CRISPR/Cas9 is a system found in bacteria and involved in immune defence. Bacteria use CRISPR/Cas9 to cut up the DNA of invading bacterial viruses that might otherwise kill them.
Today we’ve adapted this molecular machinery for an entirely different purpose – to change any chosen letter(s) in an organism’s DNA code.
The Cas9 protein is commercially available and works in in vitro reactions.
A couple drawbacks though, the specificity is not perfect, sometimes even with up to 3/4 mismatches, efficient cut can occur and what is responsible for this lack of specificity is not yet fully understood. your cut site has to be adjacent to a PAM motif (NGG with the classically used Cas9 from S. pyogenes) and you would likely have to produce an sgRNA for each of the cut site you want to use.
We use this technique for modifying yeast strain for stress tolerance. Please consult our review paper " Genome engineering for breaking barriers in lignocellulosic bioethanol production " Article Genome engineering for breaking barriers in lignocellulosic ...
Dear Mushtak
We use the CRISPR/Cas9 system for site-directed mutation in plant target genes. Usually this technique is used for a spot mutation (e.g. substitution of a single base) and sometimes we can silent the gene via incorporating a premature stop codon. Hence, finally the gene function will be altered and we can see its effect on phenotype. Plz find practical examples via simple search in google.
Please refer to my review article "Genome editing in crop improvement: Present status and future prospects" published in Journal of Crop Improvement 2017 Volume 31 No. 4 pages 453-559.
Please refer to my review article ''CRISPR/Cas approach: A new way of looking at plant-abiotic interactions'' published in Journal of Plant Physiology''.
The Cas9 protein is commercially available and works in in vitro reactions.
A couple drawbacks though, the specificity is not perfect, sometimes even with up to 3/4 mismatches, efficient cut can occur and what is responsible for this lack of specificity is not yet fully understood. your cut site has to be adjacent to a PAM motif (NGG with the classically used Cas9 from S. pyogenes) and you would likely have to produce an sgRNA for each of the cut site you want to use.
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