Question

CRISPR Gene editing

What happens to a bacterium if a double-strand DNA break is not repaired?

Answer 1

CRISPR:

CRISPR is Clustered Regulatory Interspaced Short Palindromic Repeats.

CRISPR associated –Protein 9 nuclease Caspase (Cas-9) is used as a molecular tool for editing of genomes.

It acts in a similar manner as genetic engineering or construction of GMO (genetically modified organism).

CRISPR/ Cas9 system has components that help in their gene editing:

1. crRNA- guide RNA (g RNA) and sequence recognizing trac rRNA

2. trac rRNA- in stem-loop structure, bind (to be activated) to crRNA

3. Cas9-active form have nuclease, nick formation, single strand/double strand breaks, editing.

4. sgRNA-single guide RNA-with combination of other components

Process:

• The components may be introduced within the organism through plasmid.
• The plasmid may be introduced by transfection or by direct injections into the target cells.
• Also, some virus mediated vectors may be used or liposomes.
• For transgenic organism creation for mutational related studies like cancer mutations, knockout of the wild type genes is used.
• The recognition point in the target DNA is designated as the PAM (Proto-spacer adjacent motif). The sequence is at the 3’ end of the target sequence(about 20 nucleotides)
• The CAS-9 component recognizes the PAM sequence. CAS- 9 then mediates unwinding or DNA and nick formation (nick is formed at about 3 bp upstream of the PAM).
• The CAS-9 recognition is aided with crRNA.
• The gRNA thus, associated with the crRNA act as recognition sequence, changing of which alters the recognition capabilities of the CAS-9.

Application:

• For transgenic organism creation for mutational related studies like cancer mutations, knockout of the wild type genes is used.
• The alteration of genome is caused by gene knockout resulting in loss of function of genes.
• The target genes are eliminated or inactivated.
• Thus, CRISPR causes a permanent effect as gene “Knock out” is used.