Efficient Genome Editing of Genes Involved in Neural Crest Development Using the CRISPR/Cas9 System in Xenopus Embryos.

摘要

The reverse genetics approaches including Zinc finger nucleases (ZFNs) or Transcription activator-like effector nucleases (TALENs) are widely used to study gene function. Other research strategies such as gene overexpression and gene knockdown mediated by RNAi or Morpholino antisense oligonucleotides (MOs) were commonly used as well. In the last three years, a new genome editing platform called Clustered regularly interspaced short palindromic repeats (CRISPR) has received particular attention and developed rapidly in genome editing study. This genome editing tool consists of Cas9 protein and a single guide RNA (sgRNA). In theory, any sequence with the form '5'-N 20-NGG-3'' could be the target site of CRISPR/Cas9. The double strand breaks induced by Cas9 are mainly repaired through errorprone non-homologous end joining (NHEJ) pathway, resulting in the formation of insertion or deletion (indel) mutations.;Neural crest (NC) is a transient cell population which can only be found in vertebrate embryos. It can differentiate into various derivatives. Many factors are involved in the induction and specification of NC. The Xenopus is an ideal model to study NC development, however, the knowledge obtained from this model system was mainly by overexpression and MO induced gene knockdown.;We utilized CRISPR/Cas9 to disrupt 14 genes in Xenopus tropicalis by injecting Cas9 mRNA together with sgRNAs into one-cell embryos successfully. We also generated the multiplex gene disruption in Xenopus embryos by injecting Cas9 mRNA together with mixtures of sgRNAs targeting to different genes. Furthermore, we induced the long fragment deletions and inversions by injecting Cas9 mRNA together with two sgRNAs targeting two loci simultaneously in one gene. Besides, we investigated the off-target effect of CRIPSR/Cas9 system and demonstrated that it can indeed induce nonspecific DNA cleavage in vivo, although the rate of off-target cleavage was relatively low. To reduce the off-target of Cas9, we tested the Cas9-D10A, a nickase mutant version of Cas9, in which the RuvC domain is mutated. In combination with a pair of sgRNAs, the D10A could generate the indel mutations and decrease off-target cleavage notably. Moreover, we showed evidence that the gene knock-in could be achieved by using a single strand oligo or by using a vector harboring a 'bait sequence'. Finally, the Cas9 induced indel mutation efficiently passed through germline to G1 offspring.;Taken together, our results indicated the CRISPR/Cas9 system is an efficient and versatile method for gene editing in Xenopus tropicalis.