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Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

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Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.
Optimization of the knock-in efficiency by EGFP site-specific integration at the pROSA26 locus. (A) Scheme for EGFP site-specific knock-in at the pROSA26 locus. The EGFP-KI-positive PFF cell clone indicated with a red arrow. (B) The effect of the homology arm lengths on the EGFP site-specific knock-in in PFFs. EGFP fluorescence was analysed via fluorescence microscopy and FACS, at three days post-transfection. Detailed statistical analyses were showed in Fig. S6. (Remarks: the control group that only the circular EGFP targeting donor vector introduced into the PFFs and without the CRISPR/Cas9 vector). (C) The result of the genomic PCR analysis confirmed the knock-in events at the pROSA26 locus. P2 primers amplified the 5′-junction and P7 primers amplified the 3′-junction; the sequences of these primers are listed in Table S3. Lanes 1-6 represent the EGFP knock-in-positive cell clones. NC: Negative control; M: D2000. (D) The effect of different drugs and concentrations on EGFP site-specific knock-in in PFFs. (Column chart, n = 3 independent experiments). Control, DMSO control; SCR7-2, SCR7(2 μM); SCR7-5, SCR7 (5 μM); SCR7-10, SCR7 (10 μM); Noco-0.5, Nocodazole (0.5 μg/ml); Noco-0.75, Nocodazole (0.75 μg/ml); Noco-1, Nocodazole (1 μg/ml);Nu7441-1, Nu7441 (1 μM); Nu7441-2, Nu7441 (2 μM); Nu7441-3, Nu7441(3 μM); Ind-2, indirubin-3′-monoxime (2 μg/ ml); Ind-3, indirubin-3′-monoxime (3 μg/ml); Ind-4, indirubin-3′-monoxime (4 μg/ml); P-RAD51, pig RAD51 over expression plasmid. (E) The effect of different concentrations of SCR7 on PFFs proliferation. (Line chart. n = 3. Graphs show the mean ± S.E.M.).
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Scientific RepoRts | 7: 3036 | DOI:10.1038/s41598-017-02785-y
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Optimization of a CRISPR/Cas9-
mediated Knock-in Strategy at the
Porcine Rosa26 Locus in Porcine
Foetal Fibroblasts
Zicong Xie, Daxin Pang, Kankan Wang, Mengjing Li, Nannan Guo, Hongming Yuan, Jianing
Li, Xiaodong Zou, Huping Jiao, Hongsheng Ouyang, Zhanjun Li & Xiaochun Tang
Genetically modied pigs have important roles in agriculture and biomedicine. However, genome-
specic knock-in techniques in pigs are still in their infancy and optimal strategies have not been
extensively investigated. In this study, we performed electroporation to introduce a targeting donor
vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal
Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the eciency of the EGFP
site-specic knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP
reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting
dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study
demonstrates that we establish an ecient procedures for the exogenous gene knock-in technique and
creates a platform to eciently generate promoter-less and selectable marker-free transgenic PFFs
through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less
and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specic
genome engineering techniques for additional species.
Genetically modied pigs have provided numerous advantages to agriculture and are also widely used to study
human diseases1. Traditional homologous recombination (HR) can be used to generate transgenic pigs, but its
low eciency and laborious and time consuming properties remain problematic. us, the generation of trans-
genic pigs would benet from a more ideal gene editing tool, a “safe harbour” locus in the pig genome and e-
cient gene targeting strategies.
e Rosa26 locus is a “safe harbour” and is conserved in multiple species, including mice2, human3, rats4,
pigs5, sheep6 and rabbits7. e sequence of the pig ROSA26 (pROSA26) locus has been completely characterized,
and the pRosa26 promoter has been identied8. is porcine endogenous promoter is suitable for driving exoge-
nous gene expression in a high and stable manner by avoiding DNA methylation.
With the recent development of site-specic nucleases, including zinc nger nucleases (ZFNs), transcrip-
tion activator-like eector nucleases (TALENs) and clustered regularly interspersed short palindromic repeat
(CRISPR)/CRISPR-associated protein 9 (Cas9), it has become possible to generate transgenic animals with a high
eciency. e CRISPR/Cas9 system is an ideal gene editing tool, and its high eciency, rapid assembly, low cost
and ease of use have enabled its wide use in various species912. However, even with CRISPR/Cas9, the knock-in
eciency (particularly for pigs) remains low, and optimal strategies have not been extensively investigated. Here,
we improved upon several knock-in related conditions, including the transfection eciency and optimal trans-
fection dosage of CRISPR/Cas9 and a suitable homology arm length for ecient homologous recombination
(HR). Our results indicate that CRISPR/Cas9-mediated genome engineering can be successfully adopted to e-
ciently generate promoter-less and selectable marker-free transgenic PFFs. e purpose of this study was to serve
utilization of somatic cell nuclear transfer (SCNT) to generate transgenic pigs11, 13. So, the EGFP reporter PFFs
were further used to evaluate two key factors in the process of achieving transgenic pigs the limiting dilution
method and a strategy to evaluate the safety and feasibility of the knock-in locus. In summary, this study provides
Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University,
Changchun, Jilin Province, People’s Republic of China. Zicong Xie and Daxin Pang contributed equally to this work.
Correspondence and requests for materials should be addressed to X.T. (email: xiaochuntang@jlu.edu.cn)
Received: 14 October 2016
Accepted: 19 April 2017
Published: xx xx xxxx
OPEN
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Scientific RepoRts | 7: 3036 | DOI:10.1038/s41598-017-02785-y
a simple and ecient approach for a promoter-less and selectable marker-free site-specic knock-in strategy and
contributes towards the development of transgenic pigs.
Results
Establishing an ecient and versatile electroporation system. e transfection eciency of the
PFFs was a barrier against generating transgenic pigs by SCNT. Our group transiently transfected pEGFP-N1
plasmids into PFFs in a previous study, approximately 90% of the surviving cells displayed green uorescence14.
Aer further optimization, the electroporation eciency of the PFFs reached approximately 96.45% (Fig.1A).
Additionally, we conrmed that this electroporation system had high transfection eciency in various other
Figure 1. An ecient and versatile electroporation system. (A) Transient transfection of the pEGFP-N1 vectors
into the PFFs. e transfection eciency was analysed with uorescence microscopy and FACS at 24 hours
post-transfection. (B) Transient transfection of the pEGFP-N1 vector into the indicated cell lines. EGFP
uorescence was analysed via uorescence microscopy. (C) Transfection of the EGFP mRNA and CY3-labelled
siRNA into PFFs. e uorescence was analysed via uorescence microscopy.
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Scientific RepoRts | 7: 3036 | DOI:10.1038/s41598-017-02785-y
cell lines (Fig.1B and Supplementary Fig.S1), and the electroporation parameters for those cell lines are listed
in Table1. Interestingly, the EGFP mRNA (10 μg) and the Cy3-siRNA (Cy3 labelled siRNA, 100 nM) were also
successfully and eciently introduced into the PFFs by the electroporation system, and the EGFP and the Cy3-
positive cells were observed by uorescence microscopy at 24 hours post-transfection and 3 hours post-transfec-
tion, respectively (Fig.1C).
Optimization of the targeting/cutting eciency at the PFF pRosa26 locus. e endogenous
pRosa26 promoter was utilized to express the exogenous gene as shown in the Fig.2A. According to a previous
report on the pRosa26 gene5, three sgRNAs were designed to target intron 1 of the pRosa26 gene. To obtain an
ecient sgRNA, three sgRNA-recombined PX330 plasmids were introduced into PFFs by electroporation. Aer 3
days in culture, the targeting/cutting eciencies of the three sgRNAs were measured via T7 endonuclease I (T7EI)
assays and conrmed by sequencing; the results showed that sgRNA91 achieved a higher targeting/cutting e-
ciency than sgRNA86 and sgRNA89 (Supplementary Fig.S2). To our knowledge, dierent transfection systems/
methods have dierent requirements regarding the amounts and concentrations of the CRISPR/Cas9 vectors,
and suitable transfection dosages of the CRISPR/Cas9 vectors should achieve higher cutting/targeting eciencies
with less cytotoxicity. erefore, dierent doses of the sgRNA91/Cas9 vectors were introduced into the PFFs, and
the results of the sequencing and T7EI digestion assays showed that the 30 μg group achieved a higher targeting/
cutting eciency than that of the other groups (Fig.2B). Additionally, the TA cloning results conrmed that
the mutation eciency of sgRNA91/Cas9 was 34.3% (Fig.2C and Supplementary TableS1). Although the 40 μg
and 50 μg groups also achieved high targeting/cutting eciencies (Fig.2B), they produced higher cytotoxicities
(Supplementary Fig.S3). Several groups have eciently obtained genetically modied mice and pigs by injecting
the DNA or mRNA of site-specic nucleases into zygotes to achieve targeted mutations15, 16. Because of the low
cost, ease and rapidity of the parthenogenote injection, we planned to evaluate the targeting/cutting eciencies
of three sgRNAs (sgRNA91, sgRNA86 and sgRNA89) and reconrm the higher targeting/cutting eciency of
sgRNA91 for the following experiments. SgRNA-91 (20 ng/µL) and the Cas9 mRNA (40 ng/µL) were injected into
the cytoplasms of parthenogenetic embryos; the results showed that the mutation eciency of the parthenoge-
netic embryos was only 14. 8% (8/54), and the development rate of the parthenogenetic embryos was only 8.3%
(34/410) (Supplementary Fig.S4). e development rate of the parthenogenetic embryos was lower than that of
the control group, which was 11% (44/396).
Optimization of the site-specic EGFP insertion at the PFF pRosa26 locus. On the basis of our
preceding work mentioned above, to achieve a one-step transgenic strategy, a non-linearized targeting donor
vector (PUC57-pRosa26-EGFP) that did not contain a promoter or a selectable marker was used in this study
(Supplementary Fig.S5). e EGFP knock-in strategy is shown in Fig.3A. First, the homology arm length was
evaluated for ecient homologous recombination (HR) in PFFs. e donor vectors that contained dierent
homologous arm lengths (0.5 kb, 0.8 kb, 0.9 kb and 1.5 kb) were constructed and introduced into PFFs along
with the sgRNA91/Cas9 vector. On the basis of the control group (only non-linearized targeting donor vec-
tors were introduced into the PFFs and without the CRISPR/Cas9 vectors), the uorescence microscopy and
FACS results showed that all four donors produced site-specic EGFP insertion into the PFF pRosa26, and
the 1.5 kb group (10.4%) had a higher knock-in eciency than did the other groups (3.1%, 4.3% and 5.5%)
(Fig.3B, Supplementary Fig.S6). e site-specically inserted EGFP was conrmed by PCR with specic primers
(Fig.3C), and the sequencing results are shown in Supplementary Fig.S6. As indicated above, our electropo-
ration system was also suitable for mRNA transfections. erefore, the in vitro transcribed Cas9 mRNA and
sgRNA91 were introduced into the PFFs with the EGFP donor vector by electroporation. ree days later, the
EGFP-positive cells were observed by uorescence microscopy and analysed by FACS. Interestingly, the results
conrmed that the EGFP site-specic knock-in was also achieved in PFFs by electroporation of the Cas9 mRNA
and sgRNA (6.5%) (Fig.3B, Supplementary Fig.S6).
e SCR7 ligase IV inhibitor reportedly increases the eciency of HDR-mediated genome editing in several
mammalian cell lines2, 17. Given this observation, we combined multiple SCR7 doses with the electroporation
buer solution (the electroporation system) and cell culture medium (the cell culture system). First, we combined
dierent doses of SCR7 (1 μM, 2 μM, 5 μM and 10 μM) and the pEGFP-N1 plasmids (30 μg) into the electropora-
tion system, and the uorescence microscopy analysis results at 24 hours post-transfection showed that the 2 μM
Cell lines
Parameters For Electroporation(BTX)
Set Voltage(V) Pulse Length
(ms) Number of
Pulses
PFF 340 1 3
PK-15 300 1 3
Ver o 280 1 3
HUVEC 220 1 3
HepG2 240 1 3
293T 220 1 3
C2C12 250 1 3
SP20 220 1 3
Table 1. Parameters of various cell lines for electroporation (BTX-ECM2001).
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group, compared with other group, was introduced into the PFFs with a high eciency and no apparent cytotox-
icity (Supplementary Fig.S7). Our results demonstrated that no noticeable enhancement to the HDR eciency.
However, the CCK-8 analysis conrmed that SCR7 (5 μM) could signicantly promoted cell proliferation in the
cell culture system (Fig.3E and Supplementary Fig.S8). On the basis of these results, the 2 μM SCR7 parameter
from the transfection system and the 5 μM SCR7 parameter from the culture system, those were selected to
explore the SCR7 eectiveness with the EGFP site-specic knock-in events. However, there was no noticeable
enhancement of the HDR eciency in our system.
We next wished to directly compare the knock-in eciency stimulated by SCR7 to that of other drugs recently
reported, including the cyclin-dependent kinase (CDK) inhibitor indirubin-3-monoxime18, DNA-PKcs inhibi-
tors NU744119 and microtubule polymerization inhibitor Nocodazole20. Aer transfection, PFFs were incubated
for 6 hours in drug-free medium, and then, replaced the drug-free medium with dierent concentrations of drug
medium to allow the knock-in of the EGFP-KI donor. 72 hours later, the EGFP-positive cells were observed by
Figure 2. Optimization of the mutation/cutting eciency at the pROSA26 locus in PFFs. (A) Scheme for site-
specic targeting/cutting of the pROSA26 locus on chromosome 13 by CRISPR/Cas9. (B) Chromatograms of
the PCR amplicons for each CRISPR/Cas9 dose group (0 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg and 50 μg). e red
arrow indicates the cleavage site, and the PAM is underlined in red. e mutation eciency for each CRISPR/
Cas9 dose (5 µg, 10 µg, 20 μg and 30 µg) was determined by using the T7E1 cleavage assay. M: 100 bp DNA
ladder. NC: Negative control, transfection dose of CRISPR/Cas9 was 0 µg. e blue arrows indicated wild type
bands and the red arrows indicated cleaved amplicons. (C) e TA cloning and sanger sequences were analysed
for indels. e wild-type sequence is located on the rst line (WT), and the mutated sequences from the TA
cloning are arranged below (T1~T12). e target site is indicated in green; the PAM is indicated in red.
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Scientific RepoRts | 7: 3036 | DOI:10.1038/s41598-017-02785-y
Figure 3. Optimization of the knock-in eciency by EGFP site-specic integration at the pROSA26 locus. (A)
Scheme for EGFP site-specic knock-in at the pROSA26 locus. e EGFP-KI- positive PFF cell clone indicated
with a red arrow. (B) e eect of the homology arm lengths on the EGFP site-specic knock-in in PFFs. EGFP
uorescence was analysed via uorescence microscopy and FACS, at three days post-transfection. Detailed
statistical analyses were showed in Fig.S6. (Remarks: the control group that only the circular EGFP targeting
donor vector introduced into the PFFs and without the CRISPR/Cas9 vector). (C) e result of the genomic
PCR analysis conrmed the knock-in events at the pROSA26 locus. P2 primers amplied the 5- junction and
P7 primers amplied the 3- junction; the sequences of these primers are listed in TableS3. Lanes 1–6 represent
the EGFP knock-in-positive cell clones. NC: Negative control; M: D2000. (D) e eect of dierent drugs
and concentrations on EGFP site-specic knock-in in PFFs. (Column chart, n = 3 independent experiments).
Control, DMSO control; SCR7-2, SCR7(2 μM); SCR7-5, SCR7 (5 μM); SCR7-10, SCR7 (10 μM); Noco-0.5,
Nocodazole (0.5 μg/ml); Noco-0.75, Nocodazole (0.75 μg/ml); Noco-1, Nocodazole (1 μg/ml);Nu7441-1,
Nu7441 (1 μM); Nu7441-2, Nu7441 (2 μM); Nu7441-3, Nu7441(3 μM); Ind-2, indirubin-3-monoxime (2 μg/
ml); Ind-3, indirubin-3-monoxime (3 μg/ml); Ind-4, indirubin-3-monoxime (4 μg/ml); P-RAD51, pig RAD51
over expression plasmid. (E) e eect of dierent concentrations of SCR7 on PFFs proliferation. (Line chart.
n = 3. Graphs show the mean ± S.E.M.).
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uorescence microscopy and analysed by FACS. Our result showed that synchronization with Nocodazole (1 µg/
ml) resulted in a 2.8-fold increase (29.6%) (Fig.3D), indirubin-3-monoxime (4 µg/ml) treatment had a 1.9-fold
increase (19.7%) (Fig.3D). However, the SCR7 and NU7441 treatment did not show increase of knock-in e-
ciency compared to control (Fig.3D).
Additionally, previous study showed that RAD51 recombinase was a key player for HR and the repair of DNA
DSBs21 and the cDNA sequence of pig RAD51 gene was successfully isolated by PCR-based methods22. In order to
investigate RAD51 eects on the indicated pROSA26 site insertion, we constructed pig RAD51-over expression
plasmid (Supplementary Fig.S9), and then introduced the plasmid with sgRNA91/Cas9 and the EGFP donor
vector into PFFs, 72 hours later, the EGFP-positive cells were observed by uorescence microscopy and analysed
by FACS. e results showed that the RAD51-over expression plasmid could result in a 1.8-fold increase (18.4%)
in the indicated PFFs system (Fig.3D).
O-target analysis. O-target eects by CRISPR/Cas9 have been reported, owing to the system’s ability
to tolerate the sequence mismatches15, 23. O-target eects may aect the development of the blastocyst and the
generation of transgenic pigs. erefore, 10 potential o-target sites were selected according to the online CRISPR
design (Fig.4A), and 10 random EGFP-positive cell clones were evaluated (Fig.4B). e DNA sequencing and
Figure 4. O-target analysis. (A) e target sequence and ten of the predicted o-target sites for sgRNA91/
Cas9. e PAM sequence is labeled in blue. (B) A total of ten potential o target loci (Lanes 1–10) were
selected to examine o target eects in EGFP site-specic knock-in cell clone. In order to further conrm the
specicity of sgRNA91/Cas9, ten EGFP site-specic knock-in cell clones (EK1-EK10) were selected randomly
were evaluated by PCR and sequencing. WT: Wild-type. (C) T7E1 assays for the ten potential o-target sites.
OT1-OT10 represents 10 potential o-target sites, and T represents target site.
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T7E1 assay showed that no o-target mutations were detectable in any of the potential o-target sites or these
EGFP-positive cell clones (Fig.4B,C and Supplementary Fig.S10).
Application of the EGFP knock-in reporter PFFs. As described above, our primary goals involved the
selection of genome-modied PFFs to generate transgenic pigs through SCNT. During our screening of the cell
clones with the limiting dilution method, we notice that the cell inoculation number had an important eect on
the purity and activity of the nuclear donor cell clone that would directly inuence the positivity rate, the activity
of the embryos and of transgenic pig birth rates. In this study, we found that the purities of the EGFP site-specic
Figure 5. Application of the EGFP-KI reporter PFFs. (A) e purity of each EGFP gene site-specic knock-in
cell clones was analysed via uorescence microscopy and conrmed via FACS. (B) To conrm of the purity of
each cell clone by western blotting (shown on the le); Genomic PCR analysis of the blastocysts (shown on the
right); these 23 nuclear donor cells were derived from cell clone-EK4. e results show the high purity of cell
clone-EK4. e red arrow indicates the knock-in bands and the blue arrow indicates the wild-type bands. NC:
negative control. (C) e proportion of mixed nuclear donor cells was analysed via uorescence microscopy
and conrmed via FACS. (D) Genomic PCR analysis of the blastocysts (upper). Nuclear donor cells were
derived from the mixed nuclear donor cells in g. C . e red arrow indicates the knock-in bands. Aer 7 days
of culture, the mixed blastocysts were observed by uorescence microscopy (lower). M: D2000. (E) Generation
of the EGFP reporter PK-15 cell lines was conrmed via uorescence microscopy.
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knock-in cell clones were strongly aected by the degree of the cell dilution; PFF cell clones with dierent purities
displayed diering apparent EGFP uorescence intensities via uorescence microscopy and conrmed by FACS
(Fig.5A). e purities of the cell clones were also conrmed by western blotting (Fig.5B). e higher-purity
EK4 cell clone was used as the donor cell for SCNT, recombinant cells were cultured until blastocyst stage, and
23 blastocysts were harvested for PCR analysis. e results conrmed that all 23 blastocysts were positive for the
EGFP knock-in (Fig.5B).
e visible EGFP site-specic knock-in PFFs can be used to optimize the limiting dilution method. To obtain
donor cells with highest purities and activities, dierent cell numbers (1,500, 2,500, 3,500, 4,500, 5,500, 6,500 and
7,500) were inoculated into 100 mm cell culture dishes. Aer 9 days, the cell clones in the 100 mm dishes were
counted and statistically analysed, and the results showed that the inoculation of 3,500 cells in the 100 mm dish
produced the highest purity and activity of the EGFP knock-in positive cell clones (Supplementary Fig.S11).
e EGFP site-specic knock-in PFFs can also be used to accurately evaluate the safety/feasibility of the
knock-in site. e EGFP knock-in-positive and wild type (WT) cells were mixed in a specic proportion (e.g.,
56%) (Fig.5C), and the mixed cells were randomly selected for SCNT. Aer 7 days, the mixed blastocysts were
observed by uorescense microscopy and PCR (Fig.5D), and the results showed that both the overall blastocyst
development rate (12%) and the EGFP-positive blastocyst development rate (11%) were similar to those of the
WT group (10%) (Fig.5D, Table2).
Discussion
Transefection eciency has always been a key step in the generation of genetically modied large animals,
particularly those in which a precise modication is achieved by homology-directed repair. In this study, we
improved the electroporation eciency in PFFs and conrmed that this electroporation system would also obtain
high transfection eciencies in various cell lines. Additionally, we demonstrated that the electroporation system
can be used for mRNA and siRNA transfections. ese results indicate that electroporation is a versatile trans-
fection system that may be used to explore the biological functions of mRNAs, siRNAs and various cell lines. For
example, siRNAs could be designed to suppress several NHEJ key molecules, including DNA ligase IV, KU70,
KU80 and XLF24, 25, and mRNAs could be designed that increase the expression of key HDR key molecules,
such as RAD51 and RAD5221, 22, 26. en, corresponding siRNAs or mRNAs could be introduced into the PFFs
with CRISPR/Cas9 and the donor vector by using this transfection system, which may be used to enhance the
site-specic knock-in eciency. Here, we conrmed that the heights and complexities of the multi-peaks in the
chromatogram were consistent with the results of the TA cloning experiments and T7E1 assays. is method
provides a convenient means of estimating the mutation/cutting eciency. Several recent studies have shown
that highly ecient homologous recombination can occur in pig zygotes through direct zygote injections of Cas9/
sgRNA16, 27, 28. However, our results showed that the mutation eciency was only 14% when the Cas9 mRNA and
sgRNA were microinjected into parthenogenetic embryos. ese results indicated that the injection dose of the
sgRNA and Cas9 RNA needed to be optimized further and also highlighted the dierence between zygotes and
parthenogenetic embryos.
Although the indel eciency of CRISPR/Cas9 is high, low knock-in eciency has presented a major bot-
tleneck in the technique’s broad application. ere are numerous opportunities and challenges in the eld of
improving the knock-in eciencies. Although the positive and negative selection method can be used to improve
the knock-in eciency, the adverse eects of introducing drug selectable marker genes into cells and animals
have not been addressed. e possibility that drug selection may harm cells and animals in ways that are currently
not understood or that cannot be detected should not be dismissed. Moreover, expression of a drug selecta-
ble marker gene in genetically modied cells or species may also interfere with internal gene expression13, 29.
Selectable marker genes have traditionally been inserted into the genome between loxP sites and removed by
using Cre. However, deleting a selectable marker is a laborious process. In this study, we optimized the conditions
for a site-specic gene integration at the pRosa26 locus in PFFs with the CRISPR/Cas9 vector and an EGFP tar-
geting donor vector (a non-linearized vector, that does not contain a promoter or selectable marker), for a nal
eciency of approximately 29.6%. Furthermore, the promoter-less transgenic method averts several problems,
such as unstable phenotypes, unpredictable gene expression and oncogene activation. Our results demonstrate
that the CRISPR/Cas9 system can eciently generate promoter-less and selectable marker-free transgenic PFFs in
one step, and it saves time and money whilst favouring promoter-less and selectable marker-free transgenic pigs.
We have also shown that EGFP site-specic knock-in PFFs can be successfully produced by co-electroporation
of the Cas9 mRNA, sgRNA and donor vector, thus reducing the cytotoxicity and random integration risk that
oen accompanies sgRNA/Cas9 DNA-plasmids, because the Cas9-mRNA is easier to be degraded compared
with Cas9-plasmids aer the gene of interest is targeted/cut. However, the knock-in eciency was lower than
that of the sgRNA/Cas9 DNA-plasmids; this result suggested that an appropriate transfection dose, transfection
concentration, timing of transfection and sgRNA-Cas9 mRNA proportion would need to be established to further
improve the knock-in eciency in future investigations.
Total Nuclear Donor Cell Numbers Blastocyst Numbers Blastocyst Development
Rate
EGFP Positive
(219 * 56%) WT
(219 * 44%) Tot al EGFP
Positive WT Tot al EGFP
Positive WT Tot al
~120 ~99 219 14 10 24 12% 10% 11%
Table 2. e safety evaluation of the gene target site.
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Previous reports have shown that SCR7, an NHEJ inhibitor and NU7441, a DNA-PKcs inhibitor increase the
HDR frequency2, 19, 30. However, in our study, the addition of the SCR7 and NU7441 showed no signicant eects
on the HDR eciency at the indicated pROSA26 locus in PFFs. e discrepancy between our work and these
reports suggest that dierent species may respond dierently to these drugs or alternative NHEJ mechanisms
that are not SCR7 orNU7441 sensitive exist in PFFs. Additionally, the size of DNA donors, the timing of drug
treatment, the culture environmental conditions and genetic factors may appear to inuence the knock-in events
and it remains to be tested whether SCR7 has eects when double stranded oligodeoxynucleotide (dsODN) or
single stranded oligodexynucleotide (ssODN) is used as the donor. Surely, additional work is needed to conrm
the eects of SCR7 and NU7441 in PFFs system because only a single locus was targeted in our present work.
Interestingly, we found that SCR7 significantly promoted PFFs proliferation when the culture concen-
tration was 5 μΜ (Fig.3E). Additionally, we identied two drugs the cyclin-dependent kinase (CDK) inhib-
itor, indirubin-3-monoxime and microtubule polymerization inhibitor, Nocodazole, they could improve the
knock-in eciency at the indicated pROSA26 locus in PFFs. Our results showed that a 2.8-fold increase and a
1.9-fold increase in HDR with Nocodazole and indirubin-3-monoxime at the indicated pROSA26 locus. ese
results may indicate that HDR is the major DNA repair mechanism aer G2/M phase arrest in PFFs. To further
increase the knock-in eciency, we demonstrate Nocodazole, indirubin-3-monoxime and RAD51-over expres-
sion plasmid as separate tools to eciently lead to an increase in knock-in eciency, with highest rates of HDR up
to 29.6% in PFFs. Further investigations should be done to optimize the timing of drug treatment, the concentra-
tion and combination of these drugs to obtain a higher frequency of homozygous targeting in pROSA26 locus or
others, and further rening the process. However, the eects of these drugs treatment on blastocyst development
and birth rate of piglets should be performed comprehensively and systematically in subsequent research.
Additionally, aer combining dierent doses of SCR7 and the pEGFP-N1 plasmids (30 μg) into the electro-
poration system, SCR7 can be quickly introduced into PFFs along with pEGFP-N1 plasmids. We were able to
quickly and conveniently evaluate the eect of SCR7 on the electroporation system; we also conveniently assessed
SCR7’s cytotoxicity by monitoring numbers and proportions of EGFP-positive cells by uorescence microscopy.
e o-target mutation is a major concern with the Cas9-mediated gene editing system, thus indicating that
CRISPR/Cas9 can tolerate small numbers of mismatches between sgRNA and the target region, particularly
when the mismatch is 8–12 bases away from the protospacer adjacent motif (PAM)31. Our results indicated that
sgRNA91/Cas9 did not induce detectable o-target mutations in the 10 EGFP transgenic PFF cell clones, thus
possibly suggesting that the o-target eect of the CRISPR/Cas9 is site-dependent and that CRISPR/Cas9 may be
a reliable gene targeting tool for genome modication. Nevertheless, to thoroughly detect a potential o-target
eect, whole-genome sequencing analyses or other testing methods should be used for further conrmation.
Additionally, DNA nicks32, fCas933, truncated gRNA34 and RFNs35 can be used for gene editing to avoid o-target
eects in the future.
Transgenic pigs can be generated by SCNT, but the purity and activity of the nuclear donor cell is key to this
process. When the limiting dilution method is used to select the transgenic PFF cell clones, too many inoculated
cells lead to a coalescence or cross-linking of the cell clones and reduce the purity of the cell clones. However, too
few inoculated cells will weaken the interactions among the cells and decrease their proliferation rate, thereby
aecting the quality or activity of the donor cell. e EGFP reporter cell lines enabled us to improve the limit-
ing dilution method and conveniently evaluate the activity and purity of each cell clone through uorescence
microscopy. In this study, we successfully selected the optimal cell inoculation density in 100 mm culture dishes
(3,500 cells/dish on average) by using the EGFP reporter PFFs. ese cells may provide a reference for other
genetically modied animals that are produced by SCNT.
Using the EGFP reporter PFFs, we developed a strategy to evaluate the feasibility and safety of the “safe har-
bour” locus. We believed that the hybrid transplantation strategy might further exclude human inuence; it was
more accurate than the separate and individual transplantation method.
Additionally, we introduced sgRNA91/Cas9 into the PK-15 cell line with the EGFP-KI donor and obtained
the PK-15-EGFP-reporter cell lines, which stably expressed the EGFP protein (Fig.5E). It is well known that, a
limitation of transgenic PFFs is their lower viability and proliferation rate through serial cell passages. However,
cell passaging has a weak inuence on the viability and proliferation ability of the PK-15-EGFP-reporter cell line.
Compared with cells with transiently co-transfected EGFP plasmids or EGFP random integration cell lines, the
PK-15-EGFP-reporter cell line has the follow advantageous features: a precise integration site, a specic copy
number, and a higher controllability. With these advantages, the PK-15-EGFP-reporter cell line can be readily
used to optimize gene editing tools or to screen recombinants, such as CRISPR/Cpf136, C2c29 and others. e
gene editing tools described above can be designed to target EGFP or its expression products, and the factors and
conditions that inuence these tools can be conveniently analysed via FACS and uorescence microscopy.
In summary, we conrmed the eciency and versatility of the transfection system. We also optimized the
transfection dosage of the CRISPR/Cas9 and a suitable homology arm length for ecient homologous recom-
bination (HR). With this strategy, we demonstrated that the CRISPR/Cas9 system can efficiently generate
promoter-less and selectable marker-free transgenic PFFs in one step, and it saves time and money whilst gen-
erating promoter-less and selectable marker-free transgenic pigs. Using the EGFP reporter PFFs, we further
optimized the limiting dilution method and established strategies to evaluate the safety and feasibility of the
integration site; these analyses established an evaluation system and contributed to the process of generating
transgenic pigs. ese methods and their parameters can be directly adapted to other mammalian species, and
they provide a reference for more sophisticated genome modications.
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Scientific RepoRts | 7: 3036 | DOI:10.1038/s41598-017-02785-y
Materials and Methods
Ethics statement. All animal studies were approved by the Animal Welfare and Research Ethics Committee
at Jilin University (Approval ID: 20150315), and all procedures were conducted strictly in accordance with the
Guide for the Care and Use of Laboratory Animals. All surgeries were performed under anesthesia, and every
eort was made to minimize animal suering.
Plasmid construction. e donor vector contained a 0.5 Kb le homologous arm (HA) and a 1.0 kb right HA
(Fig.S5). e HAs were amplied by genomic PCR and cloned into the PUC57 vector. e SA sequence and the
EGFP gene were subsequently inserted between the right and le arms.
sgRNAs that targeted the pROSA26 locus were designed using online soware, and sgRNA oligonucleotides
were annealed and cloned into the PX330 vector (Addgene) using the method described by Zhang at the Broad
Institute of MIT.
In vitro transcription. e T7 promoter was added to sgRNA and Cas9 by PCR amplication. PCR products
were used as templates for in vitro transcription (IVT) with a MEGAshortscript T7 kit (Life Technologies) and
the mMESSAGEmMACHINE T7 ULTRA kit (Life Technologies) according to the manufacturer’s instructions.
e sgRNA and Cas9 mRNA were puried using a MEGAclear kit (Life Technologies) according to the manu-
facturer’s instructions and were eluted in RNase-free water. e concentration and quality of the mRNA were
determined by using a NanoDrop 2000 spectrophotometer and by agarose gel electrophoresis.
Isolation and culture of PFFs. Twelve 33-day-old fetuses were separated from Large White sows in gestation
period, and primary PFFs were isolated from these 33-day-old foetuses of Large White pigs. Aer removal of the
head, tail, limb bones and viscera from the foetal body, the fetuses were cut into little small pieces, digested with a
sterile collagenase solution and cultured in Dulbeccos modied Eagle’s medium (DMEM, GIBCO) supplemented
with 10% foetal bovine serum (FBS) at 39 °C and 5% CO2 in a humidied incubator.
SCNT. e EGFP-KI-positive PFF cells line that were selected by the limiting dilution method were injected
into the perivitelline cytoplasms of enucleated oocytes. e reconstructed embryos were activated and cultured
to develop into blastocysts. e blastocysts were analysed by uorescence microscopy (Nikon ts100). High quality
blastocysts were collected into a cell lysis solution that contained 0.45% NP40 and 0.6% proteinase K, and their
genomic DNA was extracted at 56 °C for 1 h and 95 °C for ten minutes in BIO-RAD PCR machine. e lysate was
used as the PCR template. To conrm the cell clones and blastocysts for the site-specic knock-in, we assessed the
5-junction and the 3-junction by PCR, the primers and sequences are shown in TableS3.
T7EI assay. e T7E1 assay was performed as previously described. Briey, PCR products were puried with
a TIANgelMidi purication Kit (TIANGEN, Beijing, China) and were denatured and annealed in NEBuer 2
(NEB) in a thermo cycler. Hybridized PCR products were digested with T7E1 (NEB, M0302L) for 30 minutes at
37 °Cand subjected to 2% agarose gel electrophoresis.
Western blotting. e Cell clones were solubilized in lysis buer. e protein concentrations were measured
with a BCA protein assay kit (Beyotime, Haimen, China); the proteins were separated on 10% SDS-PAGE gels
and transferred to nitrocellulose membranes. e membranes were subsequently blocked for 2 h in 5% low-fat
milk in PBST. e membranes were incubated with anti-GFP antibody (1:200, Beyotime) for 1 h, washed 3 times
with a TBST buer, and incubated for 1 h with a horseradish peroxidase-conjugated anti-goat secondary antibody
(1:1000, Beyotime). Aer three washes in PBST, the signals on the membranes were acquired with ECL-Plus
western blotting reagent.
O-target analysis. Potential o-target sites of the pRosa26 site were detected by PCR and DNA sequencing. e
PCR programme was as follows: 94 °C for 5 min; 94 °C for 30 sec; 32cycles of 58 °C for 30 sec and 72 °C for 40 sec;
72 °C for 5 min; storage at 4 °C. e primer sequences are listed in supplementary TableS4. e PCR products
that surrounded the o-target sites were puried and digested with T7E1 for 30 min at 37 °C and subjected to 2%
agarose gel electrophoresis.
Electroporation of PFFs. Approximately 3 × 106 PFFs and 30–60 µg of the corresponding plasmids (25 µg of
EGFP-N1, 30 µg of PX330 and 30 µg of PX330 plus 30 µg of donor vector) were suspended in 300 µL of Opti-MEM
(Gibco, Grand Island, New York, USA) in 2 mm gap cuvettes, and electroporated by using specied parameters
with a BTX-ECM 2001.
Selection of PFF cell clones. e cells were inoculated into ten 100 mm dishes at 48 h post-transfection, and the
cell inoculation density per 100 mm dishes was 3,500 cells/dish on average. e cell clones were picked and cul-
tured into 24-well plates. Aer a conuence of 80% or more was reached, 15% of each cell clone was digested and
lysed with 10 μl NP40 lysis buer (0.45% NP40 plus 0.6% proteinase K) for 1 h at 56 °C and 10 min at 95 °C. e the
lysate was used as the PCR template and was subjected to 1% agarose gel electrophoresis. Additionally, the PCR
products were sequenced to conrm the knock-in events. e positive cell clones were thawed and cultured in
12-well plates before SCNT.
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PCR detection. To test cell clones for site-specic insertion of the EGFP gene, we performed a 5-junction and
3-junction PCR reaction. ese primers are listed in TableS3.
Fluorescence detection and flow cytometric analysis. PFFs were electroporated with pEGFP-N1;
EGFP-KI-positive cell clones and EGFP-KI-positive embryos were assessed by fluorescence microscopy
(Olympus BX51). e harvested cells were washed twice, resuspended in 300 µL of DPBS and analysed using a
BD Accuri C6 ow cytometer.
Statistical analysis. The data were statistically analysed with GraphPad Prism software (t-test), and a p
value < 0.05 was considered statistically signicant.
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Acknowledgements
We thank the members of the Animal Biotechnology laboratory, College of Animal Science, Jilin University.
is work was supported by Special Funds for Cultivation and Breeding of New Transgenic Organisms (Nos
2016ZX08006003 & 2014ZX0800604B) and the China National Natural Science Foundation (31472053 and
31572345).
www.nature.com/scientificreports/
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Scientific RepoRts | 7: 3036 | DOI:10.1038/s41598-017-02785-y
Author Contributions
Conceived and designed the experiments: Z.X., D.P., H.J., H.O. and X.T. performed the experiments: Z.X., K.W.,
M.L., H.Y., J.L., X.Z. and N.G. Wrote the paper: Z.X., Z.L., H.O., H.J. and X.T.
Additional Information
Supplementary information accompanies this paper at doi:10.1038/s41598-017-02785-y
Competing Interests: e authors declare that they have no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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© e Author(s) 2017

Supplementary resource (1)

... Treatment with nocodazole leads to a reversible cell cycle synchronization in various cell cultures [19,22] but not in primary neonatal fibroblasts or embryonic stem cells [19]. In porcine fetal fibroblasts, nocodazole (1 µg/mL) resulted in a 2.8-fold increase in HDR (up to 29.6%) in GFP knock-in experiments using CRISPR-Cas9 [24]. In HEK293T, nocodazole increased the HDR rate up to 6-fold with CRISPR-Cas9 and ssODN as a template, depending on the target locus. ...
... Several cyclin-dependent kinases can be inhibited by various molecules to achieve cell cycle synchronization in the G1/S or G2/M phases, for example, by indirubins [41]. In GFP knock-in experiments using CRISPR-Cas9, treatment of porcine fetal fibroblasts with indirubin-3′-monoxime (4 µg/mL), an inhibitor of cyclin-dependent kinase 1 (CDK1), increased HDR by 1.9 times (up to 19.7%) [24]. Similar results were obtained for HeLa, HT-1080, and U-2 OS cells: an increase in the HDR rate by 2-5 times using transfection with Several cyclin-dependent kinases can be inhibited by various molecules to achieve cell cycle synchronization in the G1/S or G2/M phases, for example, by indirubins [41]. ...
... Similar results were obtained for HeLa, HT-1080, and U-2 OS cells: an increase in the HDR rate by 2-5 times using transfection with Several cyclin-dependent kinases can be inhibited by various molecules to achieve cell cycle synchronization in the G1/S or G2/M phases, for example, by indirubins [41]. In GFP knock-in experiments using CRISPR-Cas9, treatment of porcine fetal fibroblasts with indirubin-3 -monoxime (4 µg/mL), an inhibitor of cyclin-dependent kinase 1 (CDK1), increased HDR by 1.9 times (up to 19.7%) [24]. Similar results were obtained for HeLa, HT-1080, and U-2 OS cells: an increase in the HDR rate by 2-5 times using transfection with expression vectors, coding meganuclease I-SceI, or ZFNs. ...
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Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation.
... Up to a 6-fold increase in the KI rate was demonstrated by combining the modified Cas9/donor with compounds that promote HDR. Additionally, promoterless EGFP reporters targeting other endogenous loci have been used to evaluate the HDR efficiency of different strategies in different cells (Table S1) [18][19][20][21][22][23][24]. ...
... These data suggested EGFP in different forms of the promoterless EGFP reporter could be expressed in the absence of the Cas9/sgRNA. Besides, a similar phenomenon was also found when the promoterless EGFP reporter targeting endogenous ROSA26 locus [20] was transfected into PFF cells alone ( Figure S3). ...
... Intriguingly, Mohammadi et al. also recently reported that an EGFP coding sequence could be expressed at up to 50% of the level as CMV-driven cassettes in 293T cells [38]. Furthermore, the established promoterless reporter contains an ATG initiation codon just like those in many published papers [7,12,[19][20][21]. After the unexpected EGFP expression was detected, ATG initiation codon is suspect to help the expression. ...
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An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.
... This method is used to evaluate large insertions or deletions for example when the strategy is to generate large deletions and two sgRNAs are designed to remove part of an exon or even complete exons Wu et al. 2017;Hirata et al. 2020;Koppes et al. 2020). PCR product digestion with mismatch-sense S. Navarro-Serna et al. endonucleases, such as T7 endonuclease I are also used to detect mutations produced by CRISPR/Cas9 and other programmable endonucleases (Wang et al. 2015a;Kang et al. 2016;Bloom et al. 2017;Li et al. 2017a;Xie et al. 2017). This method involves performing a PCR of the target region of the embryo/animal sample. ...
... 24,50-56 other studies reported poor or lack of activity. [57][58][59][60][61][62][63][64][65] It might be the case that SCR7 is functional only in specific cell lines or specific experimental conditions and thus might not be sufficiently robust to be widely applicable. Confusion also arose regarding the actual chemical structure of the active compound form. ...
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CRISPR technologies are increasingly being investigated and utilized for the treatment of human genetic diseases via genome editing. CRISPR-Cas9 first generates a targeted DNA double-stranded break, and a functional gene can then be introduced to replace the defective copy in a precise manner by templated repair via the homology-directed repair (HDR) pathway. However, this is challenging owing to the relatively low efficiency of the HDR pathway compared with a rival random repair pathway known as non-homologous end joining (NHEJ). Small molecules can be employed to increase the efficiency of HDR and decrease that of NHEJ to improve the efficiency of precise knock-in genome editing. This review discusses the potential usage of such small molecules in the context of gene therapy and their drug-likeness, from a medicinal chemist’s perspective.
... In 2018, Ma et al. successfully identified a Pifs501 locus in porcine genome and confirmed its reliability as a novel SH locus, although the expression of the integrated EGFP at this locus was lower than that at pROSA26 (Ma et al. 2018). To date, only a few SH loci of other species origin have been applied to genomic modifications in pigs, such as pROSA26 (Xie et al. 2017) and pH11 (Ruan et al. 2015). However, there is a lack of systematic comparison of different SH loci in the porcine genome to examine the safety and expression efficiency of these loci for transgenesis. ...
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Transgenic technology is now widely used in biomedical and agricultural fields. Transgenesis is commonly achieved through random integration which might cause some uncertain consequences. The site-specific integration could avoid this disadvantage. This study aimed to screen and validate the best safe harbor (SH) locus for efficient porcine transgenesis. First, the cells carrying the EGFP reporter construct at four different SH loci (ROSA26, AAVS1, H11 and COL1A1) were achieved through CRSIPR/Cas9-mediated HDR. At the COL1A1 and ROSA26 loci, a higher mRNA and protein expression of EGFP was detected, and it was correlated with a lower level of DNA methylation of the EGFP promoter, hEF1α. A decreased H3K27me3 modification of the hEF1α promoter at the COL1A1 locus was also detected. For the safety of transgenesis at different SH locus, we found that transgenesis could relatively alter the expression of the adjacent endogenous genes, but the influence was limited. We also did not observe any off-target cleavage for the selected sgRNAs of the COL1A1 and ROSA26 loci. In conclusion, the COL1A1 and ROSA26 were confirmed to be the best two SH loci with the COL1A1 being more competitive for porcine transgenesis. This work would greatly facilitate porcine genome engineering and transgenic pig production.
... The above findings suggested that generation of a transgenic pigs expressing the HLA-G1 gene from the porcine ROSA-26 locus using HDR-mediated KI by CRISPR/Cas9 gene editing would improve xenograft survival. We optimized the length of left and right homologous arms to improve KI efficiency at the porcine ROSA26 locus (52,53). The sequence for synthesis of HDR and ROSA26 was first confirmed by Sanger sequencing. ...
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The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1 ⁻ /HLAG1 ⁺ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1 ⁺ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger’s sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous β2-microglobulin (β2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1 ⁺ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4 ⁺ and CD8 ⁺ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1 ⁺ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1 ⁺ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1 ⁺ may extend survival of porcine pancreatic islet and organ xenografts.
... The insertion of the transgene into Rosa 26 locus allows site-specific integration of a single copy of transgene resulting in its stable and optimized expression without causing any aberrant physiological consequences (Dulauroy et al., 2012;Sadelain et al., 2012). Rosa 26 locus has also been identified and successfully targeted in rat (Kobayashi et al., 2012), rabbit (Yang et al., 2016), pig (Li et al., 2014;Xie et al., 2017), sheep (Wu et al., 2016), cattle (Wang et al., 2018), and human (Irion et al., 2007). ...
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Transgenic goats are ideal bioreactors for the production of therapeutic proteins in their mammary glands. However, random integration of the transgene within-host genome often culminates in unstable expression and unpredictable phenotypes. Targeting desired genes to a safe locus in the goat genome using advanced targeted genome-editing tools, such as transcription activator-like effector nucleases (TALENs) might assist in overcoming these hurdles. We identified Rosa 26 locus, a safe harbor for transgene integration, on chromosome 22 in the goat genome for the first time. We further demonstrate that TALEN-mediated targeting of GFP gene cassette at Rosa 26 locus exhibited stable and ubiquitous expression of GFP gene in goat fetal fibroblasts (GFFs) and after that, transgenic cloned embryos generated by handmade cloning (HMC). The transfection of GFFs by the TALEN pair resulted in 13.30% indel frequency at the target site. Upon cotransfection with TALEN and donor vectors, four correctly targeted cell colonies were obtained and all of them showed monoallelic gene insertions. The blastocyst rate for transgenic cloned embryos (3.92% ± 1.12%) was significantly (p < 0.05) lower than cloned embryos (7.84% ± 0.68%) used as control. Concomitantly, 2 out of 15 embryos of morulae and blastocyst stage (13.30%) exhibited site-specific integration. In conclusion, the present study demonstrates TALEN-mediated transgene integration at Rosa 26 locus in caprine fetal fibroblasts and the generation of transgenic cloned embryos using HMC.
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Efficient, stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR associated protein9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative polymerase chain reaction (qPCR), western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e-16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P24h=0.31, P48h=0.96, P72h=0.24, P96h=0.17, P120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.
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CRISPR/Cas9 is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert anti‐tumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding of protective immune response in vivo. In this study, we used CRISPR/Cas9 mediated DNA cleavage and homologous recombination to develop a novel knock‐in tool which expresses the ultra‐bright fluorescent probe ZsGreen in YUMM 1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and were preferentially trafficking tumor antigens to draining lymph nodes. Additionally, we also used this knock‐in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment. Our results demonstrate that the ZsGreen expressing YUMM1.7 melanoma model provides a valuable tool for study of phagocytosis in vivo.
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The CRISPR-Cas nuclease has emerged as a powerful genome-editing tool in recent years. The CRISPR-Cas system induces double-strand breaks that can be repaired via the non-homologous end joining or homology-directed repair (HDR) pathway. Compared to non-homologous end joining, HDR can be used for the treatment of incurable monogenetic diseases. Therefore, remarkable efforts have been dedicated to enhancing the efficacy of HDR. In this review, we summarize the currently used strategies for enhancing the HDR efficiency of CRISPR-Cas systems based on three factors: (1) regulation of the key factors in the DNA repair pathways, (2) modulation of the components in the CRISPR machinery, and (3) alteration of the intracellular environment around double-strand breaks. Representative cases and potential solutions for further improving HDR efficiency are also discussed, facilitating the development of new CRISPR technologies to achieve highly precise genetic manipulation in the future.
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INTRODUCTION Almost all archaea and about half of bacteria possess clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune systems, which protect microbes against viruses and other foreign DNA. All functionally characterized CRISPR systems have been reported to target DNA, with some multicomponent type III systems also targeting RNA. The putative class 2 type VI system, which has not been functionally characterized, encompasses the single-effector protein C2c2, which contains two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains commonly associated with ribonucleases (RNases), suggesting RNA-guided RNA-targeting function. RATIONALE Existing studies have only established a role for RNA interference, in addition to DNA interference, in the multicomponent type III-A and III-B systems. We investigated the possibility of C2c2-mediated RNA inference by heterologously expressing C2c2 locus from Leptotrichia shahii (LshC2c2) in the model system Escherichia coli. The ability of LshC2c2 to protect against MS2 single-stranded RNA (ssRNA) phage infection was assessed by using every possible spacer sequence against the phage genome. We next developed protocols to reconstitute purified recombinant LshC2c2 protein and test its biochemical activity when incubated with its mature CRISPR RNA (crRNA) and target ssRNA. We systematically evaluated the parameters necessary for cleavage. Last, to demonstrate the potential utility of the LshC2c2 complex for RNA targeting in living bacterial cells, we guided it to knockdown red fluorescent protein (RFP) mRNA in vivo. RESULTS This work demonstrates the RNA-guided RNase activity of the putative type VI CRISPR-effector LshC2c2. Heterologously expressed C2c2 can protect E. coli from MS2 phage, and by screening against the MS2 genome, we identified a H (non-G) protospacer flanking site (PFS) following the RNA target site, which was confirmed by targeting a complementary sequence in the β-lactamase transcript followed by a degenerate nucleotide sequence. Using purified LshC2c2 protein, we demonstrate that C2c2 and crRNA are sufficient in vitro to achieve RNA-guided, PFS-dependent RNA cleavage. This cleavage preferentially occurs at uracil residues in ssRNA regions and depends on conserved catalytic residues in the two HEPN domains. Mutation of these residues yields a catalytically inactive RNA-binding protein. The secondary structure of the crRNA direct repeat (DR) stem is required for LshC2c2 activity, and mutations in the 3′ region of the DR eliminate cleavage activity. Targeting is also sensitive to multiple or consecutive mismatches in the spacer:protospacer duplex. C2c2 targeting of RFP mRNA in vivo results in reduced fluorescence. The knockdown of the RFP mRNA by C2c2 slowed E. coli growth, and in agreement with this finding, in vitro cleavage of the target RNA results in “collateral,” nonspecific cleavage of other RNAs present in the reaction mix. CONCLUSION LshC2c2 is a RNA-guided RNase which requires the activity of its two HEPN domains, suggesting previously unidentified mechanisms of RNA targeting and degradation by CRISPR systems. Promiscuous RNase activity of C2c2 after activation by the target slows bacterial growth and suggests that C2c2 could protect bacteria from virus spread via programmed cell death and dormancy induction. A single-effector RNA targeting system has the potential to serve as a general chassis for molecular tools for visualizing, degrading, or binding RNA in a programmable, multiplexed fashion. C2c2 is an RNA-guided RNase that provides protection against RNA phage CRISPR-C2c2 from L. shahii can be reconstituted in E. coli to mediate RNA-guided interference of the RNA phage MS2. Biochemical characterization of C2c2 reveals crRNA-guided RNA cleavage facilitated by the two HEPN nuclease domains. Binding of the target RNA by C2c2-crRNA also activates a nonspecific RNase activity, which may lead to promiscuous cleavage of RNAs without complementarity to the crRNA guide sequence.
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The UAF1-USP1 complex deubiquitinates FANCD2 during execution of the Fanconi anemia DNA damage response pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1-deficient cells are also impaired for DNA repair by homologous recombination. Herein, we show that UAF1 binds DNA and forms a dimeric complex with RAD51AP1, an accessory factor of the RAD51 recombinase, and a trimeric complex with RAD51 through RAD51AP1. Two small ubiquitin-like modifier (SUMO)-like domains in UAF1 and a SUMO-interacting motif in RAD51AP1 mediate complex formation. Importantly, UAF1 enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1 but independent of USP1. Mechanistically, RAD51AP1-UAF1 co-operates with RAD51 to assemble the synaptic complex, a critical nucleoprotein intermediate in homologous recombination, and cellular studies reveal the biological significance of the RAD51AP1-UAF1 protein complex. Our findings provide insights into an apparently USP1-independent role of UAF1 in genome maintenance.
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The laboratory rabbit has been a valuable model system for human disease studies. To make the rabbit model more amendable to targeted gene knockin and stable gene over-expression, we identified a rabbit orthologue of the mouse Rosa26 locus through genomic sequence homology analysis. Real-time PCR and 5′ RACE and 3′ RACE experiments revealed that this locus encodes two transcript variants of a long noncoding RNA (lncRNA) (rbRosaV1 and rbRosaV2). Both variants are expressed ubiquitously and stably in different tissues. We next targeted the rabbit Rosa26 (rbRosa26) locus using CRISPR/Cas9 and produced two lines of knock-in rabbits (rbRosa26-EGFP, and rbRosa26-Cre-reporter). In both lines, all the founders and their offspring appear healthy and reproduce normally. In F1 generation animals, the rbRosa26-EGFP rabbits express EGFP, and the rbRosa26-Cre-reporter rabbits express tdTomato ubiquitously in all the tissues examined. Furthermore, disruption of rbRosa26 locus does not adversely impact the animal health and reproduction. Therefore, our work establishes rbRosa26 as a safe harbor suitable for nuclease mediated gene targeting. The addition of rbRosa26 to the tool box of transgenic research is expected to allow diverse genetic manipulations, including gain-of function, conditional knock out and lineage-tracing studies in rabbits.
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Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.
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Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies.
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The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.
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Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety.
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Precise genome modification in large domesticated animals is desirable under many circumstances. In the past it is only possible through lengthy and burdensome cloning procedures. Here we attempted to achieve that goal through the use of the newest genome-modifying tool CRISPR/Cas9. We set out to knockin human albumin cDNA into pig Alb locus for the production of recombinant human serum albumin (rHSA). HSA is a widely used human blood product and is in high demand. We show that homologous recombination can occur highly efficiently in swine zygotes. All 16 piglets born from the manipulated zygotes carry the expected knockin allele and we demonstrated the presence of human albumin in the blood of these piglets. Furthermore, the knockin allele was successfully transmitted through germline. This success in precision genomic engineering is expected to spur exploration of pigs and other large domesticated animals to be used as bioreactors for the production of biomedical products or creation of livestock strains with more desirable traits.
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Transgenic pigs play an important role in producing higher quality food in agriculture and improving human health when used as animal models for various human diseases in biomedicine. Production of transgenic pigs, however, is a lengthy and inefficient process that hinders research using pig models. Recent applications of the CRISPR/Cas9 system for generating site-specific gene knockout/knockin models, including a knockout pig model, have significantly accelerated the animal model field. However, a knockin pig model containing a site-specific transgene insertion that can be passed on to its offspring remains lacking. Here, we describe for the first time the generation of a site-specific knockin pig model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. We also report a new genomic "safe harbor" locus, named pH11, which enables stable and robust transgene expression. Our results indicate that our CRISPR/Cas9 knockin system allows highly efficient gene insertion at the pH11 locus of up to 54% using drug selection and 6% without drug selection. We successfully inserted a gene fragment larger than 9 kb at the pH11 locus using the CRISPR/Cas9 system. Our data also confirm that the gene inserted into the pH11 locus is highly expressed in cells, embryos and animals.
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CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN-3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR-Cas systems so far described.