Cite as: P. A. Rice et al., Science
10.1126/science.abb2022 (2020).TECHNICAL COMMENTSPublication date: 5 June 2020 http://www.sciencemag.org 1The Scytonema hofmanni CRISPR-associated transposase
(ShCAST) system described by Strecker et al. ( 1 ) is based on
a Tn7-like transposon that carries its own nuclease-defective
CRISPR-Cas system, which it uses for RNA-guided target
choice ( 2 ). The canonical Escherichia coli transposon Tn7
uses two enzymes to create double-strand breaks at the do-
nor-transposon junctions: TnsB to cleave one strand (and
subsequently attach it to target DNA) and TnsA to cleave
the other (second) DNA strand, leading to excision of the
transposon from a donor plasmid followed by its insertion
into a target plasmid. When TnsA is mutated, however, the
products are not these simple insertions of the transposon,
but rather co-integrates in which the entire donor plasmid,
flanked by copies of the transposon, is inserted into the tar-
get ( 3 ). Similar co-integrate products are also created by
systems such as Mu transposase, which is a TnsB homolog
that lacks a TnsA partner ( 4 ).
The ShCAST Tn7-like transposon used by Strecker et al.
is naturally lacking in TnsA. Although some DDE transposa-
ses harbor insertion domains that trigger other mechanisms
for cleavage of the second strand, ShCAST TnsB does not.
Homologies to the other systems noted above therefore sug-
gest that their products may not be solely simple insertions
as cartooned, but instead may include fusions of the donor
plasmid and target. Unfortunately, no experiments were
reported that would distinguish between the two possibili-
ties, such as measuring the size of purified “pInsert” product
plasmids containing the transposon or testing for the pres-
ence of donor DNA in the final products. Although the
ShCAST integration system is suggested to bypass the need
for homologous recombination–mediated DNA repair nor-
mally found in some CRISPR/Cas applications, processing ofco-integrate products to remove the donor DNA would likely
still require these recombination functions unless another
nuclease processed the second strand at the donor site.REFERENCES- J. Strecker, A. Ladha, Z. Gardner, J. L. Schmid-Burgk, K. S. Makarova, E. V. Koonin,
F. Zhang, RNA-guided DNA insertion with CRISPR-associated transposases.
Science 365 , 48–53 (2019). doi:10.1126/science.aax9181 Medline - J. E. Peters, K. S. Makarova, S. Shmakov, E. V. Koonin, Recruitment of CRISPR-Cas
systems by Tn7-like transposons. Proc. Natl. Acad. Sci. U.S.A. 114 , E7358–E7366
(2017). doi:10.1073/pnas.1709035114 Medline - E. W. May, N. L. Craig, Switching from cut-and-paste to replicative Tn7
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Medline - A. B. Hickman, F. Dyda, DNA Transposition at Work. Chem. Rev. 116 , 12758–12784
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6 February 2020; accepted 11 May 2020
Published online 5 June 2020
10.1126/science.abb2022Comment on “RNA-guided DNA insertion with CRISPR-
associated transposases”
Phoebe A. Rice^1 *, Nancy L. Craig^2 , Fred Dyda^3
(^1) Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. (^2) Department of Molecular Biology and Genetics, Johns Hopkins
University School of Medicine, Baltimore, MD, USA.^3 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda,
MD, USA.
*Corresponding author. Email: [email protected]
Strecker et al. (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type
transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this
system has great promise, we note that the well-established biochemistry of Tn7 suggests that the
particular system used may insert not only the transposon but also the entire donor plasmid.