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Mechanisms of Precise Genome Editing using Oligonucleotide Donors
Yinan Kan1, Brian Ruis2, Taylor Takasugi3 and Eric A. Hendrickson2, 4
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Minnesota Medical School, Minneapolis, MN 55455
Department of Biochemistry, Molecular Biology, and Biophysics, University of
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Avenue Louis Pasteur, Room 238, Boston, MA 02115.
Current Address:
Department of Genetics, New Research Building, 77
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333 Cedar St., New Haven, CT 06510.
Current Address:
Department of Medicine, Yale University Medical School,
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Minnesota Medical School, 6-155 Jackson Hall, 321 Church St., SE.
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Minneapolis, MN 55455. E-mail:
[email protected].
Corresponding author: Eric A. Hendrickson, BMBB Department, University of
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Abstract
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The use of programmable meganucleases is transforming genome editing and
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functional genomics.
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genomic lesions could be introduced in vivo with unprecedented ease.
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presence of homology donors, these lesions facilitate high-efficiency precise
25
genome editing (PGE) via homology-directed repair (HDR) pathways.
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However, the identity and hierarchy of the HDR (sub)pathways leading to the
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formation of PGE products remain elusive.
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blue fluorescent protein conversion system to systematically characterize
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oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase
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variants in human cells. We demonstrate that, unlike double-stranded DNA
31
(dsDNA) donors with central heterologies, ODNs generated short conversion
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tracts with Gaussian-like distributions. Interestingly, single nick-induced PGE
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using ODN donors produced conversion tracts biased either mostly uni- or
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bi-directional depending on the relative strandedness of the ODNs and the nick.
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Moreover, the ODNs were physically incorporated into the genome only in the
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bidirectional, but not in the unidirectional, conversion pathway.
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presence of double-stranded genomic lesions, the unidirectional conversion
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pathway was preferentially utilized even though the knock-in mutation could
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theoretically have been converted by both pathways.
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suggest that ODN-mediated PGE utilizes synthesis-dependent strand
41
annealing and single-stranded DNA incorporation pathways.
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pathways generate short conversion tracts with Gaussian-like distributions.
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Although synthesis-dependent strand annealing is preferentially utilized, our
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work unequivocally establishes the existence of a single-stranded DNA
45
incorporation pathway in human cells.
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HDR-mediated gene conversion and establishes guidelines for PGE in human
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cells.
CRISPR/Cas9 was developed such that targeted
2
In the
Here we established a green to
In the
Collectively, our results
Both of these
This work extends the paradigms of
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Introduction
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Genome editing is the intentional alteration of the genetic information in living
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cells or organisms. Since Clustered Regularly Interspaced Short Palindromic
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Repeats/CRISPR-associated 9 (CRISPR/Cas9) (Jinek et al. 2012) was
52
repurposed for genome editing (Cong et al. 2013; DiCarlo et al. 2013; Jinek et
53
al. 2013), the speed of CRISPR methodology is quickly bridging the genotype
54
and phenotype worlds and facilitating high-throughput reverse genetic studies
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(Wright et al. 2016). In the CRISPR age, targeted genomic lesions can be
56
introduced with unprecedented ease, which facilitate either high-efficiency
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gene disruption by non-homologous end joining (NHEJ) or, in the presence of
58
donor DNA, PGE by HDR (Carroll 2014).
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genotype can be expeditiously generated for most genetically-tractable
60
organisms (Bassett et al. 2013; DiCarlo et al. 2013; Liu et al. 2014; Yang et al.
61
2014; Reardon 2016), and within one round of subcloning of cultured cells
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(Ran et al. 2013b; Byrne et al. 2014). Although HDR is the more desirable
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pathway for genome editing and gene therapy, its activity is much lower than
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NHEJ and dependent on the cell cycle status in human cells (Mao et al. 2008b;
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Mao et al. 2008a; Lin et al. 2014; Orthwein et al. 2015). Thus, marker-free
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PGE still requires the laborious screening of hundreds of subclones, especially
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in hard-to-transfect cells or those with low HDR activity or limited tolerance of
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genomic lesions. Moreover, bi-allelic and multiplexed PGE is still challenging
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in human cells.
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Research models of the desired
Complicating our understanding of PGE is the fact that HDR has multiple
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sub-pathways in eukaryotic cells (San Filippo et al. 2008).
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sub-pathways can lead to PGE without significantly sacrificing genomic
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stability: the double-strand break repair (DSBR, aka, Holliday junction
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resolution) pathway (Orr-Weaver et al. 1981; Orr-Weaver and Szostak 1983),
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Holliday junction dissolution (Wu and Hickson 2003; Bizard and Hickson 2014;
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Four of these
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Swuec and Costa 2014), synthesis-dependent strand annealing (SDSA)
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(Strathern et al. 1982; Hastings 1988; Larocque and Jasin 2010) and
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single-strand DNA incorporation (ssDI, also sometimes referred to as
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single-strand assimilation) (Radecke et al. 2006b; Storici et al. 2006; Davis and
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Maizels 2014) pathways (Supplemental Fig. S1).
81
fraction of HDR events leading to the conversion of desired knock-in mutations
82
using exogenous homology donors. Importantly, the identity and hierarchy of
83
usage of the HDR sub-pathways leading to the formation of PGE products
84
remains a knowledge barrier that hinders the improvement of PGE.
PGE is defined by the
85
In a previous study we demonstrated that in human somatic cells the
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introduction of large knock-in mutations with dsDNA donors (including
87
transfected plasmids or viruses with dsDNA intermediates) preferentially
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engages the DSBR sub-pathway for PGE (Kan et al. 2014). Although ODNs
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probably do not serve as natural HDR donors, many laboratories have
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reported that PGE can be routinely obtained by exogenously transfecting ODN
91
donors into cells (Chen et al. 2015; Richardson et al. 2016). Since ODN
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donors are likely unable to physically form Holliday junctions and therefore
93
cannot engage DSBR, they must perforce utilize some form of the SDSA or
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ssDI pathways during PGE (Storici et al. 2006; Davis and Maizels 2014).
95
the absence of a targeted meganuclease like Cas9, the ODN donors may use
96
spontaneously occurring genomic lesions in order to perform PGE. Indeed, it
97
was proposed that ODNs could be incorporated into the gaps generated by
98
nucleotide excision repair (Faruqi et al. 2000; Kuan and Glazer 2004) or
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lagging strand synthesis during DNA replication (Ferrara and Kmiec 2004; Wu
100
et al. 2005; Huen et al. 2006), although the specific mechanisms are still
101
disputed (Suzuki 2008; Jensen et al. 2011).
102
models involve the physical incorporation of the ODNs into the genome via an
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ssDI-like mechanism; a prediction that was later supported by experiments
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carried out by Radecke et al. (Radecke et al. 2006b). 4
In
Interestingly, both of these
Confusingly, however,
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the same group of authors also demonstrated that ODNs were not physically
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incorporated into the genome during double-strand break (DSB)-induced PGE,
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suggesting the existence of an alternative mechanism(s) (Radecke et al.
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2006a).
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single-strand nicks, it was proposed by Davis and Maizels that certain forms of
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the SDSA and the ssDI pathways could be utilized depending on the
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strandedness of the donor ODNs (Davis and Maizels 2011; Davis and Maizels
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2014), a hypothesis that was supported by recent work from the Corn
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laboratory (Richardson et al. 2016).
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conversion tracts and hierarchy of these pathways have remained elusive.
Finally, in the presence of meganuclease-induced targeted
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However, the exact mechanisms,
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Results
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The enhanced green fluorescent protein (EGFP) to blue fluorescent
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protein (BFP) conversion system
119
To
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meganuclease-induced PGE using ODN donors, we established the EGFP to
121
BFP conversion system (Fig. 1A).
122
sequence similarity of EGFP and BFP.
123
mutations into the chromophore domain, EGFP can be completely converted
124
into BFP (Katada et al. 2008).
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adeno-associated virus (rAAV)-mediated PGE (Khan et al. 2011; Kan et al.
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2014), we introduced the CMV-EGFP-pA expression cassette into the
127
hypoxanthine
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chromosome of human HCT116 cells, in both sense (S) and antisense (AS)
129
directions with respect to the direction of the HPRT1 gene (Supplemental Fig.
130
S2A, B).
131
expression and the inactivation of the HPRT1 gene. After rAAV infection, the
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HPRT1-negative cells were enriched using 6-thioguanine selection, and
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individual subclones with bright EGFP expression were screened using
134
designated primers (Supplemental Fig. S2A, B).
135
precisely knocked into the HPRT1 gene in 1 out of 4 subclones in the sense
136
orientation and 2 out of 4 subclones in the antisense orientation (Supplemental
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Fig. S2C, D). One individual subclone from each orientation was designated
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as HPRT1-EGFP (S) and (AS) cell lines, respectively, and used for
139
subsequent studies. Targeted genomic lesions such as nicks and DSBs were
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subsequently introduced into these reporter cell lines within the chromophore
141
sequence using Cas9 or nCas9 from S. pyogenes and sgRNAs in both S and
142
AS orientations with respect to EGFP (Fig. 1A). In the presence of ODN
143
donors bearing the SH mutations (ACCTAC to TCTCAT), EGFP could
systematically
investigate
the
molecular
mechanisms
of
This system takes advantage of the By introducing T65S and Y66H
To this end, using recombinant
phosphoribosyltransferase 1 (HPRT1) locus
on
the X
Targeted integration in either direction results in mono-allelic EGFP
6
The EGFP cassette was
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efficiently be converted into BFP, which generated distinguishable spectra
145
under flow cytometry (Supplemental Fig. S3B, C).
146 147
ODNs stimulate efficient PGE in the presence of genomic lesions
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We first compared the efficiency of ODN-mediated PGE using nCas9 with
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ODNs (BFP_S160 and BFP_AS160, see Supplemental Fig. S10 for more
150
details) and sgRNAs in both S and AS orientations with respect to EGFP.
151
transfecting the HPRT1-EGFP cell lines with the nCas9 and sgRNA
152
expression vectors and ODN donors all in a DNA format, we could routinely
153
induce efficient EGFP to BFP conversion as quantitated using flow cytometry
154
(Fig. 1; Supplemental Fig. S3). In contrast, the omission of the Cas9 and/or
155
sgRNA resulted in 7 nt)
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resection of the 3’-end was required for PGE to occur and we believe that
575
these two observations are connected given the sharp drop-off in the SDSA
576
conversion tracts (Fig. 2A, B).
577
that double-strand lesions in general loosen the local chromatin architecture
578
more than single-strand nicks and thus permit relevant co-factors (such as
579
resection nucleases) easier access to the 3’-end, which ultimately facilitates
580
more vigorous homology searches.
581
The only instance where
If this exception is illustrative, it may suggest
In our nick-induced PGE experiments, no significant strand bias for either
582
the nick or the ODN donors was observed (Fig. 1B).
583
were consistent with a previous report using zinc-finger nickases at three
584
independent chromosomal loci (Ramirez et al. 2012) but inconsistent with a
585
more recent one (Davis and Maizels 2014).
586
explained by the fact that the recent report employed ODN donors with a large
587
(17 bp) heterology flanking the genomic lesion (Davis and Maizels 2014).
588
discussed above, a sizable heterology on both sides of the genomic lesion
589
could strongly favor the use of ssDI as a bi-directional gene conversion
590
pathway.
591
resection of at least 8 to 9 nt would be required and that likely occurs at a much
592
lower frequency.
593
transactions such as transcription and DNA replication are unlikely to have a
594
major influence on PGE mediated by nicks. With that said, it is important to
595
note that while no strand-specific biases were noted in any of our experiments,
These observations
The latter difference might be
As
In contrast, in order to be converted by the SDSA pathway, 3’
Collectively, these observations suggest that strand-specific
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a significant 2- to 4-fold higher frequency of PGE was noted for the BFP
597
reporter in the anti-sense orientation than in the sense orientation (Fig. 1 and
598
Supplemental Fig. S3). The reason for this bias is not completely clear, but it
599
is unlikely to be due to conflicting transcription between the EGFP reporter and
600
the endogenous HPRT1 locus because EGFP expression was very high in
601
these cells. Indeed, we believe the bias was technical (and not biological) in
602
nature since the higher EGFP (and consequently BFP) expression may have
603
simply allowed us to more easily isolate correctly targeted cells.
604
also note that a strand bias was observed when PGE was mediated by nicks
605
and same sense ODN donors in RPE+hTERT cells (Supplemental Fig. S8B).
606
As discussed previously, we do not believe that this is related to transcription
607
nor DNA replication issues, but to the fact that these cells are MMR proficient,
608
which is especially inhibitory to the strand annealing step required for ssDI.
Finally, we
609
PAM-in paired-nickases are significantly less effective in producing
610
NHEJ-related mutations than their PAM-out counterparts, which has mainly
611
been attributed to the steric hindrance of the nCas9 proteins in the PAM-in
612
configuration and/or the displacement loops formed the by sgRNAs (Ran et al.
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2013a; Cho et al. 2014; Shen et al. 2014).
614
additional or alternative explanation (Fig. 4A, 5A): PAM-out paired-nickases
615
generate 5’-overhangs that are subject to exonuclease resection (Symington
616
and Gautier 2011).
617
normally engage the error-prone NHEJ pathway and produce deletions in
618
between.
619
may remain largely as separated nicks (Fig. 4B, 5B), which may be precisely
620
repaired by standard single-strand nick repair without engaging the NHEJ
621
pathway.
622
at a comparable efficiency to their PAM-out counterparts (Fig. 1B), at least
623
when the PAM sequences are placed far enough apart from each other to
624
avoid steric hindrance.
Our results may provide an
The resection creates a double-strand gap that will
In contrast, the 3’ overhangs produced by PAM-in paired-nickases
Importantly, however, the PAM-in paired-nickases can induce PGE
Thus, we propose that PAM-in paired-nickases are 23
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less efficient in NHEJ-mediated gene disruption because the 3’ overhangs
626
rarely form double-strand gaps due to the lack of 3’ to 5’ resection (Symington
627
and Gautier 2011).
628
often used for PGE, they may actually be advantageous because they may
629
induce similar levels of HDR with less NHEJ mutations compared to their
630
PAM-out counterparts.
631
efficient generation of short 3’ overhangs with PAM-out sgRNAs, minimizing
632
the effect of steric hindrance (Nishimasu et al. 2014).
Consequently, although PAM-in paired nickases are not
The use of the Cas9 N863A variant further allows the
633
Although paired-nickases induced PGE with a higher frequency compared
634
to a single nickase (Fig. 1B), we noticed that paired-nickases (in both PAM
635
configurations) occasionally produced imprecise HDR products using ODN
636
donors (Supplemental Tables S6 to S9).
637
contained NHEJ-like mutations near the position of the distal nick with respect
638
to the knock-in mutations. Since a single nickase can also generate NHEJ
639
mutations (Davis and Maizels 2011; Davis and Maizels 2014), we propose that
640
the imprecise HDR was generated by the repeated nicking of the distal nickase
641
in the PGE products.
642
the proximal nicks because, in our system, the conversion of the knock-in
643
mutations would destroy the binding site of the proximal nickase and prevent
644
further nicking in the PGE products. If this hypothesis is correct, an important
645
guideline of ODN-mediated PGE would be to introduce an additional silent
646
mutation in the PAM sequence of the ODN donors to prevent the further
647
binding of Cas9 or nCas9 in the PGE products. Alternatively, these nicks
648
might be converted to frank DSBs, which through canonical end joining might
649
yield NHEJ-like mutations.
Most of the imprecise products
These mutations were not found near the position of
650
Our conversion tract data also provides insight into a recent paradox:
651
although SDSA is the more efficient form of meganuclease-induced HDR (Li et
652
al. 2001; Heyer et al. 2010), the majority of the PGE products are actually
653
generated by the DSBR pathway when dsDNA donors with long central 24
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654
heterologies are employed (Li and Baker 2000; Li et al. 2001; Langston and
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Symington 2004; Kan et al. 2014). Our results and other’s demonstrate that
656
SDSA is apparently a short-tract gene conversion pathway using chromosomal
657
(Elliott et al. 1998; Larocque and Jasin 2010) and ODN donors (Fig. 2A, B).
658
Also, the Gaussian-like nature of the associated conversion tracts makes it
659
extremely inefficient in producing gene conversions longer than 100 bp (Fig.
660
2A, B).
661
engage
662
meganuclease-induced
663
generated unidirectional long-tract gene conversion products (Elliott et al. 1998;
664
Larocque and Jasin 2010), which were believed to be produced by
665
break-induced replication (BIR) (Borts and Haber 1987; Kraus et al. 2001;
666
Llorente et al. 2008). In the case of PGE, however, BIR using exogenous
667
homology donors leads to the formation of chromosome:donor fusions and
668
severely compromises genomic stability (Borts and Haber 1987; Kraus et al.
669
2001; Llorente et al. 2008). In contrast, our previous work demonstrated that
670
the DSBR pathway is a second (and precise) long-tract gene conversion
671
pathway (Kan et al. 2014), which generates bi-directional conversion tracts
672
with a linear distribution.
673
SDSA is the more efficient HDR pathway for short-tract gene-conversion, the
674
DSBR pathway is the predominant pathway of PGE using dsDNA donors with
675
long central heterologies.
Thus, dsDNA donors with long central heterologies would perforce a
long-tract
gene
HDR
conversion
using
pathway.
chromosomal
donors
Notably, occasionally
Collectively, these results indicate that although
676
dsDNA donors can mediate robust PGE. We can routinely achieve about
677
20% marker-free PGE using Cas9-2A-GFP with associated FACS enrichment
678
(Duda et al. 2014) and circular dsDNA donors with long central heterologies
679
(data not shown).
680
should be utilized in the presence of DSBs and/or paired-nicks for converting
681
large knock-in mutations (Fig. 7). One of the reasons that these donors work
682
so well is that they may have a longer half-life than ODN donors, which better
As a practical guideline, we propose that circular dsDNA
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683
coincides with the kinetics of the Cas9 expressed in a DNA format.
684
contrast, ODN donors should be utilized for converting small modifications.
685
Although ODN donors are optimally used for introducing SNPs, they can also
686
be used to introduce more complex genomic lesions by producing slightly
687
longer conversion tracts.
688
knock-in mutation(s) needs to be placed within the effective conversion zones
689
of the SDSA or ssDI pathways, or it will not be incorporated into the genome
690
(Fig. 7).
691
accommodate the shorter half-life of ODN donors and increase the efficiency
692
of PGE.
In
Regardless, what is critical is that the desired
Cas9 expressed in mRNA or protein formats may be used to
693
Finally, our results demonstrated that the ODN donors are not physical
694
incorporated into the genome in the SDSA pathway, making this pathway
695
particularly valuable for agricultural engineering.
696
livestock containing CRISPR-mediated gene disruption are generally not
697
categorized as genetically modified organisms (GMOs), PGE using exogenous
698
HDR donors is under more stringent regulatory scrutiny (Wolt et al. 2016). An
699
empirical distinction lies in whether the engineering process leads to the
700
incorporation of foreign DNA into the genome (Wolt et al. 2016).
701
established that when an nCas9 and ODNs complementary to the strand of the
702
nick are used in combination, the genomic modification is introduced via the
703
endogenous DNA repair process of SDSA, whereas the ODN donors merely
704
serve as repair templates (Fig. 2, 4, 6). These findings extend the scope of
705
non-GMO plants and livestock to ODN-mediated PGE.
26
Although plants and
We
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706
Methods
707
Nucleotide sequences
708
All sgRNA targets, ODN donors, primers and the relevant plasmid sequences
709
can be found in Supplemental Fig. S10.
710 711
Cell culture
712
All cell lines utilized in this study were purchased from the American Type
713
Culture Collection.
714
McCoy’s 5A medium supplemented with 10% FBS, 1% L-glutamine, 1%
715
penicillin/streptomycin.
716
Dulbecco’s modified Eagle medium supplemented with 10% FBS, and 1%
717
penicillin/streptomycin. All cells were grown with 5% CO2 at 37 oC.
The human HCT116 and DLD-1 cell lines were cultured in
The human RPE+hTERT cell line was cultured in
718 719
The HPRT1-EGFP cell lines
720
The rAAV EGFP knock-in vectors were constructed using an unpublished
721
method (Kan et al., manuscript in preparation).
722
cassette was amplified from EGFP-N2 (Clontech), and the homology arms
723
flanking
724
(Supplementary Fig. S10).
725
were ligated into a modified version of the pAAV-MCS vector in both sense and
726
antisense orientations with respect to HPRT1. rAAV packaging and infections
727
were performed as described (Khan et al. 2011).
728
were seeded into 10 cm tissue culture dishes and selected with 5 μg/mL 6-TG
729
for 14 days.
730
under a fluorescence microscope, and subsequently screened by PCR using
731
the indicated primers (Supplemental Fig. S2A, B). One of the targeted clones
732
with EGFP in either sense or antisense directions in the HPRT1 locus were
733
flow sorted for high EGFP expression using a FACSAria II cell sorter (BD
HPRT1
exon
3
were
amplified
Basically, the CMV-EGFP-pA
using
designated
primers
The CMV-EGFP-pA cassette and homology arms
The infected HCT116 cells
Individual colonies were initially scanned for EGFP expression
27
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734
Biosciences) and used for subsequent studies.
735 736
The EGFP to BFP conversion system
737
The EGFP reporter cells were seeded at ~50% confluency. The next day, 5 x
738
105 cells were transfected with a Cas9 or nCas9 expression plasmid (10 μg;
739
#41815 and #41816, respectively, Addgene), the MLM3636 plasmid containing
740
the designated sgRNA expression cassette (10 μg; #43860, Addgene) and the
741
relevant ODNs (10 μg) using a Neon Transfection System (Invitrogen).
742
paired-nickases, cells were transfected with 7.5 μg of each sgRNA plasmid,
743
the Cas9 expression plasmid and ODNs. All transfections were performed
744
using 100 μL tips under elevated conditions compared to the manufacturer’s
745
protocol: 1530V, a 10 msec pulse width, and 3 pulses.
746
experiment, cells were transfected using ODNs without flanking SNPs
747
(Supplementary Fig. 10), and the percentage of BFP-positive cells were
748
quantitated 2 days after transfection using a LSRFortessa cell analyzer (BD
749
Biosciences).
750
using ODNs containing flanking SNPs.
751
were combined, cultured for 2 days and sorted for BFP expression using a
752
FACSAria II cell sorter (BD Biosciences).
For
For the PGE efficiency
For the conversion tracts experiment, cells were transfected Cells from 5 individual transfections
753 754
Conversion tracts analysis
755
The BFP-positive cells were single-cell subcloned into 96-well-plates at a
756
concentration of 3 to 10 cells per well.
757
trypsinized, transferred into new 96-well plates and grown to confluency.
758
Genomic DNA was prepared using DirectPCR Lysis Reagent (Viagene).
759
BFP fragments containing all the potential SNPs were amplified and confirmed
760
by PCR using primers BFP_EF X BFP_ER and BFP_CF X BFP_ER,
761
respectively.
762
purification kit.
14 days later, single colonies were
The
The PCR products were purified using a Qiaquick PCR The retention of vector-borne SNPs was analyzed by Sanger 28
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763
sequencing.
764 765
Statistics
766
Kolmogorov-Smirnov tests were performed to determine the degree of
767
symmetry around the central dinucleotides of the ODN donors for SNP
768
retention curves induced by single-nicks. All reads from Sanger and Illumina
769
sequencing were randomly divided into three groups.
770
frequency of all SNPs with approximately equal distance to the central
771
dinucleotides
772
Kolmogorov-Smirnov equations in pairs.
773
the corresponding graphs.
774
the distributions around the Y-axis, which is the central heterology of the ODN
775
donors.
was
compiled
in
each
group
and
The retention
entered
into
the
The resultant p-value is shown on
The p-value reflects the degree of symmetry of
776 777
The PIGA conversion system
778
0.5 million DLD-1 and RPE+hTERT cells were transfected with 15 μg
779
PIGA_KO ODNs (Supplemental Fig. S6A, S8A, S9) and 15 μg Cas9
780
D10A-2A-mCherry with the respective S or AS sgRNA expression cassette in
781
one backbone (adapted from PX461).
782
each sgRNA, recovered for 14 days, stained with the FLAER reagent
783
(Pinewood Scientific Services) at a concentration of 5 X 10-9 M and FACS
784
sorted for PIGA negativity.
785
expression were collected for S and AS sgRNA, respectively, using a
786
non-stringent gate (Supplemental Fig. S7).
787
of HDR and NHEJ events and some wild type cells. Then the genomic DNA
788
was prepared using DirectPCR lysis (Viagen), and the target locus was
789
amplified using flanking Nextera compatible primers (NextF & NextR,
790
Supplemental Fig. S10). The amplicons were gel purified and sequenced
791
using Nextera amplicon sequencing (paired-end 2 x 125 bp).
10 transfections were combined for
5.6% and 6.5% of the cells with the lowest PIGA
29
These cells contain the majority
The HDR
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792
products were characterized as the reads containing the central GT
793
dinucleotide.
794
retention frequency of flanking SNPs in the HDR products versus the position
795
of the SNPs in reference to the GT dinucleotide.
The SNP retention curves were compiled by plotting the
796 797
The biotin incorporation assay
798
The biotin incorporation assay was performed as described (Radecke et al.
799
2006b) with minor modifications. The EGFP reporter cells were transfected
800
with internal biotin-labeled ODNs (BFP_S90_Biotin) and flow sorted as
801
described above. The genomic DNA from 1 x 105 BFP-positive cells was
802
prepared using a Puregene Cell kit (Gentra) with a 60 min centrifugation step
803
after isopropanol precipitation.
804
completion with XhoI and XbaI restriction enzymes (New England Biolabs).
805
The biotinylated DNA fragments were isolated using a Dynabeads Kilobase
806
BINDER kit (Life Technologies) according to manufacturer’s protocol, except
807
that all reagents were supplemented with 0.1% BSA, and two additional
808
washes using 0.1 M NaOH and 0.05 M NaCl at room temperature and one
809
more rinse with 95 oC water were performed to remove the non-covalently
810
linked genomic fragments.
811
detected with a 40-cycle PCR using Phusion Hot Start II High-Fidelity DNA
812
polymerase (Thermo Scientific).
813
Dynabeads purification was used as control in a 25-cycle reaction.
The genomic DNA was digested to
The biotinylated genomic fragments were
The genomic DNA preparation prior to
814 815
Author’s contributions
816
Y. K. and E. A. H. designed the study, analyzed the data and wrote the
817
manuscript. Y. K. performed the bulk of the experiments and T. T. and B. R.
818
assisted during the latter stages of the project.
819
30
Downloaded from genome.cshlp.org on April 20, 2017 - Published by Cold Spring Harbor Laboratory Press
820
Acknowledgements
821
We thank Drs. Feng Zhang, Keith Joung and George Church for sharing
822
plasmids through Addgene.
823
University of Minnesota Flow Cytometry Core for cell sorting.
824
supported by grants from the National Institutes of General Medical Sciences
825
(GM088351) and the National Cancer Institute (CA154461 and CA190492).
We thank Jason Motl and Nisha Shah at This work was
826 827
Competing Interests
828
E.A.H. declares that he is a member of the scientific advisory boards of
829
Horizon Discovery, Ltd. and Intellia Therapeutics, companies that specialize in
830
applying gene editing technology to basic research and therapeutics.
831
31
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832 833
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1027
1956-1968.
1028
38
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1029
Figure Legends
1030
Figure 1.
1031
antisense cell line. (A) Schematics of the EGFP to BFP conversion system in
1032
the HPRT1-EGFP antisense cell line. An EGFP expression cassette was
1033
knocked into the HPRT1 locus of the HCT116 cell line (only the antisense
1034
orientation is diagrammed here for the sake of ease of presentation).
1035
Genomic lesions could then be introduced near the chromophore (TY residues)
1036
of the EGFP sequence using CRISPR/Cas9 cleavase, nickases or dual
1037
nickases.
1038
the sequence of the SH residues leads to the conversion of EGFP to BFP.
1039
The ODN donors also contained synonymous SNPs that could be
1040
co-converted with the sequence of the SH residues. Schematic elements:
1041
EGFP, green boxes; BFP, blue boxes; HPRT1, inverted yellow boxes;
1042
sequences of the chromophore residues, TY and SH; the cytomegalovirus
1043
promoter, CMV; the polyadenylation sequence, pA; Cas9 variants, red ovals
1044
with scissors; genomic lesions, lightning bolts; ODN donors, horizontal red
1045
lines; synonymous SNPs, vertical blue hashmarks.
1046
conversion in the HPRT1-EGFP antisense cell line.
1047
D10A variant are labeled as Cas9 and nCas9, respectively.
1048
strandedness (S, sense; AS, antisense) of the sgRNA and ODNs (for this
1049
experiment without synonymous SNPs) are labeled with respect to the coding
1050
sequence of EGFP.
1051
complementary to that of the sgRNA. All data are shown as the mean ± SEM
1052
of three biological replicates.
1053
images of each bar shown in (B).
The EGFP to BFP conversion system in the HPRT1-EGFP
HDR repair of the genomic lesions using ODN donors containing
(B) The efficiency of BFP The wildtype Cas9 and The
Note that the Cas9 D10A variant nicks the strand
(C) Single representative flow cytometry
1054 1055
Figure 2. Conversion tracts of single-nick and DSB-induced HDR using ODN
1056
donors.
(A-E) The conversion tracts of single-nick-induced HDR using ODN
39
Downloaded from genome.cshlp.org on April 20, 2017 - Published by Cold Spring Harbor Laboratory Press
1057
donors complementary to (A, B) or the same strand of (C, D) the nick, and
1058
DSB-induced HDR (E).
1059
the retention frequency of each SNP in both 6SNP_A and 6SNP_B donors.
1060
The positions of the SNPs and predicted genomic lesions are labeled in
1061
reference to the center of the chromophore sequence. Schematic elements
1062
are colored as follows: genomic DNA, black; genomic lesions, orange; ODNs,
1063
red; chromophore sequences, TY and SH; SNPs on the 6SNP_A ODNs, blue;
1064
SNPs on the 6SNP_B ODNs, green; homology regions, dashed silver crosses;
1065
strandedness of DNA, S and AS.
1066
synonymous SNPs.
1067
the tri-nucleotides in the S orientation, and the distance between the SNPs and
1068
the center of the SH sequence (TCTCAT) is labeled. Because the last T in
1069
the SH sequence is a wobble nucleotide it could be used as SNP as well and is
1070
represented as the ATG at position +3. ODNs with six distributed or clustered
1071
SNPs are labeled as 6SNP_A (a) and 6SNP_B (b), respectively.
1072
sequences shown at the bottom of the panel emphasize that the S nick is
1073
made at position -1 and the AS nick at position -2.
1074
the ODNs can be found in Supplemental Fig. S8.
The conversion tracts were compiled by overlaying
(F) Schematics of the ODN donors with
The SNPs are represented as the central nucleotide of
The
The actual sequences of
1075 1076
Figure 3.
1077
Repair of complementary strand nicks via SDSA. (B) Repair of nicks on the
1078
same strand via ssDI. Schematic elements are labeled as follows: Top —
1079
genomic DNA, black; genomic lesions and DNA ends, hatched orange lines;
1080
ODNs, red; knock-in mutations, green; resection nucleases, yellow PAC
1081
ManTM; base pairing, purple vertical lines; DNA synthesis, red dashed arrows;
1082
processing nucleases, yellow lightning bolts; Bottom — predicted SNP
1083
retention curves, red dashed line; predicted position of genomic lesions,
1084
orange vertical dashed line; position of the knock-in mutation selected for,
1085
green vertical solid line; regions with more than 50% co-conversion frequency,
Mechanisms of single-nick-induced PGE using ODN donors.
40
(A)
Downloaded from genome.cshlp.org on April 20, 2017 - Published by Cold Spring Harbor Laboratory Press
1086
black double-headed arrows, strandedness of DNA, S and AS; orientations of
1087
the DNA ends, 5’ and 3’.
1088 1089
Figure 4. The conversion tracts of paired-nicks-induced HDR.
1090
conversion tracts of paired-nicks-induced HDR using S (A, B) and AS ODNs (C,
1091
D).
1092
frequency of each SNP in both 6SNP_A and 6SNP_B donors (Fig. 2F).
1093
positions of the SNPs and predicted genomic lesions are labeled in reference
1094
to the center of the chromophore sequence.
1095
colored as in the legend of Fig. 2.
(A-D) The
The conversion tracts were compiled by overlaying the retention The
All schematic elements are
1096 1097
Figure 5.
1098
Repair of the PAM-out double nicks via SDSA.
1099
double nicks via ssDI. All schematic elements are colored and defined as in
1100
the legend to Fig. 3.
Mechanisms of paired-nick-induced PGE using the S ODNs.
(A)
(B) Repair of the PAM-in
1101 1102
Figure 6. The biotin pull-down assay. (A, B) Schematic illustration of the
1103
biotin pull-down assay via the SDSA (A) and ssDI (B) pathways. In the case
1104
of ssDI (B) the biotinylated ODN is predicted to be incorporated into the target
1105
genomic locus whereas in SDSA (A) it should not.
1106
digested genomic fragments covalently linked to biotin can be enriched using
1107
streptavidin beads under denaturing conditions.
1108
BFP_QR can specifically amplify these genomic fragments with ODN
1109
incorporation but not free ODN donors.
1110
orange ovals; genomic DNA, black lines; ODN sequence, solid red lines; DNA
1111
synthesis, dashed red lines; chromophore sequence, TY and SH; genomic
1112
lesions, hatched yellow lines; homology regions, dashed silver crosses;
1113
restriction sites, XhoI and XbaI; PCR primers, horizontal arrows.
1114
of the biotin pull-down assay.
The XhoI and XbaI
The primers BFP_QF and
Biotin, yellow circle; streptavidin,
(C) Results
After transfecting with biotinylated ODNs 41
Downloaded from genome.cshlp.org on April 20, 2017 - Published by Cold Spring Harbor Laboratory Press
1115
(BFP_S90_Biotin; Supplemental Fig. S8), the BFP-positive cells were
1116
enriched using FACS sorting.
1117
was digested with XhoI and XbaI.
1118
were pulled down with streptavidin beads in denaturing conditions, PCR
1119
amplified using one internal primer and one flanking primer of the ODN donors
1120
(BFP_QF and BFP_QR; Supplemental Fig. S8) and detected on an agarose
1121
gel.
The genomic DNA of the BFP-positive cells The fragments covalently linked to biotin
1122 1123
Figure 7.
1124
PGE is defined as the fraction of HDR leading to the conversion of the desired
1125
knock-in mutations using exogenous homology donors.
1126
retrospectively, the hierarchy of the HDR pathway is determined primarily by
1127
the types of homology donors and secondarily by the genomic lesions.
1128
dsDNA donors containing a sizable central heterology must be converted via
1129
the DSBR model, which generates long conversion tracts with a linear
1130
distribution.
1131
depending on the strandedness of the ODNs and the relative position of the
1132
knock-in mutation to the genomic lesion.
1133
conversion tracts in normal distributions.
1134
presence of double-stranded genomic lesions when both pathways can
1135
convert the knock-in mutation effectively.
The hierarchy of meganuclease-induced HDR leading to PGE.
In the PGE products,
ODN donors can utilize both SDSA and ssDI pathways,
42
These pathways generate short
SDSA is preferentially utilized in the
CMV
HP
B
HP
TY CY EGFP EGFP CY TY
pA
RT
HP
RT
CMV
HP
TY CY pA EGFP RT EGFP CY TY Sense nickase nCas9
RT
nCas9 Antisense nickase Sense oligonucleotides CMV (synonymous SNPs) SH
HP
RT
HP
3
Single nick DSB Paired nicks
2
1
TY CY EGFP pA RT EGFP CY TY 0 (synonymous SNPs) SH Antisense oligonucleotides Cas9: nCas9 nCas9 nCas9 nCas9 Cas9 Cas9 nCas9 nCas9 nCas9 nCas9 CMV sgRNA*: S SH AS S AS S AS PAM- PAM- PAM- PAMCY out in out in pA BFP RT EGFP CY ODNs: S S AS AS AS AS S S AS AS SH *nCas9 (D10A) nicks com plem entary to the strand of the sgRNA Conversion tracts
HP
HP
BFP conversion efficiency (%)
A
RT
A
BS
SH
S
TY
S AS
TY
AS
SH
AS
TCTCAT
100
TCTCAT
ATG
CGC
80 TCA CAC
-60
-40
-20
0
20
40
60
Distance to the central heterology (bp)
C
80
-80
-60
D
-40
-20
CGC
80 TCC
60 TCA
-80
E
-60
-40
-20
100
40
TCA
-80
TCTCAT
100
-60
ATG
80 TCA 60 CAC TCC AAC AAT
-60
0
20
40
SH
(21 bp) GTC
TCTCAT(G) (0 bp) (3 bp)
TCC
GTT GTC CTG AAC CTG
0
20
40
60
Distance to the central heterology (bp)
80
60
6SNP_A (-19 bp) CAC
AAC (42 bp)
6SNP_B
TCC
40 20
-20
-20
(-31 bp) (-7 bp) (9 bp) (30 bp)
(-2 bp)
-40
-40
Distance to the central heterology (bp)
AAC (-40 bp)
DSB 6SNP_A 6SNP_B
GTC CTG AAC CTG
(-2 bp)
F
S AS
-80
AAT
(-61 bp) AAT
CGC
GTT
20
AAC
80
80
TCC
40
TCC
SH
S
60
CAC
60
60
Nick 6SNP_A 6SNP_B
80
CTG
20
ATG
CGC
AAC
0
40
TY
Nick 6SNP_A 6SNP_B
Distance to the central heterology (bp)
20
TCTCAT
GTC CTG
CAC 20 TCC AAT AAC (-1 bp)
CTG
SH
GTT
40
0
S S AS
ATG
AAC
Distance to the central heterology (bp)
TCTCAT
100
CTG
AAT AACTCC CAC (-2 bp)
SH
AS
GTC
TCA 20
TY
S AS
GTT
40
TCC
Nick 6SNP_A 6SNP_B
TCC
60
GTT CTGAAC CTG (-1 bp) GTC
AAT
ATG
80
20
AAC
-80
60 40
TCC
100
CGC
Nick 6SNP_A 6SNP_B
CGC
SH
TCA (-13 bp)
TCC CTG GTT (15 bp)
sgRNA_S: TC//TCAT sgRNA_AS: T//CTCAT
(-1 bp) (-2 bp)
(63 bp) CTG
80
A
Synthesis-Dependent Strand Annealing
S AS
B
Single-Strand DNA Incorporation
S AS
Futile homology search
Minimal 3’ processing S AS
S
5’
3’
3’
5’
S AS
S
Single-strand DNA Incorporation
Strand invasion and synthesis S AS
S
3’
5’ 3’
5’
S AS
S
Strand annealing
Strand displacement
S AS
S
S AS
S
Flap cleavage and ligation
Flap cleavage and ligation S AS
S AS (1) Predicted SNP retention
20 bp (3’)
(2) Predicted SNP retention
10 bp (5’)
20 bp (3’)
10 bp (5’)
A
B
SH
S
TY
S AS CAC
S AS
ATG
80 CAC
6SNP_A 6SNP_B
TCC
AAC
GTT (-1 bp) GTC CTG AAC CTG
(-41 bp)
-60
-40
-20
0
20
40
60
Distance to the central heterology (bp)
C
AAT
80
-80
AAC
TCC
40
-60
(-41 bp)
-40
-20
(-1 bp)
0
0
20
40
60
80
SH
TCTCAT ATG TCC CGC 80
6SNP_A
TCA
6SNP_B
GTT
20 AAT
-20
TY
Paired nicks (PAM-out)
80
TCC 60
-40
Distance to the central heterology (bp)
AS
CGC TCTCAT ATG TCA CAC
(-30 bp)
S AS
SH
AS
-60
GTC CTG AAC CTG (4 bp)
20
D
TY
S AS
GTT 6SNP_B
40
20
AAT
Paired nicks (PAM-in) 6SNP_A
60
TCC
40
-80
TCTCAT CGC ATG TCC TCA
Paired nicks (PAM-out)
60
-80
TY
CGC TCTCAT
TCA TCC 80
AAC
SH
S
60
40
60
CTG
Distance to the central heterology (bp)
80
AAT
-80
-60
GTC CTG
20 CAC TCC AAC (-30 bp)
-40
-20
6SNP_A 6SNP_B
40
GTC CTG AAC
20
GTT
Paired nicks (PAM-in)
0
AAC (4 bp)
20
CTG
40
60
Distance to the central heterology (bp)
80
A
Synthesis-Dependent Strand Annealing Paired nicking (PAM-out)
S AS 3’
S AS
5’
Single-Strand DNA Incorporation Paired nicking (PAM-in)
S AS
Extensive 5’ resection
Futile homology search
5’
S
3’
3’
5’
S AS
S 3’
S AS
B
Strand invasion, 3’ processing and synthesis
Single-strand DNA incorporation
5’
S
5’
3’ 3’
5’
S AS
S Strand annealing
Strand displacement
S AS
S
S Flap cleavage, ligation and single-strand gap repair
S AS
S AS
Flap cleavage, ligation and single-strand nick repair
S AS (3) Predicted conversion tracts
(4) Predicted conversion tracts
A
B
Biotin SH
XhoI
XbaI
TY
XhoI
XhoI
XbaI
BFP_CF
C
ssDI
XhoI
SH SH
SH
XbaI
TY
SDSA
Digest Pull-down Denature
Biotin SH
XbaI
SH SH
Digest Pull-down Denature
Strepavidin
BFP_CF
BFP_QR
SH
Cas9
nCas9
nCas9
Cas9
nCas9
nCas9
sgRNA*
S
AS
AS
PAM-out
PAM-in
ODNs
S
S
S
S
S
Model
SDSA
ssDI
SDSA
SDSA
ssDI
Input (25 cycles) Output (40 cycles) *nCas9 (D10A) nicks com plem entary to the strand of the sgRNA
Strepavidin BFP_QR
Meganuclease-induced precise genome engineering
dsDNA donors (for large mutations)
ODN donors (for small modifications)
including viruses with dsDNA intermediates
Homology
Heterology
DSBs Double-strand break repair (0) Conversion tracts
Homology Effective complementary Effective same strand lesion strand lesion
Nicks Inefficient
Synthesis-dependent strand annealing (1) Conversion tracts
-20 bp Effective conversion zone(~kb)
Single-strand DNA Incorporation (2) Conversion tracts
+10 bp
Effective conversion zone (~30 bp)
-20 bp
+10 bp
Effective conversion zone (~30 bp)
Downloaded from genome.cshlp.org on April 20, 2017 - Published by Cold Spring Harbor Laboratory Press
Mechanisms of precise genome editing using oligonucleotide donors Yinan Kan, Brian Ruis, Taylor Takasugi, et al. Genome Res. published online March 29, 2017 Access the most recent version at doi:10.1101/gr.214775.116
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