Optimization of Genome Editing with the CRISPR/Cas9 System

Optimization of genome editing needs to be performed at severalsteps, from guide RNA design to delivery of CRISPR/Cas9 reagents.In treated cells, genome editing per se takes place during repair ofDNA double-strand breaks made by CRISPR/Cas9 nucleases. SeveralDNA repair pathways can process may be involved and influence theeventual outcome of including classical non-homologous end-joining (NHEJ), micro-homology mediated end-joining (MMEJ) and homologousrecombination (HR). Our lab has developed strategies of genome editingthat take advantage of these different possibilities in order to increase theefficiency of genome editing with the CRISPR/Cas9 system.For example, when developing gene editing in Clytia hemisphericajellyfish, we observed that unexpectedly deletions made in targetgenes were often flanked by short stretches of identical sequence. Thisis characteristic of DNA repair by MMEJ which therefore appears to bethe predominant end-joining pathway for introduction of indels duringgenome editing with CRISPR-Cas9. This unexpected finding is in factcommon to many experimental systems and needs to be taken intoaccount when designing guide RNAs.Gene inactivation by the introduction of small deletions or insertionsis highly efficient with the CRISPR/Cas9 system. In contrast, makingprecise, programmed modifications such as transgene insertions oftenremains challenging. We have developed several strategies to addressthis challenge, including homology-independent transgene integrationin zebrafish and using chemically modified oligonucleotide donors. Wealso recently developed a novel approach for increasing the efficiency oftransgene integration by homology-dependent repair (HDR).

کلیدواژه ها:

CRISPR/Cas9 ، Genome Editing