CRISPR Gene Editing in Stem Cells

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DMSO (Sigma). The committee on Human Research at the University
of California, San Francisco approved the iPSC research protocol (#10-
02521).
Generation of Stable CRISPRi and CRISPRn iPSC Lines
iPSCs were singularized with accutase, resuspended in PBS, and counted
with a Countess automated cell counter (Life Technologies). For plasmid
transfections, the human stem cell nucleofector kit 1 solution was used on
the Amaxa nucleofector 2b device (program A-23; Lonza). To generate the
CRISPRi and CRISPRn iPSC lines, two million WTC or WTB iPSCs were
nucleofected with the appropriate knockin vector (5 mg) and each AAVS1
TALEN pair (2 mg). Cells were then seeded in six-well plates in serial dilu-
tions in mTeSR supplemented with Y-27632 (10 mM). Selection was applied
3 days post-nucleofection with the appropriate antibiotic in mTeSR plus
Y-27632 (10 mM). To knock in the CRISPRi construct (carrying the Neomycin
resistance gene cassette), Geneticin (Life Technologies) was applied at
100 m g/ml. To knock in the CRISPRn and GCaMP constructs (carrying the
Puromycin resistance gene cassette), 0.5 mg/ml Puromycin (Life Technolo-
gies) was added. Selection was maintained for 10 days until stable colonies
appeared. Colonies with a diameter of greater than 500 mm were manually
picked using a P200 pipette tip under an EVOS FL picking microscope (Life
Technologies) and transferred to individual wells of a 24-well plate containing
mTeSR medium supplemented with Y-27632 (10 mM). Clones were then
expanded into larger vessel formats.
Generation of CEM CRISPRi Cell Line
CEM CRISPRi cells were generated by electroporation of 0.5 mg of each
AAVS1 TALEN pair and 1 mg of the Gen1 CRISPRi vector with an Amaxa nucle-
ofector 2b device and Amaxa cell line nucleofector kit C (Lonza). Cells were
selected in 1 mg/ml G418, and clonal lines were generated by dilution in
96-well plates. Clonal populations were selected based on doxycycline induc-
tion of mCherry expression. Oligos encoding the CD4 protospacer were an-
nealed and cloned into the pSLQ1371 vector using restriction sites BstXI
and BlpI, and lentivirus was produced in HEK293T cells (Gilbert et al., 2014).
To compare performance of CD4 gRNAs, each was transduced into CEM-
CRISPRi cells. Transduced populations were incubated for 96 hr with doxycy-
cline (2 mM). Knockdown efficiency was calculated by gating all mCherry-ex-
pressing cells, and comparing cell-surface CD4 expression in the presence
or absence of gRNA-expressing cells (BFP+). Three independent stable CEM
CRISPRi clones were selected with 0.6 mg/ml Puromycin and incubated in
the presence or absence of doxycycline (2 mM) for 14 days to assess maximal
CD4 knockdown. Cells were stained using anti-CD4 APC-conjugated antibody
and cell surface CD4 staining was quantified using a BD LSRII flow cytometer.
CD4 knockdown was quantified as percent reduction relative to no doxycy-
cline treatment condition.
gRNA Design and Cloning into the gRNA-Expression Vector
For CRISPRi, three to five gRNAs were designed to target near the TSS of
the gene of interest (250 bp upstream and downstream, respectively). The
location of the TSS was determined using NCBI
gRNA oligos were designed, phosphorylated, annealed, and cloned
into the pgRNA-CKB vector using BsmBI ligation strategy. Additional details
and a list of gRNA sequences are listed in supplemental experimental
procedures.
gRNA Nucleofection and Selection of Stable CRISPRi and CRISPRn
Clones
The gRNA-expression vector (pgRNA-CKB) was transfected into either the
CRISPRi or CRISPRn cells with the human stem cell nucleofector kit 1 solution
on the Amaxa nucleofector 2b device (program A-23; Lonza). Two million
CRISPRi or CRISPRn iPSCs and 5 mg of the circular gRNA-expression plasmid
were used per nucleofection. Nucleofected cells were then seeded in a single
well of a six-well plate in mTeSR supplemented with Y-27632 (10 mM). Blasti-
cidin selection (10 mg/ml) was applied 24 hr post-nucleofection in mTeSR
supplemented with Y-27632 (10 mM) for 7–10 days, until stable colonies
appeared. Stable colonies were then pooled and passaged at least three times
in mTeSR plus Blasticidin and Y-27632 to enrich for cells with integration at
transcriptionally active sites (Figure S3).
RNA Sequencing
For each sample, 1 mg of total RNA was prepared using TRIzol as previously
described. Strand-specific mRNaseq libraries were prepared using TruSeq
Stranded mRNA Library Prep Kit (Illumina). Upon completion, libraries were
quantified and pooled using Qubit dsDNA HS assay and Agilent’s Bioanalyzer
high-sensitivity DNA assay. The indexed libraries were pooled and sequenced
on Illumina HiSeq 4000 as 50-bp single-end reads. Reads were aligned to the
hg19 genome assembly using the Ensembl 75 reference transcriptome
customized to include the GCaMP6f constructs using TopHat2 (Kim et al.,
2013a). Unaligned reads were subsequently aligned to the CRISPRi or
CRISPRn knockin constructs where appropriate. Transcript alignments were
then counted using SubRead v1.4.6 and analyzed with custom scripts written
in Python (Liao et al., 2013). All data are displayed as reads per million (RPM)
with a pseudocount of 0.075.
iPS-CM Differentiation and Lactate Purification
iPSCs were differentiated into iPS-CMs using the WNT modulation-differenti-
ation method (Lian et al., 2012) (Figure S5A). iPS-CMs were purified via a modi-
fied version of the lactate metabolic-selection method (Tohyama et al., 2013).
Additional details are outlined in Supplemental Experimental Procedures.
ACCESSION NUMBERS
The accession number for the RNA-seq data reported in this paper is GEO:
PRJNA307261.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
six figures, and one movie and can be found with this article online at
.
AUTHOR CONTRIBUTIONS
M.A.M. and B.R.C. were primarily responsible for conception, design, and
interpretation of the experiments. M.A.M. conducted most experiments with
help from N.H., E.F., E.S., A.T., M.P.O., T.V.E., K.H., and L.M.J. Y.M. and
A.H.C. generated the CRISPRn Gen1C iPSC line. C.I.S. performed electro-
physiology experiments. D.E.G. generated the CEM CRISPRi cell line and pro-
vided knockdown analysis. L.A.G., J.S.W., and L.S.Q. provided technical
expertise, the CRISPRi fusion cassette, and gRNA expression constructs.
J.E.V. and M.A.H. conducted and analyzed the RNA-seq experiments.
M.A.M., P.L.S., and B.R.C. wrote the manuscript with support from all authors.
ACKNOWLEDGMENTS
We thank members of the Conklin laboratory, Gladstone Institute of Cardio-
vascular Disease, Roddenberry Stem Cell Core, BioFulcrum, a Gladstone
Institutes Enterprise, and Innovative Genomics Institute for technical assis-
tance and helpful comments on the manuscript. We thank Tim Rand and
Knut Woltjen for valuable discussions and helpful comments on the manu-
script. We thank S. John Liu for RNA-seq analysis advice. We thank Jen Ber-
man and Samantha Cooper at Bio-Rad for assistance with designing ddPCR
probe-primer sets. Summer students Matthew Keller and Monique Morrison
assisted with preliminary experiments. CEM CD4+ cells were obtained from
Dr. J.P. Jacobs through the AIDS Reagent Program, Division of AIDS, NIAID,
NIH. M.A.M. is supported by the Canadian Institutes of Health Research post-
doctoral fellowship 129844. N.H. was supported by the CIRM training program
TG2-01160 and T32 HL007544. E.F. was supported by a Bridges to Stem
Cell Training grant TB1-01188 from CIRM. L.M.J. is supported by the
CIRM Training Grant TG2-01160 and NICHD Career Development Award
1K12HD072222. Y.M. received fellowships from the Uehara Memorial Foun-
dation Research and Gladstone-CIRM. D.G. is supported by UCSF-Gladstone
Center for AIDS Research (CFAR), an NIH-funded program (P30 AI027763).
T.V.E. was supported by Carlsberg Travel Grant (2013-01-0423), The Lund-
beck Foundation (R140-2013-13348), and OUH Internationalisation Founda-
tion. J.E.V., M.A.H., L.A.G., and J.S.W. were supported by the Howard Hughes
Cell Stem Cell 18, 541–553, April 7, 2016 ª2016 Elsevier Inc. 551