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2017 ; 28
(7-8
): 247-261
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CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and
delivery of genome engineering tools
#MMPMID28634692
Cebrian-Serrano A
; Davies B
Mamm Genome
2017[Aug]; 28
(7-8
): 247-261
PMID28634692
show ga
Robust and cost-effective genome editing in a diverse array of cells and model
organisms is now possible thanks to the discovery of the RNA-guided endonucleases
of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows
high levels of activity but, depending on the application, has been associated
with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis
at genomic sequences resembling the target sequence. Secondly, the stringent
requirement for a specific motif adjacent to the selected target site can limit
the target range of this enzyme. Lastly, the physical size of Cas9 challenges the
efficient delivery of genomic engineering tools based on this enzyme as viral
particles for potential therapeutic applications. Related and parallel strategies
have been employed to address these issues. Taking advantage of the wealth of
structural information that is becoming available for CRISPR-Cas effector
proteins, Cas9 has been redesigned by mutagenizing key residues contributing to
activity and target recognition. The protein has also been shortened and
redesigned into component subunits in an attempt to facilitate its efficient
delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the
properties of Cas9 orthologues and other related effector proteins from diverse
bacterial species, some of which exhibit different target site specificities and
reduced molecular size. It is hoped that the improvements in accuracy, target
range and efficiency of delivery will facilitate the therapeutic application of
these site-specific nucleases.
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