A plasmid can carry selected DNA into a host cell. Restriction enzymes cut particular recognition sequences, ligase joins DNA backbones, and a suitable promoter supports expression. A probe recognises a complementary sequence. A selectable marker can identify cells carrying a construct, but does not by itself establish production of the intended functional protein.
CRISPR tools use guide RNA to target a nuclease to a matching sequence; repair can disrupt or alter that sequence. Editing is distinct from PCR amplification and from transferring a whole gene. Somatic changes affect body-cell lineages, while germline changes can be inherited. Host processing, folding, incomplete editing and off-target changes can limit intended results.
Example 1
A plasmid contains an insulin coding sequence but no suitable promoter. Why may no insulin be made?
The host may not transcribe the inserted sequence effectively. Gene presence alone does not establish gene expression.
Example 2
A guide RNA matches a target but one DNA base differs in a second site. What does off-target editing mean?
It means an unintended site is altered. A near match does not guarantee cutting, but unintended editing is distinct from the intended target change.
Watch out: Expression, RNA processing, translation and folding must also work in the selected host.