CRISPR-Cas9 Genome Editing – a Game Changer in Genetics

CRISPR-Cas9 Genome Editing – a Game Changer in Genetics
Stefan Mundlos
Institut für Medizinischen Genetik und Humangenetik, Charité, Berlin
Max Planck Institut für Molekulare Genetik, Berlin
The type II clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9
(Cas9) system is an efficient tool for genome editing. Properly designed short guide RNAs are used to
direct Cas9 precisely to target sites, where it functions as an endonuclease to efficiently produce
double strand breaks in DNA. These breaks are repaired by two distinct mechanisms, non
homologous end joining (NHEJ) and homologous directed repair (HDR). Because NHEJ is error
prone, it produces unpredictable alterations in sequence, in particular small indels, and is therefore
suitable to induce random mutations. HDR as an alternative pathway can be used for precise
sequence editing via the incorporation of an exogenous DNA fragment, commonly a single or double
stranded DNA template. CRISPR/Cas9 can be used in embryonic stem cells (ESCs) or directly in
fertilized oocytes for the generation of mutant mice. The advantages of producing SVs in ESCs are
easy selection of the desired mutation and the direct use of these cells in culture to investigate the
effects of induced mutations. The CRISPR/Cas technology can also be used to generate large
structural variations. We applied the CRISPR technology in mouse embryonic stem cells (ESCs) and
developed a 10-week protocol that we named CRISVar (CRISPR/Cas-induced structural variants),
targeted at two different positions of a chromosome, to efficiently produce deletions, inversions, and
duplications in mice. We were able to rearrange targeted genomic intervals ranging from 1 kb to 1.6
Mb using the CRISPR/Cas system in mouse embryonic stem cells (ESCs). ESCs harboring these
mutations can be used to create chimeric animals. The efficiency of CRISPR/Cas in introducing
mutations can be considered a game changer in genetics. Variants or unknown significance can now
be tested quickly in vitro or in vivo. This is of particular importance for extremely rare diseases/variants
for which a second patient/variant is difficult to find.