TY - JOUR
T1 - Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex
AU - Wang, Joy Y.
AU - Hoel, Christopher M.
AU - Al-Shayeb, Basem
AU - Banfield, Jillian F.
AU - Brohawn, Stephen G.
AU - Doudna, Jennifer A.
N1 - Funding Information:
We thank Paul Tobias for computational resources at the Cal-Cryo EM facility, and Dr. James Hurley and Dr. Eva Nogales for supporting the microscopy work. This material is based upon work supported by the National Science Foundation under award number 1817593. J.Y.W. is supported by the US National Science Foundation Graduate Fellowship and previously by the Berkeley Graduate Fellowship. B.A.-S. is supported by the US National Science Foundation Graduate Fellowship. S.G.B. is a New York Stem Cell Foundation Robertson Neuroscience Investigator. J.A.D. is an investigator of the Howard Hughes Medical Institute. We thank A.V. Wright for input on the manuscript, C. Alza and M. Jain for technical assistance, and G.J. Knott, J.-J. Liu, A. Lapinaite, J. Cofsky, K.M. Soczek, P. Pausch and members of the Doudna laboratory and Brohawn laboratory for comments and discussions.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/5/6
Y1 - 2021/5/6
N2 - CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1—Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.
AB - CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1—Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.
UR - http://www.scopus.com/inward/record.url?scp=85105464985&partnerID=8YFLogxK
U2 - 10.1038/s41467-021-22900-y
DO - 10.1038/s41467-021-22900-y
M3 - Article
C2 - 33958590
AN - SCOPUS:85105464985
SN - 2041-1723
VL - 12
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 2571
ER -