Gene Editing Battle Minimizing Off Target Effects

The design of editing nucleases in a way that reduces off-target effects was a major issue not only for the basic science approaches, but also for their putative clinical and industrial use.

The delivery of ZFNs and TALENs, either in vivo or in vitro, can lead to toxicity or lethality due to binding at off-target sites and the induction of undesired DNA cleavage. Mutations in FokI in the case of ZFNs and TALENs were eventually introduced to promote FokI endonuclease activity only during heterodimerization events at sites bound by two discrete nucleases. Off-target cleavage of CRISPR-Cas9 systems mostly comes from the recognition of fully or partially complementary genomic sites by sgRNAs. Various approaches have been suggested to limit off-target cleavage, including limiting the amount and time of active Cas9 protein in cells by selective delivery methods or by altering Cas9 half-time. A number of Cas9 variants with lower off-target specificity have been developed, including HF-Cas9, eCas9, and HypaCas9. New variants of Cas9 and Cas9 homologs, such as CRISPR-Cas12 (Cpf1) and CRISPR-Cas13a (C2c2), can recognize different PAMs, not only significantly increasing options for precision genome editing, but also potentially having higher on-target specificity. An interesting alternative is a Cas9 fusion with FokI nuclease, which combines the advantages of ZFNs/TALENs and the CRISPR-Cas9 system. When fused to the FokI endonuclease, dCas9 has greater specificity compared to Cas9 alone. This specificity comes from the dCas9-FolkI cleavage activity, which depends strictly on the sgRNAs. The dCas9 protein proves to be a viable means to control gene expression by interfering with transcription.

#biology #biotechnology #science #genetics #genomics #genesis #bio_facts #dna #rna #protein #proteomics #molecular #molecular_biology #molecularbiology #molecularorbitaltheory #moleculargenetics #molecular_basis_of_inheritance #sciencefacts #scienceexperiment #scienceproject #crisprcas9 #crispr #geneediting #sequence #sequencing #rtpcr #pcr #ngs #aminoacids12 #aminoacidos #aminoacid #nucleotides #nucleotide