| Autor: |
Dalvie NC; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States., Lorgeree T; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States., Biedermann AM; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States., Love KR; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States., Love JC; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States. |
| Abstrakt: |
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. |
