nocodazole-treated mice, the extent of rRNA polarization was smaller compared to that in vehicle-treated controls (fig. S10). To assess whether the
difference in polarization of apical mRNA upon
nocodazole treatment could stem from coordinated
movement of ribosomes and their retained mRNA
to the basal side, we measured the combined polarization of both rRNA and of four apical genes
with high TE in nocodazole-treated mice and in
controls, and stratified the results according to
the single-cell rRNA polarization. Microtubule
disruption decreased the apical polarization of
these genes even when controlling for rRNA
polarization changes (figs. S10 and S11A). Thus,
the microtubule network mediates the asymmetric
localization of these and potentially other transcripts in the intestinal epithelium, whereas additional anchoring mechanisms seem to render
the apical localization of ribosomes less sensitive to microtubule disruption. Selective inhibition of kinesin 5 in intestinal organoids (19, 20)
using ispinesib (21) decreased the polarization
of Apob and Net1 (Fig. 4, C to E) but not of
Cdh17 and Pigr (fig. S9, E and F).
Given the transient nature of inputs in the gut,
consistently high translation rates could be energetically inefficient (22). When nutrients arrive,
however, there is a short temporal window in
which they must be efficiently absorbed (9).
Nutrient availability was shown to stimulate
rapid ribosome biogenesis in other contexts
(23, 24) via activation of translation initiation
factors, rRNA transcription, and translation of
RNAs that encode the translational machinery
(24). Here, we found a similar burst of protein
translation upon refeeding, associated with a
rapid translocation of ribosomal-protein encoding transcripts, the nature of which is yet to be
determined, into the translationally active apical
side (Fig. 4F). The burst of ribosomal protein
translation upon refeeding resembles a “
bang-bang” control strategy, in which resources are
first invested in making the ribosomal “machines,”
to facilitate a rapid increase in total protein output
(25, 26). Our approach, combining laser-capture
microdissection and whole-transcriptome sequencing with smFISH, can be readily applied to characterize mRNA polarization in other tissues
and organisms. Future studies will determine if
mRNA mislocalizations are causatively involved
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We thank K. Bahar Halpern, B. Toth, and S. Ben-Moshe for
valuable comments on the manuscript. We thank the L Lokey
animal facility (Weizmann Institute of Science, Rehovot, Israel), the
Crown Institute for Genomics (Weizmann Institute of Science,
Rehovot, Israel), and the Smoler Protein Research Center
(Technion, Haifa, Israel) for help with experimental procedures.
A.E.M. is supported by the Swiss National Science Foundation
(grant 158999) and the European Molecular Biology Organization
(EMBO) Long-Term Fellowship program (ALTF 306-2016).
N.S.-G. research is funded by the European Research Council
starting grant (StG-2014-638142). N.S.-G. is incumbent of the
Skirball Career Development Chair in New Scientists. S.I. is
supported by the Henry Chanoch Krenter Institute for Biomedical
Imaging and Genomics, The Leir Charitable Foundations,
Richard Jakubskind Laboratory of Systems Biology,
Cymerman-Jakubskind Prize, The Lord Sieff of Brimpton Memorial
Fund, the I-CORE program of the Planning and Budgeting
Committee and the Israel Science Foundation (grants 1902/12
and 1796/12), the Israel Science Foundation grant no. 1486/16,
the EMBO Young Investigator Program, and the European
Research Council under the European Union’s Seventh Framework
Programme (FP7/2007-2013)–ERC grant agreement no. 335122.
S.I. is the incumbent of the Philip Harris and Gerald Ronson
Career Development Chair. All data supporting the findings
of this study and all analysis codes are available within the
article and its supplementary materials or from the corresponding
author upon request. The generated sequencing data have
been deposited in the GenBank Gene Expression Omnibus database
( www.ncbi.nlm.nih.gov/geo/) under accession code GSE95416.
Materials and Methods
Figs. S1 to S11
Tables S1 to S7
16 March 2017; resubmitted 3 July 2017
Accepted 1 August 2017
Published online 10 August 2017
Inactivation of porcine endogenous
retrovirus in pigs using CRISPR-Cas9
Dong Niu,1,2 Hong-Jiang Wei,3,4 Lin Lin,5 Haydy George,1 Tao Wang,1*
I-Hsiu Lee,1 Hong-Ye Zhao,3 Yong Wang,6 Yinan Kan,1 Ellen Shrock,7 Emal Lesha,1
Gang Wang,1 Yonglun Luo,5 Yubo Qing,3,4 Deling Jiao,3,4 Heng Zhao,3,4
Xiaoyang Zhou,6 Shouqi Wang,8 Hong Wei,6 Marc Güell,1†
George M. Church,1,7,9† Luhan Yang1†‡
Xenotransplantation is a promising strategy to alleviate the shortage of organs for human
transplantation. In addition to the concerns about pig-to-human immunological compatibility,
the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has
impeded the clinical application of this approach. We previously demonstrated the feasibility
of inactivating PERV activity in an immortalized pig cell line. We now confirm that PERVs
infect human cells, and we observe the horizontal transfer of PERVs among human cells.
Using CRISPR-Cas9, we inactivated all of the PERVs in a porcine primary cell line and
generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlights the
value of PERV inactivation to prevent cross-species viral transmission and demonstrates
the successful production of PERV-inactivated animals to address the safety concern in
The shortage of human organs and tissues for transplantation represents one of the most substantial unmet medical needs (1). Xenotransplantation holds great promise. Porcine organs are considered to be favorable resources for xenotransplantation because they are similar to human organs in size
and function, and pigs can be bred in large
However, the clinical use of porcine organs
has been hindered by immunological incom-patibilities (2) and by the potential risk of porcine
endogenous retrovirus (PERV) transmission (3).
PERVs are gamma retroviruses found in the genome of all pig strains and can be vertically transferred through inheritance (4). Although, to date,
no study has shown PERV transmission to humans
in a clinical setting, it has been demonstrated that
PERVs can infect human cells (3, 5) and integrate
into the human genome in cell culture (6). PERV
integration could potentially lead to immunodeficiency and tumorigenesis, as reported with
other retroviruses (7, 8).
We recently demonstrated a method to inactivate all 62 copies of PERVs in an immortalized porcine cell line (PK15) and thus eliminate