What are the contributions mentioned in the paper "Targeted dna transposition using a dcas9-transposase fusion protein" ?

In this paper, the authors proposed a switchable transposase, which allows control over the timing of integration.

(Open Access) Targeted DNA transposition using a dCas9-transposase fusion protein (2019) | Shivam Bhatt

Targeted DNA transposition using a dCas9-transposase fusion

protein

Shivam Bhatt and Ronald Chalmers*

School of Life Sciences,

University of Nottingham,

Queen′s Medical Centre,

Nottingham NG7 2UH,

*To whom correspondence should be addressed.

Email: chalmers@nottingham.ac.uk

The copyright holder for this preprint (which wasthis version posted April 12, 2019. ; https://doi.org/10.1101/571653doi: bioRxiv preprint

SUMMARY

Homology directed genome engineering is limited by transgene size.

Although DNA transposons are more efficient with large transgenes, random

integrations are potentially mutagenic. Catalytically inactive Cas9 is attractive

candidate for targeting a transposase fusion-protein because of its high

specificity and affinity for its binding site. Here we demonstrate efficient Cas9

targeting of a mariner transposon. Targeted integrations were tightly

constrained at two adjacent TA dinucleotides about 20 bp to one side of the

gRNA binding site. Biochemical analysis of the nucleoprotein complexes

demonstrated that the transposase and Cas9 moieties of the fusion protein

can bind their respective substrates independently. In the presence of the

Cas9 target DNA, kinetic analysis revealed a delay between first and second

strand cleavage at the transposon end. This step involves a significant

conformational change that may be hindered by the properties of the

interdomainal linker. Otherwise, the transposase behaved normally and was

proficient for integration in vitro and in vivo.

The copyright holder for this preprint (which wasthis version posted April 12, 2019. ; https://doi.org/10.1101/571653doi: bioRxiv preprint

INTRODUCTION

Biotechnology and medicine are increasingly reliant on our ability to engineer

the mammalian genome. Lentiviruses are useful because gene-delivery

across the cell membrane is efficient and integration of the viral genome

provides long-term transgene expression. However, they are mutagenic

because they integrate at random sites using a mechanism similar to the cut-

and-paste transposons. An alternative strategy for ex vivo applications is to

establish genomic safe-havens for site-specific recombinases such as the

phage C31 integrase or the Cre recombinase (Branda and Dymecki, 2004;

Song and Palmiter, 2018). This approach lacks flexibility because it is limited

to transgene integration at a predetermined site.

Scar-less engineering of a target site must rely on the cells homologous

recombination machinery. Although desired modifications can be made very

precisely, the events are rare and difficult to recover. One way to boost the

rate of recombination by orders of magnitude is to make a DNA double strand

break at the target site (Urnov et al., 2005). This can be achieved using zinc

finger nucleases and transcription activator-like effector (TALE) nucleases,

which can themselves be engineered to target a user-defined site

(Chandrasegaran and Smith, 1999; Zhang et al., 2011). Two significant

problems are the extended lead-times and off-target cleavages. TALE and

zinc finger nucleases have now been largely superseded by the Cas9

nuclease. It can be programed to target a 20 bp recognition sequence, simply

by providing a matching guide RNA (gRNA). Off-target cleavage is relatively

low and the long recognition sequence provides ample specificity. However,

homologous recombination remains a limiting factor because the efficiency

falls off as the transgene size increases (Li et al., 2014).

Transposon vectors are widely used for gene delivery applications

(Grabundzija et al., 2010; Lamberg et al., 2002; Liang et al., 2009; Mates et

al., 2009; Way et al., 1984). Although their efficiency falls off as the cargo

The copyright holder for this preprint (which wasthis version posted April 12, 2019. ; https://doi.org/10.1101/571653doi: bioRxiv preprint

size is increased, they are less sensitive to this parameter than host-mediated

homologous recombination (Balciunas et al., 2006; Claeys Bouuaert et al.,

2013; Ding et al., 2005; Izsvak et al., 2000; Lohe and Hartl, 1996). However,

like the Lentiviruses, transposon vectors are mutagenic because the

integration sites are essentially random.

To combat random integration various groups have used site-specific DNA

binding proteins to target specific loci. Examples include Mos1, Sleeping

Beauty, piggyBac and ISY100, which were variously fused to zinc finger

proteins, TALEs and Gal4 (Feng et al., 2010; Ivics et al., 2007; Luo et al.,

2017; Maragathavally et al., 2006; Owens et al., 2013; Yant et al., 2007).

Although successful, targeting strategies have all me with significant problem:

namely, that the transposases are still capable of random integration. Since,

DNA binding proteins, such as zinc fingers and TALEs, spend most of their

time searching for their binding sites, targeted integrations are recovered

against a background of random events. To tackle this problem we are

currently developing a switchable transposase, which allows control over the

timing of integration. In parallel, we were considering the advantages of

potential targeting moieties. Cas9 is an obvious and attractive candidate

because extensive base pairing with the target provides a dwell time of

several hours (Ma et al., 2016). In contrast, DNA binding proteins remain

bound to their specific sites only for a matter of seconds or minutes.

Previously, there was an unsuccessful attempt to use a catalytically inactive

Cas9 (dCas9) to target piggyBac insertions to a hypoxanthine phosphoribosyl

transferase (HPRT) gene in human cells (Luo et al., 2017). Surprisingly,

instead of targeting the HPRT locus, the dCas9-piggyBac chimera protected it

from insertions. We therefore set out to test whether Cas9 is a general

inhibitor of transposition or whether the effect is peculiar to piggyBac. The

choice of the transposase moiety is limited by several factors. Although Tn5

is very active in vitro, and is used extensively in bacteria, it transposes poorly

in mammalian cells (Blundell-Hunter et al., 2018). Sleeping Beauty is efficient

The copyright holder for this preprint (which wasthis version posted April 12, 2019. ; https://doi.org/10.1101/571653doi: bioRxiv preprint

in vivo but lacks an in vitro system (Mates et al., 2009). While piggyBac is

probably the most efficient for gene delivery in vivo, it has a poor in vitro

system (Liang et al., 2009; Mitra et al., 2008), which precludes the

development of advanced applications such as direct delivery of

transpososomes.

Our model system is based on Hsmar1, a reconstituted mariner-family

transposon (Miskey et al., 2007; Robertson and Zumpano, 1997). Although it

is not as active as piggyBac or Sleeping Beauty in cell culture transfection

experiments, the reaction is 100% efficient in vitro (Claeys Bouuaert and

Chalmers, 2017). Furthermore, if the mariner transpososome is assembled in

vitro, it can be delivered cells directly (Trubitsyna et al., 2017). Hsmar1 also

has much lower non-specific nuclease activity than other mariner elements

such as Mos1, Mboumar1 and Himar1 (Claeys Bouuaert and Chalmers, 2010;

Dawson and Finnegan, 2003; Lampe et al., 1996; Lipkow et al., 2004; Munoz-

Lopez et al., 2008; Trubitsyna et al., 2015).

MATERIALS AND METHODS

DNA oligonucleotides and most dry chemicals were from Sigma Aldrich.

Enzymes were from New England Biolabs and DNA purification kits were from

Qiagen. The nucleotide sequences of all plasmids used in this work are given

in Supplemental Table 1. The β-galactosidase assays were as described by

Miller (Miller, 1972). LB agar indicator plates contained 40 µg/ml X-gal, when

present, .

For in vivo assays, dCas9 was expressed from derivatives of plasmid pdCas9

(Bikard et al., 2013). When present, CRISPR spacers were cloned into the

BsaI site as oligoduplexes (Supplemental Table 2). The Hsmar1 transposase

gene was added to the 5'- or 3'-end of the dCas9 gene by PCR using pdCas9

and pRC880 as templates: transposase-dCas9, pRC2302; dCas9-

transposases, pRC2303. An oligoduplex encoding spacer-7 was cloned into

The copyright holder for this preprint (which wasthis version posted April 12, 2019. ; https://doi.org/10.1101/571653doi: bioRxiv preprint

Targeted DNA transposition using a dCas9-transposase fusion protein

Figures

Citations

Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

References

Experiments in molecular genetics

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation

Highly efficient endogenous human gene correction using designed zinc-finger nucleases

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription

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