Question

Do HDR templates shelter a nuclear localization signal? Explain

Answer 1

HDR templates are used to create some specific mutations or insertion of new elements into gene which require a certain amount of homology leading to the surrounding the target sequence that will be modified.

Homology directed repair (HDR) is a naturally occurring nucleic acid repair system. It helps in modifying genomes in humans as well as other organisms. The CRISPR/Cas9 system can be used to create targeted double strand breaks. CRISPR/Cas9 technology is being used to control the specificity of HDR genome engineering techniques.Cas9 enzymes along with CRISPR sequences form the basis of this technology of CRISPR-Cas9.

Nucleocytoplasmic transport of macromolecules in the eukaryotic cells is used as a gene editing that is the intended nuclease must gain access to the interior of the nucleus and genomic content. For transit through the nuclear pore complex, this intracellular trafficking system recognizes nuclear import sequences by karyopherins .For this purpose a CRISPR/Cas system is used . This system of CRISPR/Cas causes alteration of the genome which includes appending one or more NLSs to the nuclease (e.g. Cas9) Also the alteration of the nuclear localization of Cas9 can be used as a means to control editing.Its functions include inducing many processes like the iCas system can prevent nuclear entry until addition of an external cue. Also design of a split Cas9 uses a NES sequence to restrict localization of one half of the nuclease till there is an addition of an exogenous signal. Optimization of CRISPR-based editing in various organisms has also focused on the placement, number, and identity of NLS sequence.