![]() In CRISPR activation (CRISPRa), enhancer domains (e.g., VP64 or VPR) are attached to dCas9 and upregulates gene transcription when targeted to the gene promoter. CRISPR inactivation (CRISPRi) uses a deactivated Cas9 (dCas9) to target the promoter or protein-coding region of the chosen gene and works in tandem with repressor domains (e.g., KRAB) to inhibit gene transcription. If the target site is in the exon or intron of the target gene, then this indel mutation could cause premature stop codon formation, which produces a non-sense transcript that will be degraded by non-sense-mediated decay. The CRISPR-Cas9 knockout (CRISPRko) method uses a catalytically active Cas9 to cut the target site close to the protospacer adjacent motif (PAM) within the sgRNA binding region, introducing a small indel (insertion/deletion) mutation (Fig. There are three main types of CRISPR-based genome perturbation technologies that researchers can use for pooled screens (Fig. By transducing the sgRNA lentivirus pool into desired cell types (often at a multiplicity of infection much smaller than one to ensure less than one viral particle per cell), high-throughput, parallel loss-of-function genomic perturbations can be carried out to infer their functional relevance. The oligo library can be cloned onto a lentiviral vector system to facilitate cellular delivery, integration, and expression. High-throughput DNA synthesis platforms can generate a library of oligos with various sequence features (hundred of thousands or even millions), with each oligo encoding a different sgRNA sequence, and thus a different DNA target. The intriguing programmable RNA-guided DNA targeting feature of CRISPR-Cas allows it to be scaled up to target many genomic sites in parallel in one experiment. Fusing dCas molecules to transcriptional or epigenetic effector domains allows sequence-specific gene regulation for either gene activation (CRISPRa) or repression (CRISPRi). In addition to editing, nuclease-deactivated Cas (dCas) molecules have been engineered by introducing silencing mutations to abrogate the nuclease activity. ![]() This cutting mechanism on the genomic DNA triggers host non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways, which typically leads to loss-of-gene function. Taking the most commonly used CRISPR-Cas9 system as an example, the Streptococcus pyogenes Cas9 protein can complex with a 110-nucleotide (nt) sgRNA containing a 20-nt sequence that complementarily binds to the target DNA region and induces a double-stranded break (DSB). Due to its programmable nature using customizable single-guide RNAs (sgRNAs), CRISPR-Cas has enabled powerful pooled screens to explore the function of genetic perturbations at a genome-wide scale. The Datasheet can be retrieved by clicking on the PID in the list.Clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated (Cas) proteins are a class of bacteria-encoded, RNA-guided, programmable DNA targeting and cleavage systems.
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