Cite as: J. Strecker et al., Science
10.1126/science.abb2920 (2020).TECHNICAL RESPONSESPublication date: 5 June 2020 http://www.sciencemag.org 1Rice et al. ( 1 ) raise an important point about the mechanism
of integration of the type V CRISPR-associated transposase
(CAST) systems we recently characterized ( 2 ). Although
CAST loci belong to the Tn7-like class of transposons, addi-
tional analysis indicates that type V CAST loci are most
closely related to transposons of the Tn5053 family ( 3 , 4 ), a
distinct family of transposons that has not yet been mecha-
nistically characterized. In particular, the Tn5053 family
transposons lack TnsA, the enzyme responsible for the 5′
donor cleavage in the Tn7 transposon ( 5 , 6 ), and result in a
co-integrate product that is resolved by the site-specific re-
combinase TniR ( 4 ). Thus, in CAST systems, the 5′ donor
ends might not be cleaved, resulting in a co-integrate prod-
uct containing duplicated cargo DNA and the donor back-
bone. Alternatively, a CAST protein might either cleave the
5 ′ donor end or help resolve the co-integrate to yield a sim-
ple insertion.
To investigate the exact structure of the insertion prod-
uct, we performed nanopore sequencing of 15 Scytonema
hofmanni CAST (ShCAST)–mediated genome insertions in
E. coli and found nine simple insertions and nine co-
integrates across several target sites (Fig. 1A). Similarly, a
genetic assay using a plasmid target revealed about 20% co-
integrate insertions (Fig. 1B). A tentative model is that the
initial insertion product is a co-integrate that can be re-
solved by the cellular DNA recombination and repair sys-
tems. However, we cannot rule out that CAST components
contribute to 5′ donor cleavage or co-integrate resolution in
E.coli or the native host. Notably, all instances of CAST in-
sertion in cyanobacterial genomes identified to date are
simple insertions. We will continue to investigate these as a
part of our ongoing studies.Use of linear or 5′ nicked DNA donors prevents co-
integrate formation (Fig. 1C), thereby providing an approach
for applying CAST for homologous recombination–
independent genome engineering. Continued investigation
of the mechanism of CAST is expected to yield a deeper un-
derstanding of the biology of this remarkable DNA integra-
tion system and propel its development as a molecular
technology.REFERENCES- P. A. Rice, N. L. Craig, F. Dyda, Comment on “RNA‐guided DNA insertion with
CRISPR‐associated transposases”. Science 368 , eabb2022 (2020). - 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 - G. Y. Kholodii, O. V. Yurieva, O. L. Lomovskaya, Z. Gorlenko, S. Z. Mindlin, V. G.
Nikiforov, Tn5053, a mercury resistance transposon with integron’s ends. J. Mol.
Biol. 230 , 1103–1107 (1993). doi:10.1006/jmbi.1993.1228 Medline - G. Y. Kholodii, S. Z. Mindlin, I. A. Bass, O. V. Yurieva, S. V. Minakhina, V. G.
Nikiforov, Four genes, two ends, and a res region are involved in transposition of
Tn5053: A paradigm for a novel family of transposons carrying either a mer
operon or an integron. Mol. Microbiol. 17 , 1189–1200 (1995). doi:10.1111/j.1365-
2958.1995.mmi_17061189.x Medline - M. C. Biery, M. Lopata, N. L. Craig, A minimal system for Tn7 transposition: The
transposon-encoded proteins TnsA and TnsB can execute DNA breakage and
joining reactions that generate circularized Tn7 species. J. Mol. Biol. 297 , 25–37
(2000). doi:10.1006/jmbi.2000.3558 Medline - R. J. Sarnovsky, E. W. May, N. L. Craig, The Tn7 transposase is a heteromeric
complex in which DNA breakage and joining activities are distributed between
different gene products. EMBO J. 15 , 6348–6361 (1996). doi:10.1002/j.1460-
2075.1996.tb01024.x Medline
24 March 2020; accepted 11 May 2020
Published online 5 June 2020
10.1126/science.abb2920Response to Comment on “RNA-guided DNA insertion with
CRISPR-associated transposases”
Jonathan Strecker1,2,3,4, Alim Ladha1,2,3,4, Kira S. Makarova^5 , Eugene V. Koonin^5 , Feng Zhang1,2,3,4*
(^1) Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 2 McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA
02139, USA. 3 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 4 Department of Biological
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 5 National Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Bethesda, MD 20894, USA.
*Corresponding author. Email: [email protected]
Rice et al. suggest that the CRISPR-associated transposase ShCAST system could lead to additional
insertion products beyond simple integration of the donor. We clarify the outcomes of ShCAST-mediated
insertions in Escherichia coli, which consist of both simple insertions and integration of the donor plasmid.
This latter outcome can be avoided by use of a 5′ nicked DNA donor.