Cell Stem Cell

Previews

What’s Changed with Genome Editing?

Shengdar Q. Tsai1,2 and J. Keith Joung1,2,*1Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA2Department of Pathology, Harvard Medical School, Boston, MA 02115, USA*Correspondence: jjoung@mgh.harvard.eduhttp://dx.doi.org/10.1016/j.stem.2014.06.017

Targeted genome editing using engineered nucleases can reliably generate defined genetic mutations,although the range of off-target lesions caused by these techniques is unclear. Recent papers in Cell StemCell perform high-coverage whole-genome sequencing to demonstrate the feasibility of obtaining correctlytargeted pluripotent stem cell clones with minimal unintended modifications.

Customized nucleases have enabled

efficient and targeted genome editing in

awide variety of cell types and organisms,

including human pluripotent stem cells

(PSCs). Several classes of ‘‘designer’’ nu-

cleases are commonly used for genome

editing, including zinc finger nucleases

(ZFNs) (Urnov et al., 2010), transcription

activator effector nucleases (TALENs)

(Joung and Sander, 2013), and the

CRISPR-Cas9 RNA-guided nuclease sys-

tem (RGNs) (Sander and Joung, 2014).

DNA double-stranded breaks (DSBs)

induced by these customizable nucleases

can be repaired by one of two competing

pathways in the cell: error-prone non-

homologous end-joining (NHEJ), which

leads to variable length insertion/deletion

mutations (indels), or homology-directed

repair (HDR), which can be used to intro-

duce precise alterations directed by a ho-

mologous DNA template. Several studies

appearing in Cell Stem Cell examine

whether these technologies can be used

to generate single-cell clones bearing

alterations of interest without also in-

ducing unwanted off-target mutations

(Kiskinis et al., 2014; Smith et al., 2014;

Suzuki et al., 2014; Veres et al., 2014).

When modifying the genomes of cells

or organisms, a fundamental question

arises: does a given engineered nuclease

act at genomic locations other than its

intended site? This is critically important

because unintended, off-target modifica-

tions in cell populations can lead to unex-

pected functional consequences in both

research and therapeutic contexts, where

functional consequences of even low-

frequency mutations can be of significant

concern. A more focused question is

how often different genome modification

methods yield single-cell clones bearing

on-target site modifications without addi-

tional off-target sitemutations. Theanswer

to this question is important for developing

cell-culture-based models of disease in

which one seeks to compare two isogenic

cell lines that differ only at the desired on-

target site. Smith et al., Suzuki et al., and

Veres et al. apply high-coverage whole-

genome sequencing (WGS) to provide

insights into this issue. Suzuki et al.

and Veres et al. achieved �603 genome

coverage and, together with Smith et al.,

who report �303 coverage, conclude

that mutations induced by these targeting

methodologies in individual human PSC

clones are relatively rare.

Veres et al. examined unintended

mutations arising from CRISPR-Cas9

RGN targeting of two loci (SORT1 and

LINC00116) and TALENs targeting

one locus (SORT1) in human PSCs. A

total of nine clones (three clones for

each nuclease/target combination) were

analyzed. After filtering low complexity

regions, homopolymer-associated indel

artifacts, and mutations present in the

parental cell line, they uncovered a num-

ber of single nucleotide variants (SNVs)

and indels across all nine clones, with 28

of these indels confirmed by Sanger

sequencing. In the clones that underwent

CRISPR-Cas9 targeting, no DNA se-

quences similar to the intended target

site (allowing up to six mismatches) were

found within 100 bp of these confirmed

indels. However, one clone generated

via TALEN targeting was found to contain

a likely nuclease-induced off-target

mutation at a site whose DNA sequence

contained seven mismatches relative to

the intended target sequence.

Suzuki et al. examined potential off-

target mutations generated after helper-

dependent adenoviral vector (HDAdV),

TALEN, or hybrid HDAdV/TALEN gene

Cell Stem

correction of the HBB locus in a clonal

induced pluripotent stem cell (iPSC) line

derived from a patient with sickle cell dis-

ease. After filtering their data set for direct

repeats, homopolymers, and repetitive se-

quencesandcomparingagainstcandidate

TALEN off-target sites predicted by an

in silico method, they also found SNVs in

numbers similar to those found by Veres

et al. as well as a limited number of indels

that were not localized near sites that

obviously resembled the on-target site.

They note that one technical limitation of

high-throughput short-read sequencing

technologies is the high systematic error

associated with certain repetitive, low

complexity, or homopolymer sequences.

However, the filtering methods employed,

while removing many false positives, also

removed one of two true indels confirmed

by Sanger sequencing, suggesting that

these WGS approaches may miss some

bona fide indel mutations.

Taken together, these studies demon-

strate that it is possible to isolate single

human PSC clones that appear to have

very few, if any, strictly nuclease-associ-

ated mutations. Smith et al. in this issue,

as well as Kiskinis et al. in a recent paper

utilizing ALS patient-derived iPSC lines for

diseasemodeling, arrive at similar conclu-

sions by WGS of iPSC clones bearing

alterations induced by engineered ZFNs,

TALENs, or CRISPR-Cas RGNs (Kiskinis

et al., 2014; Smith et al., 2014). Thus,

despite the fact that off-target mutations

have been previously identified in large

populations of genome-edited cells,

collectively these four studies show that

this is not an impediment to identifying

single clones that bear the intended on-

target alteration and very few identifiable

unintended nuclease-mediated changes.

However, as several of these groups

Cell 15, July 3, 2014 ª2014 Elsevier Inc. 3

Cell Stem Cell

Previews

note, these modified clones are not

strictly isogenic relative to their parental

lines because they also appear to have

acquired other genetic variation during

clonal expansion, which may account for

many of the SNVs and indels discovered.

Additional studies in which more cell

clones modified by nucleases targeted

to different sites will be needed to further

assess the generality of these findings.

Importantly, the full genome-wide

spectrum of off-target mutations induced

by engineered nucleases remains as yet

undefined by these studies. WGS is un-

likely to fully address this important issue

for at least two reasons. First, as observed

by Veres et al. and Suzuki et al., system-

atic sequencing artifacts can make it

difficult to discern nuclease-induced al-

terations, even with high fold-coverage

sequencing, and bioinformatic filtering

strategies can also remove some bona

fide mutations as well (Suzuki et al.,

2014). Second, WGS is currently imprac-

tical for identifying lower frequency off-

target mutations. For example, there is

only a 95% probability of identifying off-

target sites that are mutated with a fre-

quency of 40% when sequencing three

diploid single-cell clones (as was done

by Veres et al.), and off-target mutations

that occur with more modest frequencies,

4 Cell Stem Cell 15, July 3, 2014 ª2014 Elsev

such as 10%, would routinely be missed

in such experiments. Indeed, to have

a 95% chance of finding off-target muta-

tions that occur with frequencies of

10%, 1%, or 0.1% at least once, one

would need to sequence 15, 150, or

1,500 diploid single-cell clones, respec-

tively. Furthermore, the actual number

of genomes that would need to be

sequenced is higher because convinc-

ingly distinguishing bona fide nuclease-

induced mutations from those acquired

by routine culture of cells requires identi-

fying indels at a given site more than

once among a population of genomes.

Clearly, an unbiased, genome-wide

method that is also sensitive enough to

identify even lower frequency off-target ef-

fects is required toglobally definedesigner

nuclease specificities. While the field

awaits description of such an approach,

the findings of Veres et al., Suzuki et al.,

Smith et al., and Kiskinis et al. take an

important step forward in showing that it

is possible to identify individual nuclease-

modified cell lines that bear few, if any,

unwanted nuclease-induced alterations.

These findings will undoubtedly spur

further research on the use of genome-

edited human PSCs for disease modeling

and of genome-edited single-cell clones

for potential therapeutic applications.

ier Inc.

ACKNOWLEDGMENTS

J.K.J. is a consultant for Horizon Discovery andhas financial interests in Editas Medicine andTransposagen Biopharmaceuticals. J.K.J.’s in-terests were reviewed and are managed byMassachusetts General Hospital and PartnersHealthCare in accordance with their conflict ofinterest policies.

REFERENCES

Joung, J.K., and Sander, J.D. (2013). Nat. Rev.Mol. Cell Biol. 14, 49–55.

Kiskinis, E., Sandoe, J., Williams, L.A., Boulting,G.L., Moccia, R., Wainger, B.J., Han, S., Peng,T., Thams, S., Mikkilineni, S., et al. (2014). CellStem Cell 14, 781–795.

Sander, J.D., and Joung, J.K. (2014). Nat.Biotechnol. 32, 347–355.

Smith, C., Gore, A., Yan, W., Abalde-Atristain, L.,Li, Z., He, C., Wang, Y., Brodsky, R.A., Zhang, K.,Cheng, L., et al. (2014). Cell Stem Cell 15, thisissue, 12–13.

Suzuki, K., Chang, Y., Qu, J., Li, M., Yao, X.,Yuan, T., Goebl, A., Tang, S., Ren, R., Aizawa,E., et al. (2014). Cell Stem Cell 15, this issue,31–36.

Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang,H.S., and Gregory, P.D. (2010). Nat. Rev. Genet.11, 636–646.

Veres, A., Gosis, B.S., Ding, Q., Collins, R., Raga-vendran, A., Brand, H., Erdin, S., Talkowshi, M.E.,and Musunuru, K. (2014). Cell Stem Cell 15, thisissue, 27–30.

Human Induced Pluripotent Stem Cells:Now Open to Discovery

Omer Durak1 and Li-Huei Tsai1,*1Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology,Cambridge, MA 02139, USA*Correspondence: lhtsai@mit.eduhttp://dx.doi.org/10.1016/j.stem.2014.06.014

Human induced pluripotent stem cells represent a promising tool for investigating the underlying causes ofdisease; however, this potential currently remains unfulfilled. In this issue of Cell Stem Cell, Yoon et al. (2014)used iPSCs derived from patients harboring common genetic risk variants as the starting point to discovernovel insights into disease pathology.

The successful reprograming of somatic

cells into induced pluripotent stem cells

(iPSCs) by Takahashi and Yamanaka

in 2006 was seen as a landmark event in

disease research. This new technology

promised to provide a powerful tool for

modeling human pathology that could be

used to understand the underlying causes

of various human diseases. The following

years saw a stream of new and improved

approaches for converting somatic cells

into more differentiated cell types such