Enhanced Homology-Directed Human Genome Engineering by Controlled Timing of CRISPR/Cas9 Delivery
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Enhanced Homology-Directed Human Genome Engineering by Controlled Timing of CRISPR/Cas9 Delivery
Free PMC article
The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.
CRISPR/Cas9; cell biology; cell cycle synchronization; chromosomes; genes; genome engineering; homologous recombination; human; nocodazole; non-homologous end joining.
Conflict of interest statement
The authors declare that no competing interests exist.
Figure 1.. The effect of cell cycle synchronization on total editing and homology-directed repair frequencies in HEK293T cells.
A) Experimental schematic of timed delivery of Cas9-guide RNA
ribonucleoprotein (RNP) into human cells for genome editing.
( B) Chemical inhibitors used to arrest cells at specific phases
of cell cycle included lovastatin (Lov), which blocks at early G1 and
partially at G2/M phase; mimosine (Mim), aphidicolin (Aph), thymidine (Thy)
and hydroxyurea (HU) which arrest cells at the G1-S border prior to onset of
DNA replication; and nocodazole (Noc) which causes arrest at G2/M phase.
( C) The homology-directed repair (HDR) donor DNA is a 183 nt
ssODNA that is complementary to the target sequence (−strand) and
contains a 9 nt insertion (HindIII and SphI restriction sequences) at the
cut site and a 9 nt deletion downstream of the cut site; these modifications
are flanked by 85 nt and 55 nt asymmetrical homology arms at 5′ and
3′ ends, respectively. ( D, E) PCR-based
screening of cell cycle inhibitors for enhancement of Cas9-triggered total
editing (TE) ( D) and HDR ( E) frequencies in
HEK293T cells. For each inhibitor condition (color coded), two doses of Cas9
RNP, 30 and 100 ρmol, were transfected with 100 ρmol of HDR DNA
template; control reactions (labeled as C) contained 100 ρmol of Cas9
but no sgRNA. The TE frequency was measured using a T7 endonuclease I assay
and analyzed using a formula described in ‘Materials and
Methods’. The HDR frequency was determined directly by HindIII
digestion, which specifically cleaved the newly integrated HindIII sequence,
and calculated as the ratio of DNA product to DNA substrate. The % TE, % HDR
and standard deviation (error bars) were calculated from three
Figure 1—figure supplement 1.. FACS analysis reveals cell cycle blocks and the DNA content in the cells that are arrested at different phases of cell cycle.
Bivariate cell cycle, BrdU (S-phase), 7-AAD (DNA content) FACS analysis
reveals cells are arrested at different phases of cell cycle. Chemical
inhibitors were used to arrest cells at specific phases of cell cycle.
A) Analysis of HEK293T cells with different cell cycle
blocks and nocodazole released cells. ( B) Analysis of human
neonatal fibroblasts with different cell cycle blocks and aphidicolin
released cells. ( C) Analysis of H9 hES cells unsynchronized
(unsync), nocodazole synchronzied (Noc) and nocodazole + aphidicolin
sequential synchronized (Noc + Aph) at time of cell cycle block and
transfection or 3 days after release. Alkaline phosphatase positive and
normal ES colony morphology for all three conditions. ROCK apoptosis
inhibitor (10 μM) was required for survival of synchronized H9 ESCs
after release when cultured at low density in 6-well plates but not when
cultured at high density in 96-well plates. DOI:
Figure 2.. The enhancement of TE and HDR at the EMX1, DYRK1 and CXCR4 loci by nocodazole synchronization in HEK293T cells.
A) The effect of nocodazole on the TE and HDR frequencies at
EMX1 locus. HEK293T cells were synchronized at M phase with 200 ng/ml of
nocodazole for 17 hr before nucleofection. To determine the optimal dosage,
three concentrations of Cas9 RNP were assayed in combination with three
doses of HDR template (Figure 1C).
The TE frequencies at 10 ρmol of Cas9 RNP in the unsynchronized cells
were too low and therefore not determined (ND). ( B) The effect
of nocodazole on the TE and HDR frequencies at DYRK1 locus. The
directionality of ssODNA HDR templates, either identical (+strand) or
complementary (−strand) to the target sequence, was examined. The PAM
is highlighted in red, the target sequence in blue and the integrated
HindIII site in green. ( C) The effect of nocodazole on the TE
and HDR frequencies at the CXCR4 locus. The HDR template is ssODNA
complementary (−strand) to the target sequence, and contains a
HindIII restriction sequence flanked by 90 nt homology arms. Representative
gels from two biological replicates are shown. DOI:
Figure 2—figure supplement 1.. On-target NHEJ and HDR and off-target cleavage analyses by deep sequencing.
A) The genomic DNAs from Figure 2A,B experiments were analyzed for NHEJ and HDR
frequencies, at the on-target and off-target sites, by deep sequencing. The
TE frequency (indels/total reads) was determined at the EMX1 target, DYRK1
target and selected off-target loci as a function of Cas9 RNP dosage, (n
= 1 representative experiment). The HDR frequency (HDR/total reads)
represented specifically exogenous donor template-mediate HDR. The ratio of
Cas9 RNP-induced DSB repaired by the HDR pathway was determined as the
percentage of HDR/TE. The controls, which included the non-transfected cells
and the cells transfected with only Cas9 protein but no sgRNA, showed no
evidence of on- or off-target editing. ( B) Representative
sequences repaired by HDR and NHEJ at the EMX1 and DYRK1 loci. The Cas9
cleavage sites are marked by red triangles. DOI:
Figure 3.. Systematic investigation of DNA templates for efficient HDR at the EMX1 locus in HEK293T cells.
A) Segment of human EMX1 exon 3 shows the 20 nt target sequence
(highlighted in blue), the TGG PAM region (in red) and the Cas9 cleavage site
at three bases upstream from PAM. Seven HDR templates (color coded) were tested
for HDR efficiency. Template 1 is as described in Figure 1C. Templates 2–7 contain HindIII and BamHI
restriction sites that are flanked symmetrically by various lengths of homology
arms, ranging from 30 nt to 250 nt. Templates 2–5 are ssODNA; templates
6–7 are PCR amplified double-stranded DNA (see ‘Materials and
methods’). ( B) HDR efficiency was tested under three cell
conditions. In addition the unsynchronized and nocodazole synchronized
conditions, the cells were synchronized with nocodazole prior to nucleofection,
and immediately post nucleofection, a single dose of aphidicolin (2
μg/ml) was added to the growth media to prevent the transfected cells
from proceeding into the S phase. The purpose was to test whether blocking
passage through S phase reduces HDR efficiency, since the HDR pathway is
thought to be most active during S phase. This one-time addition of aphidicolin
is labeled as ‘Aph block’ in the third panel, as opposed to the
standard aphidicolin synchronization procedure used elsewhere in the
manuscript. Thirty ρmol of Cas9 RNP and 50 ρmol of HDR template
were used in the nucleofection reaction; the control reaction (C) contained no
HDR template. The mean % HDR and standard deviation (error bar) was determined
by HindIII digestion from three experiments. Representative gels from PCR and
HDR analyses are shown for each cell condition. Templates 6 and 7 produced
unusual banding patterns, making quantitation of DNA bands less accurate
(labeled by asterisk). DOI:
Figure 4.. The enhancement of TE and HDR frequencies at the EMX1 locus by cell cycle synchronization in human primary neonatal fibroblast and embryonic stem cells.
A) Screening of cell cycle inhibitors for enhancement of TE and
HDR frequencies in human primary neonatal fibroblast cells. For each inhibitor
condition (color coded), two doses of Cas9 RNP, 30 and 100 ρmol, were
transfected with 100 ρmol of HDR DNA template 4 from Figure 3A. A control reaction (labeled as C) contained 100
ρmol of Cas9 but no sgRNA. The % TE and % HDR were analyzed similarly as
with HEK293T cells. ( B) Three conditions were tested using hES
cells: unsynchronized, nocodazole synchronized and nocodazole-aphidicolin
sequential synchronized. The cells were treated with nocodazole for 16 hr,
washed to remove the drug and then treated with aphidicolin for 3 hr before
nucleofection. 30 or 100 ρmol of Cas9 RNP was co-transfected with 100
ρmol of HDR template 4 from Figure
3A, and cultured at high density in 96-well plates in the presence or
absence of ROCK apoptosis inhibitor (10 μM). For both experiments,
representative gels from two biological replicates are shown. The contrast of
the gel images was increased to show that no HDR was detected in other
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Research Support, Non-U.S. Gov't
Clustered Regularly Interspaced Short Palindromic Repeats*
DNA Breaks, Double-Stranded
Embryonic Stem Cells / cytology
Embryonic Stem Cells / metabolism
Endonucleases / genetics*
Endonucleases / metabolism
Genetic Engineering / methods
Recombinational DNA Repair*
Staphylococcus / chemistry
Staphylococcus / enzymology
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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