Abstract:
:The RNA-guided, sequence-specific endonuclease Cas9 has been widely adopted as genome engineering tool due to its efficiency and ease of use. Derived from the microbial CRISPR (clustered regularly interspaced short palindromic repeat) type II adaptive immune system, Cas9 has now been successfully engineered for genome editing applications in a variety of animal and plant species. To reduce potential off-target mutagenesis by wild-type Cas9, homology- and structure-guided mutagenesis of Streptococcus pyogenes Cas9 catalytic domains has produced "nicking" enzymes (Cas9n) capable of inducing single-strand nicks rather than double-strand breaks. Since nicks are generally repaired with high fidelity in eukaryotic cells, Cas9n can be leveraged to mediate highly specific genome editing, either via nonhomologous end-joining or homology-directed repair. Here we describe the preparation, testing, and application of Cas9n reagents for precision mammalian genome engineering.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Trevino AE,Zhang Fdoi
10.1016/B978-0-12-801185-0.00008-8subject
Has Abstractpub_date
2014-01-01 00:00:00pages
161-74eissn
0076-6879issn
1557-7988pii
B978-0-12-801185-0.00008-8journal_volume
546pub_type
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abstract::Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of ...
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