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CRISPR / cas9 - Based Genome Editing

One major gene editing system is CRISPR (clustered regularly interspaced short palindromic repeats) involving RNA-guided engineered nucleases. The system was first identified as an adaptive immunity pathway in prokaryotes providing resistance to bacterial phage and analogous to the RNA interference process found in eukaryotes. It has since been modified and adapted for the engineering of genomes.

 

Essentially, CRISPR may be thought of as a programmable restriction enzyme system. It utilises double-stranded sequence-specific breaks and repair using HDR (homology directed repair) via homologous recombination. The availability of a vast amount of sequence information has allowed the development of the system into an essential molecular biology technique.

 

The system consists of two parts, a short synthetic guide RNA (gRNA) and a non-specific double-stranded endonuclease termed Cas9 (Cas: CRISPR-associated). The guide RNA has a specific 20 nucleotide spacer/targeting sequence (crRNA) that identifies the genomic target to be identified and modified, linked to a trans-activating (tracrRNA) sequence necessary for the recruitment and stability of Cas9 nuclease. The

gRNA sequence and Cas9 nuclease elements are constructed into a plasmid expression vector that can be used to transfect cells under study. Transcription of the gRNA and Cas9 results in crRNA that binds to the DNA to be altered, which is held in the Cas9 nuclease with the aid of the tracrRNA. Indeed, it is the fact that the target sequence can be reprogrammed simply by changing the 20 nucleotides in the crRNA that makes the system so elegant. DNA is then digested by the Cas9 nuclease at specific points on both DNA strands. A donor DNA sequence may be included with a desired feature, such as a base change, insertion or deletion. The cell then employs homologous recombination to repair the break and incorporate the new DNA sequence. It is also possible to program Cas9 with multiple guide RNAs to allow multiplex site-specific editing for designing large deletions, inversions and translocations. Further refinements of the editing system have allowed the targeting of protein domains for transcriptional regulation and epigenetic modification.

Figure [1] - The CRISPR/Cas9 gene editing system.

The nuclease Cas9 is targeted to DNA by a guide RNA consisting of a specific 20 nucleotide spacer/targeting sequence (crRNA) that identifies the DNA to modified, linked to a trans-activating (tracrRNA) sequence necessary for the recruitment and stability of Cas9 nuclease. DNA is then digested by the Cas9 nuclease at specific points on both DNA strands. Homologous recombination to repair the break and incorporate the new DNA sequence then follows.

 

Source: Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology, Eighth Edition