CRISPR knockout & knock-in mice
CRISPR for genetically modified mice? Not so fast...
The use of genetically modified mice as model organisms for the study of human disease remains as relevant as ever1 and CRISPR-based genome editing is rapidly changing the field. However, whereas the potential of CRISPR technology is very exciting, there are some limitations to its usefulness and drawbacks inherent to the technology can easily be overlooked.
CRISPR based genome editing typically relies on the induction of a double strand break within the target genomic sequence by a nuclease, such as Cas9, as directed by a guide RNA2,3. The nuclease is delivered to a zygote or early stage embryo and may either be delivered as protein in complex with sgRNA, as mRNA that is subsequently translated, or DNA. The nuclease may remain present in cells for some time, particularly when expressed from plasmid DNA, and is not 100% efficient. Consequently, the occurrence of nuclease-mediated double strand breaks is stochastic and can occur across several early cell divisions4. Repair of these double strand DNA breaks occurs via a number of competing pathways that have evolved to maintain integrity of the genome, such as non-homologous end-joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology directed repair (HDR)5. With the exception of HDR, repair pathways are mutagenic and repair of CRISPR-mediated double stand breaks by NHEJ typically results in small insertions and deletions (INDELS) of less than 20 bp. CRISPR-mediated double strand breaks can induce large deletions (kilobases), complex genomic rearrangements, and chromosomal truncations, that are often overlooked by the genotyping methods employed6,7...Read full technical note
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