Browsing by Subject "CRISPR-Cas9"

CRISPR-Cas9 is one variant of newly emerging technologies utilizing targeted mutagenesis based on Cas family proteins and guide RNA that enable binding and modifying selected target sequence. The aim of the master’s thesis was to compare different methods of CRISPR-Cas9 induced gene editing in the genus Nicotiana and other secondary protocols necessary to identify successful mutations. PDS1 and PDS2 genes coding phytoene desaturase in plants were selected as target genes as mutant genotype produce visually identifiable photobleaching phenotype. CRISPR-Cas9 ribonucleoprotein complex mediated transformation uses separately produced Cas9 protein and guide RNA that when combined perform transient gene editing in cell. This method was planned to be used but Cas9 protein was challenging to produce in soluble form and final transformation was not achieved. This study suggests that acquiring ready-to-use Cas9 protein might be preferable choice when targeting only few transformations with CRISPR-Cas9 RNP-complex. Agrotransformation is well established method for genus Nicotiana and using Single Transcriptional Unit CRISPR-Cas9 system it is straightforward procedure from plasmid design to transformation. Successfully transformed plants were redeemed from transient agroinfiltration and stable agrotransformation experiments. Off-target mutations are possible and selective outbreeding may be needed. This method lacks the several advantages of CRISPR-Cas9 RNP-complex such as instant gene editing in cell, avoiding RNA interference and transformation over species boundaries, but is simple and functional in genus Nicotiana. Successful mutations were detected using commercial T7E1 and with natural CEL I endonuclease from celery extract. Celery extract can be used as cost-effective alternative to T7E1 for verifying or replicating previously confirmed results.