Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs

摘要

Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are new classes of genome-editing tools that target desired genomic sites in mammalian cells (Miller et al., 2011; Hockemeyer et al., 2011; Cong et al., 2013; Mali et al., 2013; Jinek et al., 2013). TALENs bind as a pair around a genomic site in which a double-strand break (DSB) is introduced by a dimer of Fokl nuclease domains. Recently published type II CRISPR/Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes a 20-nucleotide DNA sequence ("proto-spacer") beginning with G and immediately preceding an NGG motif recognized by Cas9-constituting a G(N)_19NGG target DNA sequence-resulting in a DSB three nucleotides upstream of the NGG motif (Jinek et al., 2012). However it is generated, the DSB instigates either nonhomologous end-joining (NHEJ), which is error-prone and conducive to frameshift mutations (indels) that knock out gene alleles, or homology-directed repair (HDR), which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome.