site 4 for #2 and #3, 0.02 at off-target site 6 for #2, and off-target
site 8 for #3, respectively (Table S2), which might be derived from the noise sequence.
Taken together, the above results showed that BE3 induces near perfect gene editing
in the target site with extremely low off-target mutagenesis for human embryos. Nevertheless,
more research on other aspects such as toxicity associated with the use of BE3 in
human embryos will be warranted in the future.
This study represents the first successful application of base editor in human embryos.
This exciting strategy is highly efficient and safer than editing based on DSBs. Therefore,
our line of research may have future implications considering that nearly one thousand
human genetic diseases involve T→C or A→G mutations.
FOOTNOTES
We thank members of Huang and Liu labs for helpful discussions. We are grateful to
Mr. Ming Zhao from Shanghai Institute of Hematology & State Key Laboratory of Medical
Genomics, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine for his
excellent technical assistance, and Dr. Jianghuai Liu from Nanjing University for
excellent language editing. This works is supported by the Innovation of Science and
Technology Commission of Guangzhou, China (201604020075) and National Natural Science
Foundation of China (Grant No. 31471400).
The authors declare no competing financial interests.
Electronic supplementary material
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Supplementary material 1 (PDF 1945 kb)