Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination

Genetic engineering
of industrial cell lines often requires knockout
of multiple endogenous genes. Tools like CRISPR-Cas9 have enabled
serial or parallelized gene disruption in a wide range of industrial
organisms, but common practices for the screening and validation of
genome edits are lacking. For gene disruption, DNA repair by homologous
recombination offers several advantages over nonhomologous end joining,
including more efficient screening for knockout clones and improved
genomic stability. Here we designed and characterized a knockout fragment
intended to repair Cas9-induced gene disruptions by homologous recombination.
We identified knockout clones of Komagataella phaffii with high fidelity by PCR, removing the need for Sanger sequencing.
Short overlap sequences for homologous recombination (30 bp) enabled
the generation of gene-specific knockout fragments by PCR, removing
the need for subcloning. Finally, we demonstrated that the genotype
conferred by the knockout fragment is stable under common cultivation
conditions.