CRISPR/Cas9-Directed Genome Editing UNIT 31.

1
of Cultured Cells
Luhan Yang,1,3 Joyce L. Yang,1,2,3 Susan Byrne,1,3 Joshua Pan,2
and George M. Church1
1
Department of Genetics, Harvard Medical School, Boston, Massachusetts
2
Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts
3
These authors contributed equally to this work.

ABSTRACT
Human genome engineering has been transformed by the introduction of the CRISPR
(clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated)
system found in most bacteria and archaea. Type II CRISPR/Cas systems have been
engineered to induce RNA-guided genome editing in human cells, where small RNAs
function together with Cas9 nucleases for sequence-specific cleavage of target sequences.
Here we describe the protocol for Cas9-mediated human genome engineering, including
construct building and transfection methods necessary for delivering Cas9 and guide
RNA (gRNA) into human-induced pluripotent stem cells (hiPSCs) and HEK293 cells.
Following genome editing, we also describe methods to assess genome editing effi-
ciency using next-generation sequencing and isolate monoclonal hiPSCs with the desired
modifications for downstream applications. Curr. Protoc. Mol. Biol. 107:31.1.1-31.1.17.
C 2014 by John Wiley & Sons, Inc.

Keywords: genome engineering r CRISPR r human stem cells

INTRODUCTION
Targeted human genome editing enables functional studies of genetic variation in biol-
ogy and disease, and holds tremendous potential for clinical applications. To facilitate
genome engineering, technologies such as Zinc-Finger Nucleases (ZFNs) and Transcrip-
tion Activator-Like Effector Nucleases (TALENs) have been developed to enable targeted
and programmable modification of endogenous genomic sequences (Miller et al., 2007;
Hockemeyer et al., 2011). However, the need to design new complex nucleases for each
target site limits the utility of these methods, particularly in multiplexed gene targeting
applications.

Recently, the type II bacterial CRISPR (clustered regularly interspaced short palindromic
repeats)/Cas (CRISPR-associated) system has been developed as an efficient and versatile
technology for genome editing in eukaryotic cells and whole organisms (Jinek et al., 2012,
2013; Cong et al., 2013; DiCarlo et al., 2013; Friedland et al., 2013; Gratz et al., 2013;
Hwang et al., 2013; Mali et al., 2013a; Wang et al., 2013). The CRISPR/Cas system was
first identified in bacteria and archaea as an RNA-mediated adaptive defense system that
safeguards organisms from invading viruses and plasmids (Ishino et al., 1987; Horvath
and Barrangou, 2010; Wiedenheft et al., 2012). The hallmark of the CRISPR/Cas system
consists of CRISPR arrays composed of spacers interspersed with direct repeats and cas
genes present in the operons (Bhaya et al., 2011; Terns and Terns, 2011). In CRISPR/Cas-
mediated immunity, bacteria and archaea react to viral or plasmid attack in the adaptive
phase by first integrating short fragments of foreign nucleic acid (protospacers) into the
host chromosome at the proximal end of the CRISPR array. In the expression phase,
CRISPR loci are transcribed into precursor CRISPR RNA (pre-crRNA) and further

Genome Editing

Current Protocols in Molecular Biology 31.1.1-31.1.17, July 2014 31.1.1

Published online July 2014 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/0471142727.mb3101s107 Supplement 107
Copyright C 2014 John Wiley & Sons, Inc.
 Experimental workflow
Basic Protocol 1 Basic Protocol 2 Basic Protocol 3 Basic Protocol 4

Prepare Nucleofect Assess genome Isolate genome-

Cas9 and gRNA HiPSCs editing efficiency edited cells
plasmids

U6 gRNA
+
CMV Cas

4 days 3 days 2 days 12 days

Experimental timeline
Total: 3 weeks

Figure 31.1.1 Workflow and timeline for hiPSC genome engineering. In the complete workflow,
the Cas9 and gRNA plasmid constructs are built and subsequently transfected into cells. Genome
editing efficiency is assessed using deep sequencing. Finally, monoclonal hiPSC colonies with
desired genotype can be isolated using cell sorting. The entire workflow takes 3 weeks to perform.

processed into a library of short CRISPR RNAs (crRNAs) that can recognize and pair
with complementary sequences from invading viral or plasmid targets (Carte et al., 2008;
Haurwitz et al., 2010; Deltcheva et al., 2011; Gesner et al., 2011; Sashital et al., 2011;
Wang et al., 2011). In the final interference phase, crRNAs are packaged with trans-
activating crRNA (tracrRNA) and Cas proteins to form ribonucleoprotein complexes that
together detect and destroy foreign sequences (Brouns et al., 2008; Hale et al., 2008; Jore
et al., 2011; Lintner et al., 2011; Wiedenheft et al., 2011a,b).

It has been recently demonstrated that the type II CRISPR system from Streptococcus
pyrogenes can be engineered to induce Cas9-mediated double-stranded breaks (DSBs)
in a sequence-specific manner in vitro by providing a synthetic guide RNA (gRNA)
composed of crRNA fused to tracrRNA (Jinek et al., 2012). Moreover, the system has
been successfully adapted to function in human cells with the use of human codon-
optimized Cas9 and customizable 20-nt gRNAs (Cho et al., 2013; Cong et al., 2013;
Jinek et al., 2013; Mali et al., 2013a). Once the gRNA identifies its 20-bp target followed
by a PAM (protospacer-adjacent motif) sequence–NGG, Cas9 nuclease then cleaves
the target sequence, creating a DSB (Jinek et al., 2012). The resulting DSB will either
generate nonspecific mutations knocking out a gene through the error-prone NHEJ (non-
homologous end joining) pathway, or produce specific modifications dictated by an
exogenous repair template through the HDR (homology-directed repair) pathway (Saleh-
Gohari and Helleday, 2004; Urnov et al., 2010; Chen et al., 2011). This system greatly
enhances the ease of genome engineering through the creation of desired DSBs targeted
by RNA sequences that are easy to design, synthesize, and deliver, holding great promise
for multiplexed genome editing.

With the advent of human induced pluripotent stem cells (hiPSCs) that can be re-
programmed from fibroblasts to a human embryonic stem cell (hESC)–like state with
maintained pluripotency, self-renewal, and differentiation capacity, a better understand-
ing of human biology and potential clinical applications is now possible (Takahashi and
Yamanaka, 2006; Maherali et al., 2007; Takahashi et al., 2007; Wernig et al., 2007; Yu
et al., 2007; Park et al., 2008). hiPSC technology presents a promising tool for supplying
CRISPR/Cas9 various cell types for transplantation therapy, regenerative medicine, drug testing, and
Genome Editing
developmental biology experiments. The potential of hiPSCs can be further enhanced
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by genome engineering, which may be used to study human gene function, track cells
or endogenous proteins with a knock-in reporter, and correct genetic defects for gene
therapy.

To harness the full potential of hiPSC technology, this unit provides a streamlined method
for conducting genome editing in hiPSCs (Fig. 31.1.1). Basic Protocol 1 describes
the construction of Cas9 and gRNA plasmids, including the purification of the Cas9
plasmid from stab cultures obtained from Addgene, bioinformatic analysis to determine
an appropriate target sequence, and construction of gRNA plasmid from IDT gBlocks.
Basic Protocol 2 describes the transfection of hiPSCs, while the Alternate Protocol
outlines the same process for HEK293 cells. Basic Protocol 3 describes the assessment
of genome editing efficiency in successfully transfected cells. Finally, Basic Protocol 4
describes a method to isolate monoclonal hiPSC colonies with desired genotype.

PREPARATION OF Cas9 AND gRNA PLASMIDS BASIC

PROTOCOL 1
Plasmids containing Cas9 and the guide RNA are necessary for Cas9-mediated genome
editing. This basic protocol outlines the steps necessary to prepare both plasmids for
transfection.
Materials
Cas9 plasmid (Addgene, plasmid ID 41815) as bacterial stab in agar
LB agar plate containing 100 μg/ml ampicillin (UNIT 1.1)
LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1)
HiSpeed Plasmid Maxi Kit (Qiagen)
PCR-grade sterile deionized water
PCR-Blunt II-Topo kit (Invitrogen, cat. no. K2800-20) including One Shot Top10
Chemically Competent E. coli cells (other competent cells for cloning may also
be used)
Sterilized glass beads (EMD Millipore, cat. no. 71013-3)
LB agar plate containing 50 μg/ml kanamycin (UNIT 1.1)
M13 Forward (5 -GTTTTCCCAGTCACGACG-3 ) and M13 Reverse
(5 -AACAGCTATGACCATG-3 ) universal sequencing primers
LB liquid medium containing 50 μg/ml kanamycin (UNIT 1.1)
Qiagen plasmid Mini Kit (Qiagen)

Sterile pipet tips or toothpicks for picking colonies from agar plates
37°C incubator-shaker
Nanodrop microspectrophotometer (http://www.nanodrop.com)
Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT,
LaserGene)
DNA synthesis facility
42°C incubator for heat-shocking cells
10-ml bacterial culture tubes
Access to Sanger sequencing facility

Additional reagents and equipment for DNA synthesis (UNIT 2.11) and Sanger
sequencing (UNIT 7.1)
Prepare Cas9 plasmid
1. Obtain plasmid from Addgene.
2. Use a sterile pipet tip or toothpick to scrape the bacterial stock from the Addgene
bacterial stab, and streak it onto an LB agar plate containing 100 μg/ml ampicillin.
Incubate plate at 37°C for 10 hr or overnight. Genome Editing

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 3. Once colonies are formed, pick a single colony from the plate to inoculate 200 ml of
LB liquid medium containing 100 μg/ml ampicillin. Grow overnight at 37°C with
shaking at 200 rpm.
4. Isolate plasmid DNA using a plasmid Maxiprep kit. Use Nanodrop microspectropho-
tometer to measure DNA concentration. Resuspend DNA at 1 μg/μl in water. Use
this product for transfection.
Identify appropriate gRNA targeting sequence
5. Using sequence analysis software, identify all 22-bp regions within 50 bp of the
intended genomic target in the form of 5 -N19-NGG-3 .
These 22-bp regions may be located on either strand and should ideally overlap the target
sequence.
The selected target sequence must follow the standard sequence structure of 5 -G-N19-
NGG-3 . The 5 G is necessary for the U6 promoter used on the gRNA plasmid, while the
3’ NGG is the protospacer adjacent motif (PAM) that is necessary for Cas9 recognition.
It must also be unique to the genomic target site and have minimal alternate targets on
the genome.

6. For each candidate sequence, query for alternate binding sites in the reference
genome. Because of the higher tolerance of mismatches in the first 7 bp of the target
sequence, search the reference genome for the last 13 bp of the target sequence with
the NGG protospacer adjacent motif (S13 NGG). Use NCBI BLASTN or other online
software to choose the one with minimal off-target sites at region of interest. Finalize
the design of the customized gRNA expression fragment (455 bp) by including the
selected target sequence (N19) in the gRNA expression fragment below.
This final sequence will contain everything necessary for gRNA expression, including
the U6 promoter, customized target sequence, gRNA scaffold, and termination signal, as
annotated in Figure 31.1.2.

Create gRNA plasmid construct from IDT gBlock

7. Synthesize the final gRNA expression fragment (455 bp) as a standard gBlock
without any 5 modifications from gene synthesis companies.

incorporate 19 bp of the selected target sequence (red N )

into the DNA fragment as indicated below:

U6 promoter N19 gRA backbone stop codon

Figure 31.1.2 Overview of customized gRNA expression fragment. The chosen selected tar-
get sequence is inserted in the red region of the construct above. Of note, G in green
CRISPR/Cas9 indicates the start of the U6-driven transcript. For the color version of this figure, go to
Genome Editing http://www.currentprotocols.com/protocol/mb3101.
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 8. Resuspend the gBlock (delivered at 200 ng) in 20 μl of water for a final concentration
of 10 ng/μl.
9. Pipet 1 μl gBlock, 1 μl pCRII-Blunt-TOPO vector, and 4 μl salt solution (from
PCR-Blunt II-Topo kit) in a 1.5-ml microcentrifuge tube, mixing gently. Incubate at
room temperature for at least 5 min.
10. To transform 5 μl of product into Top10 Chemically Competent E. coli cells, thaw
one aliquot of Top10 cells in ice for 10 min, add 5 μl of the TOPO cloning reaction
from the previous step, and incubate on ice for 30 min. Heat-shock the cells at 42°C,
then return to ice for 2 min. Add 250 μl of room temperature SOC medium (from
PCR-Blunt II-Topo kit) and incubate at 37°C with shaking for 1 hr.
11. Spread 100 μl of the transformation mixture using sterilized glass beads onto a
prewarmed LB agar plate containing 50 μg/ml kanamycin by gently swirling the
plate or, alternatively, using an inoculating loop for spreading. Incubate overnight at
37°C.
Expect 10 to 100 colonies, with the majority containing the desired insert.

12. After incubation, pick 5 colonies for Sanger sequencing (UNIT 7.7) using the M13
Forward and M13 Reverse universal sequencing primers.
13. After identifying the colonies with the correct sequence, grow a maxiprep culture of
the correct transformant by inoculating 200 ml of LB medium containing 50 μg/ml
kanamycin with 100 μl of the original culture (step 11). Grow overnight at 37°C
with shaking at 200 rpm.
14. Isolate plasmid DNA using a plasmid maxiprep kit. Resuspend plasmid DNA at 1
μg/ml in water. Use this product for transfection (see Basic Protocol 2 and Alternate
Protocol).

TRANSFECTION OF hiPSCs BASIC

PROTOCOL 2
Genome editing in hiPSCs holds great potential for gene therapy as well as the functional
study of genetic variation when hiPSCs differentiate into relevant cell types. While
the Cas9 and gRNA plasmids are being prepared, initiate hiPSC culture to prepare for
transfection. The proper procedure for genome editing on tissue-cultured hiPSCs is
described in this protocol.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2

incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile,
and aseptic technique should be used accordingly.
Materials
PGP1 hiPSC cells adapted for growth on Matrigel (see personal genome project
Web site: http://www.personalgenomes.org/)
Matrigel (hESC-qualified; BD Sciences, cat. no. 354277)
DMEM/F12 medium (Invitrogen)
mTeSR1 medium (StemCell Technologies, cat. no. 05850)
InSolution Rho kinase (ROCK) inhibitor (Calbiochem, cat. no. Y-27632)
P3 Primary Cell 4D-Nucleofector X kit containing P3 and Supplement 1 solutions
in addition to 16-well Nucleocuvette Strips (Lonza, cat. no. V4XP-4032)
Cas9 plasmid DNA (see Basic Protocol 1)
gRNAexpression vector (see Basic Protocol 1)
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 Phosphate-buffered saline (PBS; Life Technologies, cat. no. 20012-050)
TrypLE Express (Invitrogen, cat. no. 12604-013)

6- and 48-well tissue culture–treated plates

15- and 50-ml conical centrifuge tubes (e.g., BD Falcon)
Countess automated cell counter (Invitrogen)
Tabletop centrifuge and plate adapter
Amaxa 4D-Nucleofector System (Lonza, cat. no. CD-MN025)

Additional reagents and equipment for culture of hiPSC in mTeSR medium (see
Technical Manual Version 3.0.0 from Stem Cell Technologies;
http://www.stemcell.com/˜/media/Technical%20Resources/B/C/A/2/B/29106MAN
_3_0_0.pdf)
Prepare for transfection
1. Culture PGP1 hiPSCs using standard protocol for hiPSC in mTeSR1 medium (in
6-well Matrigel-coated plates, until the cells are 40% confluent.

To coat plates with Matrigel, do the following:

a. Thaw a vial of 300 μl Matrigel on ice.
b. Transfer 24 ml cold DMEM/F12 into a 50-ml conical polypropylene tube.
c. Transfer 300 μl Matrigel into the tube. Invert to mix.
d. Add 1 ml of this mixture per well of a 6-well plate, then leave the plate at
room temperature for 1 hr.
e. Aspirate Matrigel and replace with 2 ml cells/medium.
2. At a time point 2 hr before electroporation, replace the medium of the hiPSCs with
2 ml prewarmed mTeSR1 medium containing 2 μl/ml ROCK inhibitor.
3. At a time point 1 hr before electroporation, prepare destination wells for transfected
cells:
a. Thaw a vial of 300 μl Matrigel on ice
b. Transfer 24 ml cold DMEM/F12 into a 50-ml conical polypropylene tube.
c. Transfer 300 μl Matrigel into the tube. Invert to mix.
d. Add 500 μl of this mixture per well of 48-well plate (one well will be needed per
transfection), then leave the plate at room temperature for 1 hr.
e. Aspirate Matrigel and replace with prewarmed 500 μl mTeSR1 medium with
2 μl/ml ROCK inhibitor.
The small surface area of the wells of 48-well plates promotes high cell density and
healthy growth after transfection.

4. Prepare a transfection master mix (scale appropriately):

16.4 μl P3 and 3.6 μl Supplement 1 from Nucleofactor X kit
1 μl 1 μg/μl Cas9 plasmid
1 μl 1 μg/μl gRNA plasmid
22 μl per reaction, total.
Transfect hiPSCs
5. Aspirate the ROCK inhibitor–containing medium from the wells containing hiPSCs
and wash each well with 2 ml room temperature PBS.
6. Aspirate PBS, add 1 ml TrypLE Express, and incubate the plate at 37°C for 5 min.
7. Resuspend cells with 3 ml mTeSR1 medium and gently pipet up and down several
times to generate a single-cell suspension. Transfer disassociated cells into a 15-ml
CRISPR/Cas9
Genome Editing centrifuge tube containing 10 ml mTeSR1 medium.

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 8. Count cells with cell counter and calculate total volume required for 1 ×
106 cells/transfection, scaling as needed.
Given the toxicity of transfection, the minimum number of cells per transfection required
to isolate transfectants is 200,000. However, higher cell counts decrease the efficiency of
transfection by increasing the number of targets. A titration of cell counts ranging from
200,000 to 1 × 106 may help find the optimal balance.

9. Place desired quantity of cells (in this case 1 × 106 ) in 15-ml centrifuge tube,
centrifuge at 200 × g for 5 min at room temperature, and aspirate supernatant.
10. Resuspend each unit of 1 × 106 cells in 22 μl of the transfection master mix prepared
in step 4.
11. Quickly transfer cells into the central chamber of one well of a Nucleocuvette strip.
Place the strip into 4-D Nucleofector device.
12. Nucleofect cells using program CB150.
13. Quickly add 80 μl of prewarmed mTESR1 medium containing 2 μl/ml ROCK
inhibitor to each well of electroporated cells. Pipet up and down once or twice to
mix.
14. Transfer cells from the strip to wells of the Matrigel-coated plate containing mTeSR1
medium with 2 μl/ml ROCK inhibitor prepared in step 3.
15. Centrifuge the plate 3 min at 70 × g, room temperature. Place cells into 37°C
incubator.
16. After 24 hr, change to fresh mTeSR1 medium without ROCK inhibitor.
17. Harvest cells 3 days after electroporation. Follow protocol for assessing targeting
efficiency in Basic Protocol 3.

TRANSFECTION OF HUMAN HEK293 CELLS ALTERNATE

PROTOCOL
Genome editing in HEK293 cells is efficient and convenient, thus serving as an ideal
system to test and optimize reagent before moving to hiPSCs. Here, we describe the
transfection procedure on HEK293 cells with the Cas9/gRNA plasmids.

NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO2

incubator unless otherwise specified.

NOTE: All reagents and equipment coming into contact with live cells must be sterile,
and aseptic technique should be used accordingly.
Additional Materials (also see Basic Protocol 2)
HEK 293 cells (Invitrogen)
Complete DMEM medium (see recipe)
Lipofectamine 20000 (Invitrogen, cat. no. 11668027)
Opti-MEM medium (Invitrogen, cat. no. 31985062)
Cas9 plasmid DNA (see Basic Protocol 1)
gRNAexpression vector (see Basic Protocol 1)
12-well tissue culture treated plates
Plate 293 cells for transfection
1. Culture HEK 293 cells in complete DMEM medium in 6-well plates until the cells
are 70% confluent.
2. Aspirate medium and wash cells with 2 ml room temperature PBS. Genome Editing

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 3. Aspirate PBS, add 1 ml TrypLE Express, and incubate at 37°C for 2 min.
4. Resuspend cells with 5 ml prewarmed complete DMEM medium
5. Count cells using an automated cell counter and calculate volume required for
200,000 cells per transfection.
6. Place desired volume of cells into 15-ml centrifuge tube. Centrifuge 5 min at 200 ×
g, room temperature, and aspirate supernatant.
7. Resuspend cell pellet in 1 ml complete DMEM medium.
8. Plate cells in a 12-well tissue culture plate and return to incubator.
Transfect 293 cells
9. After a day of incubation, replace medium on cells with 1 ml fresh prewarmed
complete DMEM medium. Return to incubator and allow to incubate while preparing
DNA mix.
10. Add 5 μl Lipofectamine 2000 to 50 μl Opti-MEM in a 1.5-ml microcentrifuge tube.
Invert several times to mix. Incubate the mixture at room temperature for 5 min.
11. Add 1 μg Cas9 plasmid and 1 μg gRNA to 50 μlOpti-MEM in a 1.5-ml microcen-
trifuge tube.
12. Add diluted DNA from step 11 to diluted Lipofectamine mixture from step 10,
flicking the tube several times to mix.
13. Incubate the mixture 15 min at room temperature.
14. Add 100 μl of the mixture dropwise to the cells.
15. Replace medium after 24 hr with fresh prewarmed complete DMEM medium.
High concentrations of Lipofectamine can be toxic. Monitor cell conditions. If high cell
toxicity is observed, change to fresh DMEM medium after 8 hr.

16. Harvest cells 3 days after transfection.

BASIC GENOTYPING TRANSFECTED CELLS USING NEXT-GENERATION

PROTOCOL 3 SEQUENCING
After transfection, the targeting efficiency needs to be assessed to determine whether
isolation of genome-targeted cells from a heterogeneous population is feasible. Normally,
targeting efficiency on the order of 1% is expected for hiPSCs using Cas9-gRNA system
without selection. This basic protocol describes the assessment of the targeting efficiency
using next-generation sequencing techniques that can yield high read depths on the
targeted site from a population of nucleofected cells.
Materials
Illumina forward sequence (ACACTCTTTCCCTACACGACGCTCTTCCGATCT)
Illumina reverse sequence
(GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT)
Transfected hiPSCs (Basic Protocol 1 or Alternate Protocol) growing in culture
Phosphate-buffered saline (PBS; Life Technologies, cat. no. 20012-050)
mTeSR1 medium (StemCell Technologies, cat. no. 05850)
prepGEM gold buffer (ZyGEM)
prepGEM tissue protease enzyme (ZyGEM)
CRISPR/Cas9 KAPA Hifi Hotstart Readymix (KAPA Biosystems)
Genome Editing Illumina amplification primers (see step 3)
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 Illumina index primers (ScriptSeq Index PCR Primers)
Illumina PCR primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCC-
CTACACGACGCTCTTCCGATCT)
2-log DNA ladder (New England Biolabs)
QIAquick PCR purification kit (Qiagen)

Computer running Primer3 software (http://primer3.sourceforge.net/) for primer

identification
15-ml conical tubes (BD Falcon)
Tabletop centrifuge
Thermal cycler
Access to MiSeq sequencer

Additional reagents and equipment for agarose gel electrophoresis (UNIT 20.5A) and
measuring DNA concentration (APPENDIX 3D)
Design Illumina amplification primers for the targeting region
1. Select a 500-bp region around the targeting site.
2. Use Primer3 to identify optimal targeting primer sets that amplify 200 to 300 bp
around the targeting site.
3. Finalize the design of and order the customized Illumina amplification primers:

a. Append the Illumina forward sequence (ACACTCTTTCCCTACAC-

GACGCTCTTCCGATCT) to the 5 end of the forward primer from step 2.
b. Append the Illumina reverse sequence (GTGACTGGAGTTCAGACGTGT-
GCTCTTCCGATCT) to the 5 end of the reverse primer from step 2.
The Illumina amplification scheme is summarized in Figure 31.1.3.

Harvest cells and create sequencing library

4. Aspirate the mTeSR1 medium from the cultured, transfected hiPSCs and wash the
cells gently with PBS.
5. Aspirate PBS, add 1 ml TrypLE Express, and incubate the plate at 37°C for 5 min.
6. Transfer disassociated cells into a 15-ml conical tube containing 10 ml mTeSR1
medium and centrifuge 5 min at 200 × g, room temperature.
7. Aspirate supernatant and resuspend the cell pellet with the residual medium left in
the conical tube.
8. Prepare a 10-μl cell lysis reaction with the following reagents in a PCR strip:
8.9 μl cell pellet suspension
1 μl prepGEM gold buffer (ZyGEM)
0.1 μl of prepGEM tissue protease enzyme (ZyGEM)
9. Incubate the reaction in a thermal cycler:
75°C for 15 min
95°C for 5 min.
10. Prepare a 20-μl PCR reaction to obtain the amplicon of the targeting region.
1 μl of the reaction from step 9
10 μl 2 × KAPA Hifi Hotstart Readymix
0.2 μl 100 mM each Illumina amplification primer (see step 3)
Water to 20 μl.
11. Perform PCR with the following parameters:
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 Illumina amplification forward primer Illumina amplification reverse primer

chromosome

round 1 PCR

PCR product I

Illumina PCR primer Illumina index primer

round 2 PCR

PCR product II

Illumina targeting region index Illumina

adaptor adaptor

Figure 31.1.3 Schematic of Illumina sequencing library preparation. The first round of PCR
amplifies the targeting region with the universal forward and reverse sequences necessary to
anneal to the Illumina proprietary primers. The second round of PCR adds an index primer
necessary for deconvoluting separate sequencing pools, as well as the adaptor necessary for
attachment to the sequencing flow cell.

1 cycle: 5 min 95°C (initial denaturation)

15 to 25 cycles: 20 sec 98°C (denaturation)
20 sec 65°C (annealing)
20 sec 72°C (extension).

If you do not get clear product bands, try more cycles to amplify desired product.

12. Prepare the second round of PCR reaction to add the Illumina sequence adaptor.
5 μl of the reaction from step 11
10 μl KAPA Hifi Hotstart Readymix
1 μl Illumina index primer
0.1 μl of 100mM Illumina PCR primer
Water to 20 μl.
There are 48 orthogonal Illumina index primers from the ScriptSeq Index PCR Primers
kit. Choose independent index primers for different reactions.

13. Perform the second round of PCR with the following parameters:
1 cycle: 5 min 95°C (initial denaturation)
15 to 25 cycles: 20 sec 98°C (denaturation)
20 sec 65°C (annealing)
20 sec 72°C (extension)
1 cycle: 4 min 72°C (final extension).

If you do not get clear product bands, try more cycles to amplify desired product.
CRISPR/Cas9 14. Run PCR product on a 2% agarose gel (UNIT 20.5A) against a 2-log DNA ladder and
Genome Editing
verify the correct amplicon length.
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 The Illumina sequencing adapter adds 160 bp to the genomic amplicon.

15. PCR purify the product with QIAquick PCR purification kit. Measure concentration
of each sample (APPENDIX 3D) and pool each sample at the same concentration to
ensure equal sequencing coverage. Submit for sequencing with MiSeq Personal
Sequencer.
16. After the sequencing results arrive, analyze the results using the bioinformatics
platform of choice.
The authors recommend the CRISPR genome analyzer: http://54.80.152.219/.
The incorporation frequency is defined by the percentage of sequences that have mutated
away from the wild-type sequence from the original sample. An incorporation frequency
of 1% or more is ideal for the downstream protocols.

SINGLE-CELL ISOLATION OF GENOME-TARGETED MONOCLONAL BASIC

hiPSCs PROTOCOL 4
After a successful round of Cas9-mediated genome engineering, the next step is to
isolate monoclonal hiPSC colonies with the desired genotype. This can be accomplished
by fluorescence-activated cell sorting (FACS) and genotyping of single cell–derived
colonies. Once genome-edited monoclonal hiPSC colonies have been isolated, they can
be utilized for downstream applications—e.g., differentiating into relevant tissue types
to interrogate functionality in biology and disease.
Materials
0.1% (w/v) gelatin (StemCell Technologies, cat. no. 07903)
Irradiated CF-1 mouse embryonic fibroblasts (Michalska, 2007)
hES cell medium (see recipe)
Recombinant fibroblast growth factor (Millipore)
SMC4 (BD Biosciences; for 1× SMC4, supplement 500 ml of medium with one
vial of SMC4 purchased from BD)
Fibronectin (StemCell Technologies)
Heterogenous pool of edited hiPSC (Basic Protocol 2)
mTeSR1 medium (StemCell Technologies, cat. no. 05850) supplemented with
SMC4 (BD Biosciences) at final concentration of 1× (one vial per 500 ml
medium)
mTeSR1 medium (StemCell Technologies, cat. no. 05850), unsupplemented
Phosphate-buffered saline (PBS; Invitrogen, cat. no. 20012-050)
Accutase (Millipore)
ToPro-3 viability dye (Invitrogen)
Matrigel (hESC-qualified; BD Sciences, cat. no. 354277)

96-well plates
BD FACSAria II SORP UV (BD Biosciences) with 100-mm nozzle
Centrifuge for 96-well plates
Access to Sanger sequencing facility

Additional reagents and equipment for obtaining amplicons of the targeting region
(see Basic Protocol 3, steps 4 to 11)
1. One day before the experiment, prepare 96-well plates with CF-1 mouse embryonic
fibroblast (MEF) as follows:
a. Coat the plate by incubating 15 min with 50 μl/well of 0.1% (w/v) gelatin at
room temperature, and wash with PBS.
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 b. Thaw and plate MEF in the gelatin-coated 96-well at a concentration of 1 × 106
cells/well in complete DMEM medium.
c. Incubate the MEF plate in the 37°C incubator overnight.
d. Following the overnight incubation, change the medium to hES cell medium
supplemented with 100 ng/ml fibroblast growth factor, 1× SMC4 (one vial per
500 ml medium), and 5 mg/ml fibronectin.
2. Replace the medium on the hiPSCs in a 48-well plate from Basic Protocol 2 with
mTeSR1 medium supplemented with 1× SMC4 (one vial per 500 ml medium) for
at least 2 hr before FACS analysis is to be performed.
3. Aspirate the medium from the cultured hiPSCs, then wash the cells gently with PBS.
4. Aspirate PBS, add 200 μl/well Accutase (or enough to cover the well), and incubate
at 37° for 5 to 10 min.
5. Generate the single-cell suspension by adding 1 ml mTeSR1 (unsupplemented) to
each well and pipetting up and down gently several times.
6. Place cell suspension in a 15-ml conical tube, then centrifuge 5 min at 200 × g,
room temperature. Aspirate supernatant.
7. Resuspend the cells with 1 ml mTeSR1 and add 0.5 μl of the viability dye ToPro-3.
8. Using a BD FACSAria II SORP UV with 100-mm nozzle under sterile conditions,
sort single cells into individual wells of the 96-well plates prepared in step 1.
The 100-mm nozzle is critical for the FACS experiment, to minimize the stress on hiPSCs.

9. After collection, centrifuge plates 3 min at 70 × g, room temperature. and place the
plate into the tissue culture incubator
10. Four days after sorting, colony formation should be apparent; at this point replace
the culture medium with hES cell medium supplemented with 1× SMC4.
11. Eight days after sorting, replace medium with hES medium (unsupplemented).
SMC4 is beneficial for cell viability post sorting. However, long-duration exposure of
cells to SMC4 may lead to cell differentiation. We recommend removing SMC4 from the
culture medium once the colony formation is stable.

12. Passage the monoclonal hiPSC cells into Matrigel-coated 96-well plate and save
half of the cells for genotyping.

To coat wells with Matrigel, do the following:

a. Thaw a vial of 300 μl Matrigel on ice.
b. Transfer 24 ml cold DMEM/F12 into a 50-ml conical polypropylene tube.
c. Transfer 300 μl Matrigel into the tube. Invert to mix.
d. Add 100 μl of this mixture per well of a 96-well plate, then leave the plate
at room temperature for 1 hr.
e. Aspirate Matrigel and replace with 200 μl cells/medium.
13. Perform steps 4 to 11 in Basic Protocol 3 to obtain amplicons of the targeting region.
The amplicon produced in the first round of Illumina PCR is sufficient for Sanger se-
quencing. Either the forward or the reverse primer can be used as the sequencing primer.

14. Perform Sanger sequencing to check the genotype of the targeting region.
15. Choose a colony containing the correct mutation for downstream differentiation or
CRISPR/Cas9 processing.
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Table 31.1.1 Troubleshooting Common Problems with the CRISPR/Cas9 System

Problem Possible cause Solution

Low hiPSC viability after DNA plasmid purity is low Use maxiprep kit to generate
electroporation high-quality DNA
Too much DNA Reduce the amount of DNA
Cell density is too high before Transfect cells under
electroporation exponential growth phase
Cell number is not sufficient Use a minimum of 300,000
cells per transfection
Delay of cell recovery after Speed the recovery after
electroporation electroporation by preparing all
the necessary plates and pipets
in advance and recovering the
cells as soon as the
electroporation is complete
Cell is sensitive to trypsin Use nonenzymatic method to
treatment generate single-cell
suspension, such as EDTA
treatment
gRNA off-target effect is Try alternative gRNA targeting
prevalent and toxic to the cell site
Low genome targeting DNA transfection efficiency Use Cas9-GFP construct
efficiency is low, or cell viability is low followed by FACS to enrich
after transfection transfected cell
The targeting site is not There is no systematic
accessible/targetable knowledge yet regarding the
impact of the targeting
sequence on the targeting
efficiency. We recommend that
users design/generate/test
multiple gRNAs near the
region of interest.
Unable to obtain amplicon Primer design is not optimal Use Primer3 or other primer
of the targeting region design software to optimize the
design of primer on the
targeting region
Insufficient cell number Start with at least >1000 cells
Lysis reaction is not sufficient Elongate the prepGEM
digestion time
Too much lysis reaction in the Use no more than 1/10 volume
PCR reaction of lysis reaction in the final
PCR reaction

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see APPENDIX 4.

Complete medium for HEK 293 cells

High-glucose DMEM medium (Invitrogen) supplemented with:
10% fetal bovine serum (FBS)
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continued
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Table 31.1.2 Time Considerations for CRISPR/Cas9 Protocols

Procedure Substep Hands-on time Total experiment time Stopping point

Basic Protocol 1— 5 hr  4 days Yes
Preparation of hCas9 and
gRNA plasmids
Prepare hCas9 plasmid 2 hr 2 hr Yes
Identify appropriate gRNA 1 hr 3 days Yes
targeting sequence
Plasmid construction of 2 hr 1 day Yes
gRNA construct from IDT
gBlock
Basic Protocol 2— 3 hr 3 days (recovery after No
Transfection of Human transfection)
iPS cells
Basic Protocol 3— 4 hr 2 days Yes
Genotyping transfected
cells using next generation
sequencing
Harvest cells and create 3 hr 3 hr + 1 day (sequencing) Yes
sequencing library
Basic Protocol 4—Isolate 12 days
genome-edited hiPSCs
FACS sorting 3 hr 1 day (prepare MEF plate) Yes
+1 day (FACS) + 8 days
(colony growth)
Harvest cells and create 3 hr 3 hr + 1 day (Sanger Yes
sequencing amplicon sequencing)

1× nonessential amino acids (NEAA)

1× penicillin/streptomycin solution (pen/strep)
Store up to 3 to 6 months at 4ºC
hES cell medium
DMEM/F12 medium (e.g., Invitrogen) containing:
20% (v/v) knockout serum replacement (KOSR)
5 to 10 ng/ml bFGF
1 mM L-glutamine
100 μM nonessential amino acids
100 μM 2-mercaptoethanol
1× penicillin/streptomycin solution (pen/strep)
Store up to 3 to 6 months at 4ºC
COMMENTARY
Background Information for each new target allows for multiplexible
Cas9 is a tool for easily editing the genome genome targeting. Third, independent studies
of human cells. Compared with other genome indicate that the Cas9 system is more effi-
editing methods, such as ZFNs and TALENs, cient than other tools targeting the same region
the RNA-guided Cas9 system has certain ad- (Hwang et al., 2013; Mali et al., 2013a). How-
vantages. ever, the specificity of Cas9-mediated genome
First, the simplicity of its design and con- targeting is still under investigation. Judicious
CRISPR/Cas9 struction make the tool more accessible. Sec- selection of the targeting site is necessary to
Genome Editing ond, the mere requirement of small RNAs minimize off-target effects.
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Critical Parameters off-targeted effects. It has been shown that
As with any transfection, the quality of the the efficiency achieved by double-nickases is
DNA plasmid is paramount. We recommend comparable to that of nuclease in HEK293
plasmid maxiprep kits providing transfection- (Mali et al., 2013b; Ran et al., 2013). How-
level DNA, especially for hiPSCs. The amount ever, it is still under investigation whether the
of DNA used in the transfection is also an double-nickase strategy would work in hiP-
important parameter, since higher transfec- SCs.
tion efficiency and dosage usually yield higher
genome-targeting efficiency. When a new cell Time Considerations
line is used for the first time, the amount See Table 31.1.2 for a description of the
of DNA needed for optimal transfection ef- time required for the protocols described in
ficiency should be determined by titration. Fi- this unit.
nally, the concentration of DNA used in trans-
fection is another important parameter, since Literature Cited
the DNA volume used for hiPSC electropora- Bhaya, D., Davison, M., and Barrangou, R. 2011.
tion should be less than 1/10 of the total reac- CRISPR-Cas systems in bacteria and archaea:
tion volume to achieve effective transfection Versatile small RNAs for adaptive defense and
regulation. Annu. Rev. Genetics 45:273-297.
without incurring significant cell death. If the
concentration is too low to satisfy this require- Brouns, S.J.J., Jore, M.M., Lundgren, M., Westra,
E.R., Slijkhuis, R.J.H., Snijders, A.P.L., Dick-
ment, use a Speedvac evaporator to evaporate man, M.J., Makarova, K.S., Koonin, E.V., and
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yields higher efficiency. We recommend that generates guide RNAs for invader defense in
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M., Duda, K., Taunton, J., Collingwood,
Finally, when assessing the efficiency of T.N., Frodin, M., and Davis, G.D. 2011.
genome editing, the number of cells used in High-frequency genome editing using ssDNA
genotyping is critical for successful genotyp- oligonucleotides with zinc-finger nucleases.
ing following this protocol. We tested the sen- Nat. Methods 8:753-755.
sitivity of genotyping and found that a min- Cho, S. W., Kim, S., Kim, J. M., and Kim, J.-S.
imum of four cells is required to enable the 2013. Targeted genome engineering in human
amplification reaction. However, empirically, cells with the Cas9 RNA-guided endonuclease.
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Anticipated Results
We can achieve 2% genome targeting effi- DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X.,
Aach, J., and Church, G.M. 2013. Genome
ciency in hiPSCs and 30% genome targeting engineering in Saccharomyces cerevisiae us-
efficiency in HEK293 cells using the meth- ing CRISPR-Cas systems. Nucleic Acids Res.
ods described above. The efficiency in hiPSCs 41:4336-4343.
varies with the targeting sites and locations. Friedland, A.E., Tzur, Y.B., Esvelt, K.M., Co-
We detected 0.2% to 15% targeting efficiency laiácovo, M.P., Church, G.M., and Calarco, J.A.
in hiPSCs and 1% to >50% targeting effi- 2013. Heritable genome editing in C. elegans via
ciency in HEK 293 cells without any transfec- a CRISPR-Cas9 system. Nat. Methods 10:741-
743.
tion enrichment and selection. We recommend
that a transfection-enrichment strategy be used Gesner, E.M., Schellenberg, M.J., Garside, E.L.,
George, M.M., and Macmillan, A.M. 2011.
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approach for genome editing with mitigated Mol. Biol. 18:688-692.
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