Chapter 19
CRISPR Guide RNA Design Guidelines for Efficient Genome
Editing
Patrick Schindele, Felix Wolter, and Holger Puchta
Abstract
The simple applicability and facile target programming of the CRISPR/Cas9-system abolish the major
boundaries of previous genome editing tools, making it the tool of choice for generating site-specific
genome alterations. Its versatility and efficacy have been demonstrated in various organisms; however,
accurately predicting guide RNA efficiencies remains an organism-independent challenge. Thus, designing
optimal guide RNAs is essential to maximize the experimental outcome. Here, we summarize the current
knowledge for guide RNA design and highlight discrepancies between different experimental systems.
Key words Genome editing, CRISPR, Cas9, gRNA design, gRNA secondary structure, Mismatch
tolerance, CRISPR prediction tool
1 Introduction
1.1 Genome Editing
with CRISPR/Cas9
Targeted manipulation of DNA through site-specific double-strand
breaks (DSBs) embodies the cornerstone of modern biotechnology. The challenge to target sites of interest continuously decreased
over time with the discovery and development of novel tools, such
as engineered nucleases [1]. The CRISPR/Cas9 system with its
two-component setup accompanied by the simple target programming constitutes the current gold standard within the available
toolbox. In this system, a complex of the Cas9 nuclease and a
guide RNA (gRNA) mediates DSB induction at a selected target
site. The gRNA is of major relevance, mediating target DNA recognition and binding on the one hand and activation of target
DNA cleavage by Cas9 on the other hand [2]. The variable region
of the gRNA (guide) determines the site of target DNA binding
and can be adjusted to the sequence of interest. Employing this
system, applications such as single and multiplex editing, epigenetic
and transcriptional regulation, visualization of genomic loci, and
base editing are feasible within a large number of organisms (for
details see reviews [3–6]). The target selection solely requires an
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_19, © Springer Science+Business Media, LLC, part of Springer Nature 2020
331
CRISPR Guide RNA Design Guidelines for Efficient Genome
Editing
Patrick Schindele, Felix Wolter, and Holger Puchta
Abstract
The simple applicability and facile target programming of the CRISPR/Cas9-system abolish the major
boundaries of previous genome editing tools, making it the tool of choice for generating site-specific
genome alterations. Its versatility and efficacy have been demonstrated in various organisms; however,
accurately predicting guide RNA efficiencies remains an organism-independent challenge. Thus, designing
optimal guide RNAs is essential to maximize the experimental outcome. Here, we summarize the current
knowledge for guide RNA design and highlight discrepancies between different experimental systems.
Key words Genome editing, CRISPR, Cas9, gRNA design, gRNA secondary structure, Mismatch
tolerance, CRISPR prediction tool
1 Introduction
1.1 Genome Editing
with CRISPR/Cas9
Targeted manipulation of DNA through site-specific double-strand
breaks (DSBs) embodies the cornerstone of modern biotechnology. The challenge to target sites of interest continuously decreased
over time with the discovery and development of novel tools, such
as engineered nucleases [1]. The CRISPR/Cas9 system with its
two-component setup accompanied by the simple target programming constitutes the current gold standard within the available
toolbox. In this system, a complex of the Cas9 nuclease and a
guide RNA (gRNA) mediates DSB induction at a selected target
site. The gRNA is of major relevance, mediating target DNA recognition and binding on the one hand and activation of target
DNA cleavage by Cas9 on the other hand [2]. The variable region
of the gRNA (guide) determines the site of target DNA binding
and can be adjusted to the sequence of interest. Employing this
system, applications such as single and multiplex editing, epigenetic
and transcriptional regulation, visualization of genomic loci, and
base editing are feasible within a large number of organisms (for
details see reviews [3–6]). The target selection solely requires an
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_19, © Springer Science+Business Media, LLC, part of Springer Nature 2020
331