A P P L I C AT I O N N O T E

Knock-in a SNP
alteration in iPSCsusing Edit-Rreagents.

Introduction
An increasing number of human diseases have been found tohave a genetic component. To study the phenotypic effectsof these mutations, genome editing technologies such as theCRISPR-Cas9 gene editing system can be used to manipulatea cell’s genomic DNA (Jinek 2012, Mali 2013, Cong 2013).
By co-delivery of a Cas9 nuclease and a DNA-targeting guideRNA, a targeted double-strand DNA break is made, prompting
the cellular machinery to repair the lesion. The co-introductionof a synthetic repair template can be used by the cell during

Authors
Amanda Haupt
John SchielRevvity, Inc.

repair to incorporate precise genomic changes desired by
the researcher through the homology-directed repair (HDR)
pathway (Waddington 2016, Jehuda 2018). In this way,disease-associated mutations can be precisely introduced into
a wild-type cell background, or patient-derived disease models
can be corrected to the wild-type sequence. This generates
an otherwise isogenic pair of disease and control cell lines forstudy in the laboratory.
Human-induced pluripotent stem cells (iPSCs) are an excellent
model system for the study of genetic diseases because they
are a renewable, karyotypically normal cell type that can be
manipulated in the pluripotent state and then differentiatedinto many clinically relevant isogenic cell types. Additionally,
there are a variety of methods for generating iPSCs from patient
samples with minimally invasive procedures (Malik 2013).
Generating iPSC models remains challenging in part due todifferences in HDR-mediated editing efficiencies between celllines and locus-to-locus variability.
Here, we demonstrate the introduction of disease-associatedgenetic mutations into a wild-type human iPSC line throughthe co-delivery of Revvity Edit-R Cas9 Nuclease mRNA,synthetic sgRNA, and a single strand DNA repair template.

Knock-in a SNP alteration in iPSCs using Edit-R reagents.

These DNA-free reagents prevent potentially confounding

with mono-allelic single-nucleotide polymorphisms (SNPs)

random genomic integration, preserving the integrity of theinherited from a single parent. Therefore, to properly modeliPSC models’ genetic composition. Additionally, synthetic

the genetic disease, editing must only be made to one allele

sgRNA and Cas9 Nuclease mRNA are active in the cell forwhile maintaining a wild type allele. This type of gene editing

less time than plasmid-based reagents, which reduces thecan be particularly challenging in iPSCs where HDR is not therisk for off-target editing activity. For technical ease of use,

preferred dsDNA break repair method.

Cas9 Nuclease mRNA also precludes the need for using areagent with a cell-line specific optimized promoter.

To trigger HDR in iPSCs that would result in the introductionof a SNP, we relied on electroporation conditions previouslyoptimized to achieve high percentages of indel formation

Results

with the hypothesis that the more efficient the delivery ofTo mimic two known disease-associated genetic mutations in

HDR events (see Methods). Edit-R Cas9 Nuclease mRNA wasa healthy iPSC background, we chose two well-characterized

co-delivered with a specific sgRNA designed with the Revvity

genotypes. The R225X mutation in the LMNA gene introduces

CRISPR Design Tool to target the genome within 15 bp ofa premature stop codon resulting in various laminopathies

the desired editing location. A single strand DNA oligo was(Capell et al. 2006; Rankin et al. 2006). The MYH7 D239N

also designed for co-delivery using the Revvity HDR Donor

variant replaces an aspartic acid with an asparagine andDesigner that was complementary to the editing region andis known to be associated with early-onset hypertrophic

included the disease-associated point mutation, as well as

cardiomyopathy (Adhikari et al., 2016). Both are dominantnecessary silent mutations to prevent further CRISPR-Cas9

traits where a phenotype can be observed in patientsrecognition and cleavage after HDR repair (Figure 1).

gene editing reagents, the greater likelihood of observing rare

Figure 1: Example of the Revvity HDR Donor Designer. This tool automatically generates a recommended donor sequence designed from user
input of the modification location and sequence change desired. In the LMNA gene shown here, arginine (R) at position 225 is converted to astop codon by the insertion of a thymine (T) and removal of cytosine (C).

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2Knock-in a SNP alteration in iPSCs using Edit-R reagents.

Seventy-two hours post-electroporation, cells were imaged

and next-generation amplicon sequencing was performed.

to show that the iPSCs maintained healthy morphology

Analysis of NGS data revealed that indels were theafter manipulation (Figure 2A), and the edited and control

prominent editing outcome from this experiment, and a small

populations were lysed for PCR amplification of thefraction of alleles (3-8%) contained the disease-associatedgenomic region of interest. Amplicons were gel-purified,point mutation, as expected for this cell type (Figure 2B-C).

ABC

Figure 2: Gene Editing in iPSCs with Cas9-mRNA Nuclease, synthetic sgRNAs, and ssDNA oligo repair template delivered by nucleofection
is non-perturbing to iPSC morphology and can be used to introduce small changes to the genome. A) Phase images taken 48 hours
post-nucleofection. Scale bars are 100 μm. B-C) Analysis of the percent of total NGS sequencing reads with the specified desired edit(blue) or undesired INDELs (grey) for the LMNA locus (B) and MYH7 locus (C).

Discussion
Here we have demonstrated the ability to introduce specificmutated genotype (8% x 20% = 1.6%). Clonal iPSC lines candisease-associated genetic variants with Edit-R CRISPR

be generated through a number of techniques, including

Cas9 gene editing reagents in human iPSCs. Because iPSCs

low-density seeding of single-cell suspension iPSCs anddemonstrate low levels of HDR-mediated repair, the overallmanual clonal isolation, which does not require any special

incidence of alleles containing the disease-associatedequipment (Haupt 2018). Individual clones can then bemutation is low, and the generation of clonal cell lines isassessed for genotype through PCR amplification of therecommended to recover those with the desired genotype.

targeted loci and Sanger sequencing, high-throughput

When continuing this workflow, the researcher must consider
the prevalence of the desired mutation and the unedited
allele when determining the number of clones necessary
to screen in order to recover the desired genotype. In thisexample, where 8% of alleles have the desired mutationand 20% of alleles are wild type, an expected 1.6% ofthe population is predicted to have the mono-allelicallywww.revvity.com

droplet digital PCR techniques (Miyaoka 2018), or in somecases enzymatic evaluation through restriction fragment
length polymorphism (RFLP) analysis. This application notedemonstrates the utility of the Edit-R Cas9 Nuclease mRNA,synthetic sgRNA, and ssDNA HDR repair templates as aconvenient source of CRISPR reagents for effective genome
editing in human iPSCs.

3

Knock-in a SNP alteration in iPSCs using Edit-R reagents.

Materials and methods
Cell culture. A Human Episomal iPSC Line found to bekaryotypically normal was cultured on non-tissue culture
treated plastics (recommended for use with Vitronectincoating) coated with truncated Vitronectin (VTN-N)
Recombinant Human Protein according to manufacturer’s
recommendations. Cells were cultured in antibiotic-freemTeSR™ Plus media (Cat # 05825 StemCell Technologies).
Cultures were routinely maintained by clump passaging
two times a week with Gibco™ Versene Solution per
manufacturer’s instructions. Cells were incubated at 37 °C
and 5% CO2.
Electroporation. Prior to electroporation, cells were treatedwith 10 μM ROCK inhibitor molecule Y-27632 (Cat #72302,StemCell Technologies) for 1-4 hours to promote viability
post-electroporation. Cells were passaged into a single
cell suspension using StemPro Accutase Cell Dissociation
Reagent and resuspended in mTeSR™ Plus media
supplemented with 10 μM ROCK inhibitor molecule Y-27632.
Cell line-specific (see Cell Culture section for specificlineage) electroporation conditions, previously optimized
with pMax-GFP expression plasmid, were as follows: Lonza™
P3 Primary Cell 4D-Nucleofector™Kit (Cat # V4SP-3096)
was used to transfect 3.4 × 105 cells per reaction in anAmaxa™ 96-well Shuttle™ Device (Cat # AAM-1001S) using
setting CM-113. Each reaction contained 1 μg Edit-R EGFP
Cas9 Nuclease mRNA (Cat #CAS11860) and 2 μM custom
Edit-R synthetic sgRNA, and 0.3 μM ssDNA repair template in
20 μL reactions.
The Custom guide RNA targeting sequences:
LMNA locus• Targeting sequence 1: GCGCCGTCATGAGACCCGAC

The Custom ssDNA repair template sequences:
LMNA locus• Donor: AAACTCACGCTGCTTCCCATTGTCAATCTCGACGAGTCATGTCTCATGACGGC
GCTTGGTCTCACGCAG
MYH7 locus• Donor: AGGGACCACTCACGAAGCGGGAGGAGTTGTTGTTCCGGACTGTCTTTGCATT
GCCAAAGGCCTCCAGAGCAGGGTTG
After transfection was performed, 80 μL mTeSR™1 Without
Phenol Red media (Cat # 05876 StemCell Technologies)
supplemented with 10 μM ROCK inhibitor molecule Y-27632
was added to each reaction well, and 25 μL of eachreaction was plated into a prepared Vitronectin-coated96-well plate containing 0.2 mL pre-equilibrated mTeSR™1Without Phenol Red media supplemented with 10 μM ROCK
inhibitor molecule Y-27632. Cells were incubated at 37 °C
and 5% CO2. After 6-20 hours, cells were imaged for GFP
fluorescence from the Cas9-GFP mRNA as an indication oftransfection efficiency. Twenty-four hours post-transfection,
media was changed to mTeSR™ Plus, and cells were fed daily
until the time of harvest.
NGS analysis. On the day of harvest, replicate wells werewashed 1X in DPBS (Cat #SH30028, Fisher Scientific) andlysed in 0.1 mL/well Thermo Scientific 1× Phusion HF Bufferwith 5 μL each of Proteinase K and RNase A, and incubatedfor 1 h at 56 °C then heat-inactivated at 98 °C for 10 min.
Fifty microliter PCR reactions were carried out in 1× Phusion
HF buffer, using 0.5 μL Phusion Hot Start II DNA Polymerase,
200 μM each dNTP, 0.5 μM forward and reverse primers,
and 5 μL direct cell lysis template. Touchdown PCR cycling
was run for each sample: Denature/enzyme activation• Targeting sequence 2: CCGTCATGAGACCCGACTGG

at 98 °C for 3 min followed by 10 cycles of 98 °C for 10• Targeting sequence 3: ATTGTCAATCTCCACCAGTC

25 cycles of 98 °C for 3 min followed by 10 cycles of 98

MYH7 locuss, 72 °C for 15 s –1 °C/cycle, and 72 °C for 30 s, then°C for 10 s, 62 °C for 15 s, and 72 °C for 30 s, and a final
extension at 72 °C for 10 min. Each PCR product was gel• Targeting sequence 1: TTGGCAATGCCAAGACCGTC

purified on a 2% agarose gel and extracted with GeneJet• Targeting sequence 2: GTTGTCGTTCCGGACGGTCT

were eluted in cell culture grade water and normalized

Gel Extraction Kit per the manufacturer’s protocol. Extracts
to 20 ng/μL after quantification by Qubit™ Fluorometric
Quantification and Qubit™ dsDNA HS Assay Kit andsubmitted to Genewiz for Amplicon EZ Sequencing. Usinga custom Python script, paired-end reads were merged.

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4Knock-in a SNP alteration in iPSCs using Edit-R reagents.

Reads containing mismatches in the overlapping region

6.

N. Malik and M. Rao. A Review of the Methods forwere filtered out, while the higher Phred score for

Human iPSC Derivation. Methods in Molecular Biology.

each overlapping base was kept for matches. The non-997, 23-33 (2013).

overlapping portions of each sequence were trimmed off,and any read containing a base with a Phred score < 307.

Cardiomyopathy Mutations Significantly Increase thewere also filtered out. The remaining reads were aligned

Velocity, Force, and Actin-Activated ATPase Activity ofusing Bowtie2 (Langmead et al., 2012), and a pileup of

Human b-Cardiac Myosin. Cell Reports. 17,

reads was generated using SAMtools (Li et al., 2009).

2857-2864, (2016).

Custom Python scripts were used to summarize the editing
percentages and to generate the corresponding plots.

A. Adhikari et al. Early-Onset Hypertrophic

8.

B. C. Capell and F. S. Collins. Human laminopathies:
nuclei gone genetically awry. Nature reviews.
7:940-952 (2006).

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M. Jinek, et al. A Programmable Dual-RNA-Guided DNA
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2.

P. Mali et al. RNA-guided human genome engineering via
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3.

L. Cong, et al. Multiplex Genome Engineering UsingCRISPR/Cas Systems. Science. 339(6121), (2013).

4.

S. Waddington, et al. A Broad Overview and Review of
CRISPR-Cas Technology and Stem Cells. Current StemCell Reports. 2, 9-20 (2016).

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R. Jehuda et al. Genome Editing in Induced Pluripotent
Stem Cells using CRISPR/Cas9. Stem Cell Reviews andReports. 14, 323-336 (2018).

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J. Rankin and S. Ellard. The laminopathies: a clinicalreview. Clinical genetics. 70:261-274 (2006).

10. B. Langmead and S.L. Salzberg. Fast gapped-readalignment with Bowtie 2. Nature methods. 9,357-359, (2012).
11. A. Haupt et al. Endogenous Protein Tagging in Human
Induced Pluripotent Stem Cells Using CRISPR/Cas9.
Journal of Visualized Experiments. 138: 58130 (2018).
12. Y. Miyaoka et al. Detection and Quantification of HDR
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13. H. Li, et al. The Sequence Alignment/Map format andSAMtools. Bioinformatics. 25, 2078-2079, (2009).

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