Is it a guarantee to have a loss of expression or protein function if you have introduced an indel into a gene? In other words does a 1-3 bp indel definitely introduce a frameshift mutation or can it vary between clones? Can some clones undergo successful genome editing but still retain protein expression/function?
short answer- no
it is possible for crispr to induce a simple point mutatiom, or even an in-frame 3X bp deletion resultimg im skipped amino acids but not a true nonsense protein or early stop codon.
that being said, that is not the normal case. because the repair mechanisms for the double stranded nick caused by the cas9 are thought to be more or less random, it statistically more common for your crispr to generate an indel that does go on to caue a frameshift giving either a totally nonsense and-or early termination.
additionally there ways of further decreasing the chance of having a functinally viable mutated allele, for example by using 2 crisprs on the same gene to excise a large fragment, or by inserting oligos imto the cut site via homologous recombination, for example, which intemtionally mess up the ORF, as was done in the attached paper,in zebrafish
Article Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleoti...
Thanks Michael. I thought as much ... but then I keep getting surprised by anecdotal evidence, one of my own projects and even some published studies whereby the actual % knockout clones obtained seems lower than what would be expected based on editing efficiency or after sorting transfected cells etc.
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