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A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

12 Aug 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 732354

TL;DR: It is demonstrated that the multivalent display of ss ODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs.

AbstractGenetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic β cells to secrete protective immunomodulatory factor interleukin-10.

Summary (2 min read)

Results

  • Cas9 domains tolerate the attachment of diverse molecules.
  • The Cas9-adaptor conjugate retained the target specificity while also maintaining the on-target activity (Supplementary Fig. 4a−c).
  • To demonstrate the modular nature of their conjugation platform that should allow the rapid testing of multiple conditions and to confirm the generality of HDR enhancement by ssODN display, the authors tested the ability of the conjugates to enhance HDR under several scenarios (e.g. different genomic sites, ssODN sequences, or readouts).
  • In addition, due to the inherent high expression and secretion level of insulin in β cells, it would be easy to modulate the secretion level of exogenous gene products (Fig. 5a, b).
  • Genotyping and a glucose-stimulated IL-10 secretion test indicated that only 2% of the Ins1 gene contained Il10 knockin, though these cells still secreted on average 5.4 ng mL−1 of IL-10 per hour under the glucose-stimulated condition (Supplementary Fig. 14b and 15d), which is substantially higher than median effective doses of IL-10 in many in vitro assays35.

Discussion

  • The authors describe a simple, scalable, and modular chemical platform for site-specific Cas9 labeling with a wide range of functional molecules to expand Cas9 functionality.
  • Source data are provided as a Source Data file.
  • The use of the optimal ssODN sequence is crucial for successful HDR-based genome editing6, and the modular nature of their conjugation platform would allow rapid screening of diverse ssODNs to identify the best one.
  • Glucose responsiveness would be advantageous for cells engineered to secrete glucagon-like peptide 1 receptor agonists, which when cosecreted with insulin, would promote insulin secretion and enhance the viability of β cells49.
  • Overall, this study provides a simple and effective method for forming chemical conjugates of Cas9 to enhance its functionality, and its ease-of-use will make it convenient for scientists of all backgrounds to modify existing Cas9 tools to suit their desired applications.

Methods

  • Finally, reverse transcription was performed to obtain single-stranded donor DNAs.
  • Data acquisition and analysis was performed using MetaXpress (Molecular Devices) or Operetta Harmony 4.8 .
  • SsODN conjugates, the Cas9-adaptor was premixed with ssODN in OptiMEM and incubated at RT for 15–30 min prior to RNP formation, also known as For Cas9.
  • The next day, the cells were washed with PBS and were incubated in the KRB buffer containing 2.8 mM of glucose for 1 h.

Data availability

  • The data generated in this study are provided in the manuscript and the supplementary information, and are available from the corresponding author upon reasonable request.
  • Source data are provided with this paper.

Author contributions

  • D.L., K.J.C., and A.C. designed the conjugation platform.
  • D.L. (with assistance from B.K.L.) developed the conjugation platform.
  • D.L. and A.C. wrote the manuscript, which was edited by all the authors.

Competing interests

  • D.L. and A.C. are named inventors on Patent application no.
  • PCT/US2020/026264 filed by The Broad Institute to compositions and methods as described in this manuscript.
  • J.M.K. has been a paid consultant for companies including Stempeutics, Sanofi, Celltex, LifeVaultBio, Gecko Biomedical, Alivio Therapeutics, Skintifique, Molecular Infusions, Landsdowne Labs, Takeda, Quthero, and Mesoblast (see: https://www.karplab.net/team/ jeff-karp).
  • The interests of J.M.K. were reviewed and are subject to management plans overseen by his institution in accordance with their conflict of interest policies.
  • All other authors declare no competing interests.

Additional information

  • Reprints and permission information is available at http://www.nature.com/reprints.
  • Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
  • This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
  • If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

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ARTICLE
Engineering designer beta cells with a CRISPR-Cas9
conjugation platform
Donghyun Lim
1,2,3
, Vedagopuram Sreekanth
1,2,3
, Kurt J. Cox
1,2,3
, Benjamin K. Law
1,2,3
, Bridget K. Wagner
1
,
Jeffrey M. Karp
4,5,6,7
& Amit Choudhary
1,2,3
Genetically fusing protein domains to Cas9 has yielded several transformative technologies;
however, the genetic modications are limited to natural polypeptide chains at the Cas9
termini, which excludes a diverse array of molecules useful for gene editing. Here, we report
chemical modications that allow site-specic and multiple-site conjugation of a wide
assortment of molecules on both the termini and internal sites of Cas9, creating a platform
for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modied to more
precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN)
at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to
Cas9 signicantly increases the efciency of precision genome editing, and such a platform is
compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we
successfully engineer INS-1E, a β-cell line, to repurpose the insulin secretion machinery,
which enables the glucose-dependent secretion of protective immunomodulatory factor
interleukin-10.
https://doi.org/10.1038/s41467-020-17725-0
OPEN
1
Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
2
Department of Medicine, Harvard
Medical School, Boston, MA 02115, USA.
3
Divisions of Renal Medicine and Engineering, Brigham and Womens Hospital, Boston, MA 02115, USA.
4
Engineer ing in Medicine, Department of Medicine, Cent er for Regenerative Therapeutics, Brigham and Womens Hospital, Harvard Medical School,
Boston, MA 02115, USA.
5
Harvar d MIT Division of Health Sciences and Techno logy, MIT, Cambridge, MA 02139, USA.
6
Proteomics Platform, Broad
Institute of MIT and Harvard, Cambridge, MA 02142, USA.
7
Harvard Stem Cell Institute , Harvard University, Cambridge, MA 02138, USA.
email: achoudhary@bwh.harva rd.edu
NATURE COMMUNICATIONS | (2020) 11:4043 | https://doi.org/10.1038/s41467-020-17725-0 | www.nature.com/naturecom munications 1
1234567890():,;

C
lustered regularly interspaced short palindromic repeats
(CRISPR)-Cas9 is a DNA endonuclease that can be tar-
geted to a genomic site using a guide RNA (gRNA)
bearing sequence complementarity to the target site
1
. The genetic
fusion of Cas9 with effector domains (e.g. a transcription acti-
vator) has yielded transformative technologies
2,3
; however, this
approach is limited to fusions that are generally linear, poly-
peptidic, and located on the termini of Cas9. A covalent con-
jugation platform that allows the creation of fusions that are non-
polypeptidic (e.g. nucleic acids, small molecules, polyethylene
glycol [PEG] chains), orthogonally branched from the internal
sites of Cas9, and amenable to multiple-site attachment to give a
multivalent display of functional cargos would provide a greater
diversity of technologies and applications. For example, precise
sequence alteration at the Cas9 cleavage site requires the efcient
incorporation of exogenously supplied single-stranded oligonu-
cleotide donor DNA (ssODN)
4
via the homology-directed repair
(HDR) pathway
5,6
. However, most cells instead employ the
non-homologous end-joining (NHEJ) repair, which results in
unpredictable insertions and deletions of bases at the cleavage
site, some of which are large enough to have pathogenic con-
sequences
7,8
. Displaying ssODNs on Cas9 can increase their local
concentration around the DNA strand break site to allow
enhanced incorporation of the desired sequence. In another
application, appending PEG chains to Cas9 may reduce the
immunogenicity of this bacterial protein
9
.
An ideal conjugation platform to Cas9 should have the fol-
lowing characteristics. First, the platform should be compatible
with a diverse set of cargos and allow their multiple-site attach-
ment. Second, the platform should be robust and implementable
by nonspecialists given the diverse users of CRISPR technologies.
Third, since some of the cargos (e.g. ssODN) are only available in
small quantities and are expensive, the conjugation system should
work efciently without requiring large excesses. Ideally, the
platform should be modular and inexpensive to allow screening
of multiple conditions (e.g. ssODN sequence). Finally, for real-
world applications, the platform should allow scaled-up produc-
tion of the conjugates following good manufacturing practice
regulations
10
.
Herein, we present the development of such a platform that
relies on thiol-maleimide chemistry and DNA-base pairing,
which are both simple, well-established, scalable, and amenable to
a wide range of substrates
11
. We systematically scan the domains
of Cas9 to choose residues replaceable with engineered cysteines,
to which molecules of any size can be efciently appended
without the loss of Cas9 activity. Because many possible con-
jugates (e.g. ssODNs) are prohibitively expensive for or una-
menable to direct thiol-maleimide conjugation, we also seek to
develop a more general conjugation platform. Thus, we design a
short oligonucleotide handle adaptor, which is attached to Cas9
via thiol-maleimide chemistry and uses base pairing to anchor
any molecule containing or appended with nucleic acids (Fig. 1a).
As an example of the platforms utility, we use it to hybridize long
ssODNs that are large, expensive, and not available in sufcient
quantities for thiol-maleimide conjugation to Cas9. The resulting
Cas9:ssODN conjugates robustly enhance the precise incorpora-
tion of the desired sequence from ssODN in multiple cell types
and genomic sites. Importantly, the chemical conjugation plat-
form enables the multivalent display of ssODNs, which further
enhances the precise incorporation of the desired sequence over
that of the unitary display.
Next, we demonstrate the utility of our conjugation platform by
efciently engineering insulin-producing β cells to secrete none-
ndogenous molecules, including an immunomodulatory protein
(160 residues), without incorporating any viral or foreign
sequences (e.g. promoter) other than that of the secreted molecule.
Current β-cell transplantation therapies for type 1 diabetes suffer
from immune rejection, resulting in acute cell loss and only short-
term therapeutic effects
12,13
. The macroencapsulation of β cells
with a semipermeable membrane can protect them from the hosts
immune system, although foreign body reaction-induced
brosis can impair the mass transfer and viability of encapsu-
lated cells
1417
. Anti-inammatory cytokines, such as interleukin
10 (IL-10), can reduce brosis and promote long-term β-cell
survival and superior islet function
1820
. Therefore, engineered
β-cells that secrete anti-inammatory cytokines and antibrotic
factors can propel the development of cell-based therapeutics for
diabetes. Using our modied Cas9, we genetically repurpose the
insulin expression and secretion machinery to secrete a none-
ndogenous peptide and IL-10 in a glucose-responsive manner,
demonstrating an immediate usefulness of our genome editing
platform in developing cell-based therapeutics.
Results
Cas9 domains tolerate the attachment of diverse molecules.To
choose the sites for conjugation to Cas9, we analyzed the struc-
tures of apo-Cas9, gRNA-bound Cas9, and gRNA- and DNA-
bound Cas9 for residues that could provide a high labeling yield,
tolerate chemical modications, span all the domains of Cas9,
and were surface-exposed in various Cas9 conformations for the
efcient modications (Fig. 1b)
2123
. Using the aforementioned
criteria, we identied two sites (204, 532) on the recognition
(REC) lobe, one site (826) on the HNH domain, ve sites (1, 945,
1026, 1054, 1068) on the RuvC domain, and two sites (1153,
1207) on the protospacer adjacent motif-interacting (PI) domain.
We selected residues 558 and 1116 as controls, since modica-
tions at 558 will impede the Cas9:gRNA interaction and at 1116
will impede PAM recognition by Cas9 (Fig. 1b and Supplemen-
tary Fig. 1). We optimized the conjugation conditions for Cas9
variants using biotin-maleimide and PEG (5 kDa)-maleimide as
model compounds to ensure that modications of various sizes or
PI
HNH
RuvC
REC
BH
1207
1153
1116
826
1
1054
1068
945
1026
204
532
558
a
b
SH
SH
Cas9
Adaptor
Cas9
Cargo
Cargo
Cas9
Cas9
Cas9
Small molecule
Polymer
Fig. 1 A conjugation platform for Cas9. a A modular design strategy to
functionalize Cas9. b Structure-guided selection of chemical labeling sites.
Protospacer adjacent motif-interacting (PI) domain is in blue, HNH domain
is in yellow, RuvC domain is in cyan, recognition (REC) lobe is in magenta,
and bridge helix (BH) is in grey. Crystal structure of the Cas9-gRNA-DNA
ternary complex is used (PDB: 5F9R)
22
.
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17725-0
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natures were tolerated (Supplementary Fig. 2). The reactions were
fast and high yielding at all sites except for the 1153 C mutant
sites proximal to 1153 C (i.e. 1154 C) also yielded low conjugation
efciencies (Supplementary Fig. 2). The location of these residues
was not assigned at the crystal structure of apo-Cas9 but the
residues were assumed to be amenable to efcient conjugation
since they were expected to be surface-exposed and exible
2123
.
Our labeling results, however, indicate that the loop may have
higher-order structures that prevent efcient chemical reactions,
so we did not use those sites in future experiments. To improve
the compatibility of the system with a broader range of con-
jugates, we next utilized the optimized reaction conditions to label
Cas9 at the remaining sites with a 17-nucleotide (nt) DNA
adaptor (5-GCTTCACTCTCATCGTC-3). The conversion rates
were comparable to those of PEG labeling (Supplementary Fig. 2),
demonstrating that the efcient conjugation of multiple cargo
types can occur at these sites. Thus, the identied sites provide
high conjugation yields with diverse molecules, including small
molecule and polymers (DNA or PEG).
To identify sites tolerant to the conjugation of the DNA
adaptor without the loss of Cas9 activity, we designed an ssODN
that would insert a 33-nt DNA fragment (HiBiT sequence
24
)at
the target gene (Supplementary Fig. 3). This insertion would
result in the expression of a fusion protein with a C-terminal
HiBiT tag, which is a small fragment of the NanoLuc luciferase.
When HiBiT is complemented by LgBiT, the remainder of
NanoLuc, the full-length luciferase is reconstituted to generate a
luminescence signal proportional to the degree of knockin,
providing an easy readout for HDR (Supplementary Fig. 3a). We
chose GAPDH as the rst target gene (Supplementary Fig. 3b)
owing to its abundant expression in many cell types, which
should allow for the reliable detection of the luminescence signal.
Using the HiBiT knockin assay, we measured whether appending
the DNA adaptor to the cysteine affected Cas9 activity (Fig. 2a).
As expected, much of the Cas9 activity was lost by control
modications at residues 558 and 1116, indicating the reliability
of the HiBiT knockin assay. We identied ve sites whose activity
was largely maintained (>85% of wild-type in U2OS), even after
labeling with the 17-nt adaptor; these sites stemmed from three
regions: the REC lobe (532), the RuvC domain (1, 945, 1026), and
the PI domain (1207). To investigate the off-target prole of the
Cas9-adaptor conjugates, we used an eGFP disruption assay with
matched gRNA and mismatched gRNAs targeting the eGFP gene
of the U2OS.eGFP.PEST cells
25,26
. The Cas9-adaptor conjugate
retained the target specicity while also maintaining the on-target
activity (Supplementary Fig. 4ac). Finally, we demonstrated that
Cas9s conjugated to the long PEG chain (5 kDa, Supplementary
Fig. 2c) retained the DNA cleavage activity in the eGFP
disruption assay, assuring that Cas9 could be modied with
diverse cargos without a loss of function (Supplementary Fig. 4d).
Unitary display of ssODN enhances HDR in several cell types.
Next, we designed ssODN with a sequence complementary to the
conjugation adaptor and conrmed the binding of the ssODN
bearing the complementary sequence to the Cas9-adaptor using a
gel-shift assay (Supplementary Fig. 5). To measure the ability of
ssODN conjugates to enhance HDR and the site dependence of such
enhancements, we performed the HiBiT knockin assay in U2OS
cells. Using the luminescence signals from unconjugated ssODN as
normalization controls, we observed enhanced knockin efciency
at multiple sites (Fig. 2b and Supplementary Fig. 6a) with the
ssODN attached to Cas9. We were able to conrm such enhance-
ments in multiple cell lines, with a greater than four-fold increase in
HEK-293FT cells, around a 1.9-fold increase in human-induced
pluripotent stem cells, and a more than three-fold increase in pri-
mary br oblasts (Fig. 2cf and Supplementary Fig. 6be). For
cells with higher HiBiT signal but lower HDR enhanceme nts, we
observed site dependence, with two internal conjugation sites (532,
945) generally performing better than the terminal conjugation
0.0
0.5
1.0
1.5
Relative luminescence
a
wt
1
204
532
558
826
945
1026
1054
1068
1116
1207
U2OS
U2OS
MDA-MB-231 HEK-293FT hiPSC
Primary
fibroblast
b
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Fold knock-in enhancement
Fold knock-in enhancement
Fold knock-in enhancement
Fold knock-in enhancement
Fold knock-in enhancement
wt
1
532
945
1026
1207
f
0
1
2
3
4
5
6
wt
532
945
c
0
1
2
3
4
5
6
wt
1
532
945
1207
d
0
1
2
3
4
5
6
7
wt
1
532
945
1026
1207
e
0.0
0.5
1.0
1.5
2.0
2.5
3.0
wt
532
945
Fig. 2 Unitary display of ssODN on Cas9 domains enhances HDR in multiple cell types. a HiBiT knockin efciencies by Cas9-adaptor conjugates
compared to unlabeled wild-type Cas9 (wt) when a separate Cas9/ssODN system was used (n = 3 biologically independent experiments except for 204
where n = 2). bf ssODN display on Cas9 enhances HiBiT knockin efciency in various cells: b U2OS, c MDA-MB-231, d HEK-293FT, e human-induced
pluripotent stem cells, and f primary human neonatal dermal broblasts. Unlabeled wild-type Cas9 (wt) and Cas9-adaptor conjugates labeled at the
indicated residues were used (n = 3 biologically independent experiments). Source data are provided as a Source Data le.
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17725-0 ARTICLE
NATURE COMMUNICATIONS | (2020) 11:4043 | https://doi.org/10.1038/s41467-020-17725-0 | www.nature.com/naturecommunications 3

site (1). An examination of the c rystal structure
22
indicates that
cargos on the two internal residues are expected to align with
substrate DNA, while cargos on the terminal residue project out-
ward from the DNA, which may explain the differences in the
HDR-enhancing capacities of different ssODN-bearing sites.
ssODN display platform allows rapid and facile screening.To
demonstrate the modular nature of our conjugation platform that
should allow the rapid testing of multiple conditions and to
conrm the generality of HDR enhancement by ssODN display,
we tested the ability of the conjugates to enhance HDR under
several scenarios (e.g. different genomic sites, ssODN sequences,
or readouts). Using the HiBiT knockin assay, we conrmed HDR
enhancements at another DNA cleavage site of the GAPDH locus
in U2OS (Fig. 3a and Supplementary Fig. 7a) and at multiple
genomic loci (PPIB in U2OS, CFL1 in HEK-293FT; Fig. 3a and
Supplementary Fig. 7b, c). We then demonstrated HDR
enhancement using a uorescent readout and a longer knockin
fragment (GFP11, 57 nt). The correct incorporation of this
fragment generated detectable uorescence through the expres-
sion of a fusion protein with a C-terminal GFP11 tag, which
forms a fully functional GFP when complemented by GFP1-10
(Supplementary Fig. 8)
27
. Here as well, displaying ssODNs on
Cas9 increased the knockin efciency by more than three-fold
(Fig. 3b and Supplementary Fig. 7d). In addition to luminescence
and uorescence readouts to demonstrate HDR enhancements,
we used a restriction endonuclease site knockin assay that
quanties both NHEJ and HDR efciencies at the CXCR4 locus
by gel electrophoresis (Supplementary Fig. 9), and observed the
increase in HDR efciencies by more than two-fold when Cas9:
ssODN conjugates were employed (Fig. 3c). We then used a
previously reported droplet digital PCR (ddPCR) assay that
employs probes to distinguish between wild-type, NHEJ-edited,
and HDR-edited sequences at the RBM20 locus (Supplementary
Fig. 10a, b)
28,29
. All Cas9:ssODN conjugates increased the ratio of
HDR over NHEJ, again indicating the generality of our platform
(Fig. 3d). The conjugates also enhanced HDR when another
gRNA/ssODN pair was employed (Supplementary Fig. 10c).
Finally, we designed an assay to convert eGFP to BFP in U2OS
cells through HDR-based nucleotide exchange and found that
Cas9:ssODN conjugates enhanced precision genome editing for
this exogenous target gene as well (Supplementary Fig. 11).
Multivalent display of ssODN on Cas9 further enhances HDR.
Owing to the small size of our adaptor and the chemical nature of
our platform, multivalent displays are feasible (Fig. 4a). To
demonstrate multivalent display, we produced Cas9 double-
cysteine mutants (532 C/945 C and 532 C/1207 C) and attached
the adaptor to both sites (Supplementary Fig. 12a). Next, we
conrmed the binding of the ssODNs to Cas9 (Supplementary
Fig. 12b) and observed a boost in HDR efciency for both the 33-
nt HiBiT insertion and the two-base exchange (Fig. 4bd),
indicating that multivalent Cas9 internal modications further
improve the functionality of conjugated Cas9 proteins. Finally, to
minimize the size of the labels on Cas9, we investigated the
possibility of further decreasing the length of the adaptor. To this
effect, we found that hybridization by 13 nt or 15 nt showed a
similar HDR-enhancing effect as the standard 17-nt pairing
(Supplementary Fig. 13).
Efcient engineering of β cells to secrete IL-10. To demonstrate
the functional applicability of our chemically modied Cas9, we
a
wt
532
wt
1
wt
532
wt
532
wt
532
wt
532
wt
945
wt
532
wt
945
wt
1026
wt
1207
0
1
2
3
Fold knock-in enhancement
Fold knock-in enhancement
0.0
0.5
1.0
1.5
2.0
2.5
0
1
2
3
4
5
GAPDH PPIB CFL1
HiBiT knock-in
0
1
2
3
4
5
b
GAPDH
GFP11 knock-in
CXCR4
12-base exchange
0.00
0.02
0.04
0.06
0.00
0.02
0.04
0.06
0.00
0.02
0.04
0.06
0.00
0.02
0.04
0.06
0.00
0.02
0.04
0.06
d
p = 0.072 p = 0.015 p = 0.053
p = 0.013 p = 0.035
RBM20
2-base exchange
0.0
0.1
0.2
0.3
HDR/NHEJ
HDR/NHEJ
0.0
0.1
0.2
0.3
c
p = 0.0069 p = 0.032
Fig. 3 ssODN display platform allows facile testing of multiple conditions. a HiBiT sequence knockin efciency was increased at multiple genomic loci in
U2OS cells (GAPDH and PPIB) or HEK-293FT cells (CFL1), (n = 3 biologically independent experiments). b The GFP11 sequence insertion at the GAPDH
locus was promoted in HEK-293T cells (n = 3 biologically independent experiments). c HDR-mediated 12-base exchange efciency at the CXCR4 locus was
increased in HEK-293T cells (n = 3 biologically independent experiments). d Two-base exchange at the RBM20 locus was promoted in HEK-293FT cells.
Unlabeled wild-type Cas9 (wt) and Cas9-adaptor conjugates labeled at the indicated residues were used (n = 3 biologically independent experiments).
P-values were calculated by paired two-tailed t-test. Source data are provided as a Source Data le.
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17725-0
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used it to efciently engineer β cells to endow the cells with
immunomodulatory function. Since C-peptide is cleaved during
proinsulin processing and is cosecreted with insulin, we hypo-
thesized that knocking in the desired gene into the C-peptide
portion of the proinsulin locus would enable the secretion of the
inserted gene product. Previously, a lentiviral vector encoding a
proinsulin-luciferase fusion construct, containing a luciferase
inserted into the C-peptide, expressed functional luciferase in
levels directly proportional to insulin when stably integrated into
the INS-1E rat β-cell line and responded sensitively to external
stimuli, such as glucose concentration
30
. However, viral-vector
engineering poses safety issues such as immunogenicity to viral
components or the unintended random insertion of DNA frag-
ments into the host genome
31,32
. Direct knockin of the desired
gene fragment into the C-peptide locus using Cas9 will allow
glucose-dependent cosecretion of the target gene products with
insulin. The knockin strategy does not require long regulatory
elements (e.g. promoters), which enables footprint-free and ef-
cient HDR because of the smaller knockin size. Any viral or
foreign sequences, which are often required to drive efcient gene
expression, can be avoided to minimize the immunogenicity
issues. In addition, due to the inherent high expression and
secretion level of insulin in β cells, it would be easy to modulate
the secretion level of exogenous gene products (Fig. 5a, b).
We set out to demonstrate our β-cell engineering strategy with
the rat INS-1E β-cell line, which is widely used to study β-cell
biology. Murine cells have two insulin genes, Ins1 and Ins2, both
of which produce and secrete functional insulin; herein, we
targeted only the Ins1 gene. To identify the appropriate insertion
site in the Ins1 locus, we used HDR-mediated knockin of the
HiBiT sequence at the C-peptide portion (Fig. 5a). The target
HiBiT sequence was anked by additional prohormone con-
vertase 2 (PC2) cleavage sites
30
to ensure no extra amino acids
would be present at each end of the knockin product after
processing (Fig. 5a). We chose three gene insertion sites at the
start, middle, and terminal regions of the C-peptide locus, and
designed several gRNAs to target these sites such that insertion
sites and DNA cleavage sites would be close enough to obtain
high HDR efciency (Fig. 5a). In addition, genome-wide off-
target proles of gRNAs were considered such that potential off-
target sites had mismatches at the seed sequences or at least three
mismatches in the gene-encoding regions. When standard
genome editing was performed at the target sites using
nonconjugated Cas9 and ssODN, the HiBiT peptide was secreted
from INS-1E cells, which could be readily detected through
luminescence signals from the cell culture supernatant after
complementation by the LgBiT protein. The highest knockin
efciency was achieved by targeting the middle region of the C-
peptide (site 2) (Fig. 5c), so this insertion site was used for future
experiments. HiBiT peptide secretion was also stimulated by
glucose, consistent with the behavior expected from an insertion
at the ins1 locus (Supplementary Fig. 14a).
Based on this optimized design, we next knocked in Il10, whose
797-nt ssODN is much larger than that of HiBIT (183 nt). As our
approach leverages the insulin secretion pathway, the knockin
product will be secreted without the secretory signal peptide.
Thus, the signal peptide sequence present in the Il10 gene was
omitted when designing the knockin fragment. PC2 cleavage sites
were added at each end of Il10 to obtain intact IL-10 as the
knockin product, and the corresponding ssODN was synthesized
by reverse transcription. When INS-1E cells were transfected with
both unconjugated Cas9 and ssODN, IL-10 was secreted to the
cell culture medium as determined via enzyme-linked immuno-
sorbent assay (ELISA). No IL-10 was detected after transfection
with Cas9 or ssODN alone or in lipopolysaccharide (LPS)-treated
cells
33
(Fig. 5d). We conrmed the correct insertion of the Il10
gene at the Ins1 c-peptide region using Sanger sequencing
(Supplementary Fig. 15ac). Finally, we conjugated ssODN on
Cas9 and found that both HiBiT secretion and IL-10 secretion
were signicantly promoted by Cas9-ssODN conjugation over
that of separate Cas9 and ssODN (Fig. 5e, f and Supplementary
Fig. 16).
To further verify that the knockin products are released
through the regulated insulin secretion pathway, we investigated
the effect of insulin secretagogues
30
with distinct mode of actions.
To minimize the well-to-well signal differences originating from
the stochastic distribution of edited cells, we enriched the HiBiT
Cas9
SH
SH
ssODN
Adaptor
Cas9
a
b
532/945
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Fold knock-in enhancement
0.0
0.5
1.0
1.5
2.0
2.5
3.0
p = 0.014
p = 0.0056
wt
532
532/945
wt
945
c
0.00
0.02
0.04
0.06
HDR/NHEJ
HDR/NHEJ
0.00
0.02
0.04
0.06
532/945
wt
532
532/945
wt
945
p = 0.028
p = 0.039
d
0.00
0.02
0.04
0.06
0.08
0.00
0.02
0.04
0.06
0.08
532/1207
wt
532
532/1207
wt
1207
p = 0.015
p = 0.039
Fig. 4 Multivalent display of ssODN further enhances HDR efciency.
a Schematic illustrating the production of Cas9 double-ssODN conjugates.
b HiBiT sequence knockin at the GAPDH locus was detected in U2OS cells
(n = 4 biologically independent experiments). c, d Two-base exchange at
the RBM20 locus was detected in HEK-293FT cells. Unlabeled wild-type
Cas9 (wt) and Cas9-adaptor conjugates labeled at the indicated residues
were used (n = 3 biologically independent experiments). P-values were
calculated by paired two-tailed t -test. Source data are provided as a Source
Data le.
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17725-0 ARTICLE
NATURE COMMUNICATIONS | (2020) 11:4043 | https://doi.org/10.1038/s41467-020-17725-0 | www.nature.com/naturecommunications 5

Figures (6)
References
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Journal ArticleDOI
TL;DR: It is found that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface, and off-target cleavage of CRISPR-associated (Cas)9-based RGNs is characterized.
Abstract: Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

2,619 citations


Journal ArticleDOI
01 Jan 2016-Science
Abstract: The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

1,635 citations


Journal ArticleDOI
TL;DR: It is shown that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases, and the observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.
Abstract: CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

875 citations


Journal ArticleDOI
14 Mar 2014-Science
TL;DR: To compare the architectures and domain organization of diverse Cas9 proteins, the atomic structures of Cas9 from Streptococcus pyogenes and Actinomyces naeslundii and AnaCas9 were determined by x-ray crystallography and three-dimensional reconstructions of apo-SpyCas9, SpyCas9:RNA, and SpyCas 9:RNA:DNA were obtained by negative-stain single-particle electron microscopy.
Abstract: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

845 citations


Journal ArticleDOI
TL;DR: By rationally designing single-stranded DNA donors of the optimal length complementary to the strand that is released first, this work increases the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%.
Abstract: Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ∼6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3' end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.

755 citations


Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Engineering designer beta cells with a crispr-cas9 conjugation platform" ?

Here, the authors report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. The authors demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths.