eNeuro, Journal Year: 2024, Volume and Issue: 11(8), P. ENEURO.0264 - 24.2024
Published: Aug. 1, 2024
In 1987, while investigating the iap gene in Escherichia coli, Nakata and colleagues identified "five highly homologous sequences of 29 nucleotides were arranged as direct repeats with 32 spacing."Perplexed by this discovery, authors stated, "so far, no sequence to these has been found elsewhere prokaryotes, biological significance is not known."These few sentences launched a decades-long investigation into mystery clustered regularly interspaced short palindromic repeats, or CRISPR (Ishino et al., 1987).Since its first identification, CRISPR-an adaptive immune response prokaryotic organisms recognize combat infectious DNA-has developed tool for targeted editing variety cells (Jansen 2002).The utility lies easy targeting virtually any genomic location RNA guide.In simplest form, two components that must be expressed perform genome are Cas9 nuclease guide (gRNA; Hille Charpentier, 2016).Due simplicity, high efficiency multiplex capabilities, CRISPR-Cas become relied upon tool.As development effective, reliable, customizable vivo technologies advanced, so too did genetically encoded tools visualizing manipulating cellular function vivo.Optogenetics several other transgene technologies-fiber photometry, chemogenetics, one-photon imaging-emerged early 2000s, enabling optical recording manipulation specified cell types.While genetic reveals impact specific genes on nervous system functions diseases, like optogenetics allow functional roles circuits behavior.The next logical step progression was combine expression, facilitating groundbreaking insights how regulate circuit activity.Currently, however, platforms cell-specific expression neuronal visualization inadequate.The large Cas enzyme limited packaging space AAVs prevent reliable copackaging transgenes Cas, necessitating two-vector transduction (Swiech 2015).However, coinjection often inefficient.The recent eNeuro article Moffa al. (2024) addressed issue developing platform combines Cre-dependent mouse lines Cre-driver lines, type-specific (Platt 2014).Now, single viral vector containing gRNA choice can used achieve an edited population fully overlaps expression.This provides robust approach test novel effects synaptic transmission, activity, morphology, behavior.To single-vector CRISPR-Cas9 editing, ( 2024) carefully designed three discrete constructs where either ChRonos, GCaMp8f, mCherry inserted modified vectors along
Language: Английский