Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a

Daan C. Swarts, John van der Oost, Martin Jinek*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

92 Citations (Scopus)

Abstract

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

Original languageEnglish
Pages (from-to)221-233.e4
JournalMolecular Cell
Volume66
Issue number2
DOIs
Publication statusPublished - 2017

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Seeds
DNA
DNA Cleavage
CRISPR-Associated Proteins
Francisella
Endoribonucleases
Deoxyribonucleases
RNA Precursors
Catalytic Domain
RNA
Technology
Enzymes

Keywords

  • Cas12a
  • Cas9
  • Cpf1
  • CRISPR RNA
  • CRISPR-Cas
  • crRNA processing
  • nuclease
  • R-loop
  • seed sequence
  • target DNA cleavage

Cite this

@article{1286b4d2440441bc9cce93282faca20b,
title = "Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a",
abstract = "The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.",
keywords = "Cas12a, Cas9, Cpf1, CRISPR RNA, CRISPR-Cas, crRNA processing, nuclease, R-loop, seed sequence, target DNA cleavage",
author = "Swarts, {Daan C.} and {van der Oost}, John and Martin Jinek",
year = "2017",
doi = "10.1016/j.molcel.2017.03.016",
language = "English",
volume = "66",
pages = "221--233.e4",
journal = "Molecular Cell",
issn = "1097-2765",
publisher = "Elsevier",
number = "2",

}

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. / Swarts, Daan C.; van der Oost, John; Jinek, Martin.

In: Molecular Cell, Vol. 66, No. 2, 2017, p. 221-233.e4.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a

AU - Swarts, Daan C.

AU - van der Oost, John

AU - Jinek, Martin

PY - 2017

Y1 - 2017

N2 - The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

AB - The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

KW - Cas12a

KW - Cas9

KW - Cpf1

KW - CRISPR RNA

KW - CRISPR-Cas

KW - crRNA processing

KW - nuclease

KW - R-loop

KW - seed sequence

KW - target DNA cleavage

U2 - 10.1016/j.molcel.2017.03.016

DO - 10.1016/j.molcel.2017.03.016

M3 - Article

VL - 66

SP - 221-233.e4

JO - Molecular Cell

JF - Molecular Cell

SN - 1097-2765

IS - 2

ER -