CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks.

ELife
Joshua C CofskyJennifer A Doudna

Abstract

Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3' side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5' side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

References

Feb 1, 1996·Journal of Molecular Graphics·W HumphreyK Schulten
Oct 10, 2003·Nucleosides, Nucleotides & Nucleic Acids·Chinh T BuiRichard G H Cotton
Sep 3, 2004·Journal of Molecular Biology·Ekaterina ProtozanovaMaxim D Frank-Kamenetskii
Oct 21, 2004·Molecular Cell·Jayakrishnan Nandakumar, Stewart Shuman
Oct 14, 2005·Journal of Computational Chemistry·James C PhillipsKlaus Schulten
Mar 24, 2007·Science·Rodolphe BarrangouPhilippe Horvath
Apr 25, 2007·Nucleic Acids Research·Warren A Kibbe
Oct 22, 2008·Organic & Biomolecular Chemistry·Akiko Nomura, Akimitsu Okamoto
Apr 13, 2010·Acta Crystallographica. Section D, Biological Crystallography·P EmsleyK Cowtan
Apr 2, 2011·Nature·Elitza DeltchevaEmmanuelle Charpentier
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Sep 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Giedrius GasiunasVirginijus Siksnys
Jan 31, 2014·Nature·Samuel H SternbergJennifer A Doudna
Sep 29, 2015·Nature Reviews. Microbiology·Kira S MakarovaEugene V Koonin
Feb 4, 2016·Science·Fuguo JiangJennifer A Doudna
Apr 27, 2016·Cell·Takashi YamanoOsamu Nureki
Jul 14, 2016·Nucleic Acids Research·Florian Häse, Martin Zacharias
Nov 8, 2016·Nature Methods·Jing HuangAlexander D MacKerell
Jun 1, 2017·Nature·Stefano StellaGuillermo Montoya
Jun 13, 2017·Current Opinion in Microbiology·Eugene V KooninFeng Zhang
May 8, 2018·Proceedings of the National Academy of Sciences of the United States of America·Digvijay SinghTaekjip Ha
Jul 19, 2018·Nature Communications·Yongmoon JeonSangsu Bae
Aug 7, 2018·Molecular Cell·Isabel StrohkendlRick Russell
Dec 14, 2018·Science·Winston X YanDavid A Scott
Jan 15, 2019·Molecular Cell·Daan C Swarts, Martin Jinek
Feb 6, 2019·Nature·Jun-Jie LiuJennifer A Doudna
Feb 9, 2019·Molecular Cell·Madzia P CrossleyKarlene A Cimprich
Apr 3, 2019·Nature Structural & Molecular Biology·Gavin J KnottJennifer A Doudna
May 31, 2019·Nature Reviews. Molecular Cell Biology·Adrian Pickar-Oliver, Charles A Gersbach

❮ Previous
Next ❯

Citations

Dec 22, 2020·The CRISPR Journal·Yongqiang WuZiru Yin
Mar 5, 2021·Bio-protocol·Joshua C Cofsky, Jennifer A Doudna
Mar 20, 2021·Molecular Therapy. Nucleic Acids·Mai H TranMichael Farzan
May 1, 2021·Biomolecules·Matvey Mikhailovich MurashkoDenis Eriksonovich Demin
May 13, 2021·Analytical Chemistry·Ashwin Ramachandran, Juan G Santiago
Apr 25, 2021·Biophysical Journal·D W Bo BroadwaterHarold D Kim
Aug 13, 2021·Nature Structural & Molecular Biology·Patrick PauschJennifer A Doudna
Oct 28, 2020·Journal of Chemical Information and Modeling·Aakash SahaGiulia Palermo
Aug 22, 2021·Nucleic Acids Research·Oscar E Torres MontaguthMark D Szczelkun
Sep 11, 2021·Biotechnology Journal·Matthew S VerosloffJanice S Chen
Oct 7, 2021·Chemical Society Reviews·Yanan TangX Chris Le
Jan 20, 2022·Chemical Communications : Chem Comm·Heyjin SonSanghwa Lee

❮ Previous
Next ❯

Methods Mentioned

BETA
footprinting
electrophoresis
electrophoretic mobility shift assay
filter binding
in vitro transcription
gel filtration
PCR
dot-blot
nucleic acid spectroscopy
equilibrium titration

Software Mentioned

GraphPad
Prism
PyMOL
x3DNA
OligoCalc
Coot
ImageQuant
NAMD
XSEDE
VMD

Related Concepts

Related Feeds

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.

Related Papers

BioRxiv : the Preprint Server for Biology
John KuriyanIsaac Witte
Nucleic Acids Research
Yeraldinne Carrasco-SalasVincent Vanoosthuyse
BioRxiv : the Preprint Server for Biology
Yeraldinne Carrasco-SalasVincent Vanoosthuyse
The Journal of Biological Chemistry
Frederic Chedin, Craig J Benham
© 2022 Meta ULC. All rights reserved