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MOLECULAR BIOLOGY

Harnessing mutation: The best of two worlds
Combining vertebrate and bacterial immunity components enables targeted mutagenesis

1206

sciencemag.org SCIENCE

16 SEP TEMBER 2016 • VOL 353 ISSUE 6305

Published by AAASGRAPHIC: C. BICKEL/SCIENCEAR

in antigen receptor diversification in vertebrates—with the bacterial immune restriction system clustered regularly interspaced
short palindromic repeat (CRISPR)–Cas9
to enhance point mutation with almost
single-nucleotide precision to levels that,
if occurring genome-wide, would otherwise result in lethality. With localized mutagenesis, also achieved through a similar
approach by Komor et al. (2), comes thepromise of gene correction by means oftargeted editing. Both studies show that
not only is targeted mutation feasible butthat its efficiency, and even the type of mutation, can be driven by manipulating orengaging the various cellular pathways involved in repairing edited bases.
Targeted editing of large vertebrate genomes is a bioengineer’s dream that has
been on the horizon since the early 1970s.
But the prospects of high throughput andDownloaded from https://www.science.org at Osaka University on March 14, 2024

high versatility have only become a tantalizingly imminent possibility since theRNA-guided bacterial Cas9 entered theapid evolution requires high rates
game in 2012 (3–5). Bacteria and archaeaof mutation that come at the costacquire adaptive immunity by incorporatof reduced viability. This is true ating snippets of genetic material from forthe single-cell level in bacteria aseign DNA into their own genome during
well as in the highest complex verthe course of an infection or invasion. Thistebrates. Immunity relies on rapid
piece of foreign DNA is used to produce
evolution to enhance the recognition of
RNA guides for the Cas9 endonuclease
external threats and to neutralize them,to recognize further attacks by the sameoften by inflicting damage to the genomes
threat. The small RNA molecules associateof potentially harmful invaders. On page
with Cas9 and use homology to displace
1248 of this issue, Nishida et al. (1) report
the double helix of foreign DNA and dicombining a polynucleotide cytosine derect cleavage of the two DNA strands. Theaminase (PmCDA1)—an enzyme involved
stable displacement of one DNA strand by
the RNA guide creates a perfect substrate
for cytosine deaminases, such as AID (ac1Core Research Laboratory–Istituto Toscano Tumori,tivation-induced cytidine deaminase) or
Firenze 50134, Italy. 2MRC Laboratory of Molecular Biology,
the APOBECs (apolipoprotein B messenger
Francis Crick Avenue, Cambridge CB2 0QH, UK.
Email: car@mrc-lmb.cam.ac.uk
RNA editing enzyme, catalytic polypeptide-like), that act on single-stranded substrates (6).
The CRISPR-Cas9 comLocalized mutagenesis toolplex
has greatly facilitatedElements from bacterial and vertebrate adaptive immunity can be combined in a new approach. The bacterial endonucleasetargeted editing by means
Cas9 contributes the targeting module by using a homology-guided small RNA and by creating a single-stranded substrate forof homologous recombia vertebrate polynucleotide cytosine deaminase (AID) to initiate editing through a C-to-U change. Addition of a bacteriophagenation (4), which requires
inhibitor of uracil glycosylase (ugi) prevents the removal of the edited base, thus increasing mutagenesis efficiency. The use
exogenous addition of aof a disabled Cas9 that lacks endonuclease activity prevents unwanted collateral mutagenesis in the form of insertions andDNA template bearing thedeletions. Ig, immunoglobulin.
correction. Although thefrequency of recombination
at a specific site is increased
Bacterial immunity
Vertebrate adaptive immunity
by locally induced DNA
Bacteriophage
breaks, the process remains
Targetingrgetinefficient and at risk ofAID
Cell membranecreating genes marred withDeaminasedeletions or insertions. TheEditing
ditin
CRISPR
Cas9DNA
coupling of a catalyticallyIg genes
inactive Cas9 (dCas9) with...ACGTCAGCCAGACT...
Enhanced
ed mutagenesis
muta deaminase that can only
act on single-stranded DNA...ACGTCAGUCAGACT...
triggers localized cytosineCas9
to-uracil conversion but
Guide RNA
avoids collateral damage
Replicationand insertions or deletions
ugi
Disabled Cas9
Guide RNA/Cas9(indels) brought about by
Cleaved viral
GTCAGCthe repair of breaks.
ACCAGTCG CAG
genomeCGT G
ACCTGATTGGG TGTG
For both Nishida et al.
CCand Komor et al., tricks
TGGACTAACCCAGTUAGTCGGTCDNA...AGTCA...
learned from unraveling
repairthe mechanisms of AID (the...AGACA...
mammalian homolog of...AGCCA...
ugi
Deaminase
PmCDA1) during antibody...AGTCA...
diversification proved useLocalized mutagenesis...AGGCA...
ful (7): By inhibiting theBy Silvestro G. Conticello1 andCristina Rada2

“With localized mutagenesis...comes the promiseof gene correction...”Although testing known off-target sites ofCRISPR-Cas9 for APOBEC-dependent mutations in the study of Komor et al. suggests low collateral damage, whole-genome
or -exome sequencing will still be a necessary proposition for human gene therapy.
Physically tethering the deaminase to Cas9
might be the key strategy that allows bothgroups to minimize unwanted editing activity in mammalian cells, where transient
single-stranded DNA is unavoidably associated with transcription. It could well bethat “less is more” in this case and that theless efficient deaminase AID might be abetter choice in mammalian cells.
Transient use of dCas9-PmCDA1-ugi
practically eliminates off-target mutationsand indels but limits the genes amenable
for correction because only transitions fromGC to AT pairs can be reliably achieved.
This is not a major limitation to technological applications; the obvious combinationof dCas9-AID with yeast display immediately comes to mind as a tool for accelerated antibody evolution, but many otherexamples are ripe for its use, from genome
evolution to conventional genetics.
Aside from the technical innovation, themethods described by Nishida et al. andKomor et al. could provide insights into
the physiology of these deaminases. Coexpression of dCas9 and PmCDA1 is sufficient
to increase the frequency of localized mutation even in trans; indeed, the main re-quirement for deamination is the presenceof a small but persistent single-stranded
region of DNA (12). In vivo, other factors
contribute to editing efficiency, such as thesequence context preferences of the edited
cytosine and its position within the open
DNA. In both the Nishida et al. and Komor et al. studies, distal sites were more
frequently edited, a difference that wasexacerbated for cytosines that did not fallwithin the sequence consensus of the deaminase. As exemplified by Nishida et al., amore relaxed sequence context favored by
AID [WRC (where W is either A or T, andR is either A or G) versus NTC (where Nis any base)] can be an advantage for mutagenesis applications, whereas hybrid deaminases with different sequence context
preferences could be part of the targeted
mutation toolkit (13).
Although powerful, the system is stillshort of the promised targeted base editing.
One reason is that mutations are biased
to GC pairs and are promiscuous within
the single-stranded bubble created by theCas9-RNA guide. More importantly, theshort homology required for RNA-guided
recognition is an adaptation that provides
bacterial CRISPR defense the flexibility toaccumulate immunity against a large number of viruses or foreign plasmids while retaining a compact genome, but results inpromiscuous targeting in larger genomes.
As in the case of the deaminase component, a less efficient Cas9 with a more fastidious and longer homology requirement
would be a desirable development.
Harnessing mutation, a dream long anticipated by geneticists and pioneered byearly molecular biologists, was still haunting Michael Smith in his 1993 Nobel Prize
lecture: “The ignis fatuus of genetics has
been the specific mutagen, the reagent thatwould penetrate to a given gene, recognize
it, and modify it in a specific way.” He wasquoting Joshua Lederberg’s 1959 NobelPrize lecture. By the looks of it, the dreamt
future of genetics is now. j
REFERENCES

1. K. Nishida et al., Science 353, aaf8729 (2016).
2. A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, D. R. Liu,Nature 533, 420 (2016).
3. M. Jinek et al., Science 337, 816 (2012).
4. L. Cong et al., Science 339, 819 (2013).
5. P. Mali et al., Science 339, 823 (2013).
6. S. G. Conticello, Genome Biol. 9, 229 (2008).
7. J. Di Noia, M. S. Neuberger, Nature 419, 43 (2002).
8. I. B. Rogozin et al., Nat. Immunol. 8, 647 (2007).
9. G. Saraconi et al., Genome Biol. 15, 417 (2014).
10. C. Swanton, N. McGranahan, G. J. Starrett, R. S. Harris,
Cancer Discov. 5, 704 (2015).
11. L. Pasqualucci et al., Nature 412, 341 (2001).
12. B. J. Taylor, Y. L. Wu, C. Rada, eLife 3, e03553 (2014).
13. M. Wang, C. Rada, M. S. Neuberger, J. Exp. Med. 207, 141(2010).
10.1126/science.aai8233

SCIENCE sciencemag.org

VACCINES

Vaccine trust
and the limits
of information
Understanding trust in local
contexts is key to communication about vaccination
By Heidi J. Larson

Over the past decade, there has been
growing recognition and increasing
research around the phenomenon of
vaccine reluctance and refusal (1, 2).
More recently, there has been a flurryof articles on what is being referredto as “vaccine hesitancy,” depolarizing theearlier characterization of individuals orgroups as being outright pro- or antivaccine, and instead recognizing the liminal
state between becoming aware of, and deciding whether or not to accept, vaccination. Episodes of waning public confidence
around vaccines have become so global that
the World Health Organization’s Strategic
Advisory Group of Experts on Immunization convened a working group (3) to better understand and recommend actions toaddress this growing challenge of vaccine
hesitancy, which the group defined as “delay in acceptance or refusal of vaccination
despite availability of vaccination services.”
Indeed, vaccine hesitancy is complex andcontext specific (4). How can we better understand the circumstances that influence
this state to ensure more effective uptakeof vaccines and secure public health?
Faced with an ever-growing portfolioof new vaccines and combinations of vaccines, parents—and society more broadly—
are becoming more questioning as theyassess whether vaccines for themselves ortheir children are too many or too new,better given individually or in combination, or worth even the smallest risk. The
landscape of information and misinformation about vaccines, as well as the varied
and sometimes divisive views of legitimate
and self-espoused experts, is further complicating the public’s genuine interest inmaking the right decision. A broader environment of distrust in institutions (5) and“experts” (6) additionally prompts pubInfectious Disease Epidemiology, London School of Hygiene andTropical Medicine, London, UK. Email: heidi.larson@lshtm.ac.uk
16 SEP TEMBER 2016 • VOL 353 ISSUE 6305

Published by AAAS1207

Downloaded from https://www.science.org at Osaka University on March 14, 2024

removal of uracil with a bacteriophage
peptide (ugi) that inactivates the cellular
repair enzyme uracil glycosylase, mutation frequencies are enhanced to levels
that obviate the need for additional selection (see the figure). The sequence of suchtargeted yeast genomes contains negligible
off-target changes, but up to 10% localizedmutation. By using rat APOBEC1, a stronger DNA-RNA deaminase than lamprey
PmCDA1 (8), Komor et al. obtained mutation frequencies up to 20% in human cells,with a further increase to 37% achieved bybiasing the cellular mismatch repair towardcorrection of the unedited DNA strand—atthe slight cost of increasing indels (from<0.1 to 1.1%). APOBEC1, like the related
human APOBEC3 deaminases, is a powerful genome mutator in cancer cells (9, 10),and even in physiological conditions, AID
off-target activity is a frequent cause of oncogenic mutations and translocations (11).