Advances in Chemical Biology

0503188 - ÚMG 2019 RIV CZ eng M - Monography Chapter
Schuster, Bjorn - Chawengsaksophak, Kallayanee
CRISPR/Cas9, a bacterial immune system became the most widely used tool for genome engineering.
Advances in Chemical Biology. Praha: OPTIO CZ, 2019 - (Bartůněk, P.), s. 92-103. ISBN 978-80-88011-03-3
R&D Projects: GA MŠMT LO1220
Institutional support: RVO:68378050
Keywords : CRISPR/Cas * RNA-guided Cas9 endonuclease * PAM sequence
OECD category: Biochemistry and molecular biology

The CRISPR/Cas system has revolutionized the way we perform genomic manipulations in a wide range of cell and model organisms today and has great potential for therapeutic application. Due to its simple design and affordability it has become a versatile research tool, used in many diverse applications ranging from genomic manipulations to study the function of individual genes to high-throughput screens covering the whole genome. The major CRISPR/Cas system currently used is the RNA-guided Cas9 endonuclease which originates from Streptococcus pyogenes (SpCas9). The original CRISPR/Cas9 is composed of three components: two short guide RNAs (a crRNA which defines the target site on DNA and a tracrRNA connecting the crRNA) and the Cas9 protein. Following complex formation of the guide RNAs with the Cas9 protein, the endonuclease activity becomes activated, allowing the introduction of site-specific DNA double strand breaks for genomic manipulations. The specificity of CRISPR/Cas9 is given by a 20 nt sequence of the guide RNA, which has to be complementary to the DNA target site, and the requirement of a PAM sequence recognized by the Cas9 protein. The PAM sequence within the target DNA for Cas9 is NGG. Although the original CRISPR/Cas9 system has proven to be a very powerful and robust tool for genome engineering, it does have some drawbacks with the main concerns being the specificity, e.g. undesired side effects on off-target sites with similar sequences to the on-target site, and the restrictions due to the requirement of the PAM sequence, which limits the number of accessible target loci within the genome. In this mini review we aim to give an overview of the SpCas9 system and the improved versions of the original Cas9 protein addressing the aforementioned complications.
Permanent Link: http://hdl.handle.net/11104/0295016