Dataset Information

Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination.

ABSTRACT: 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.

SUBMITTER: Dalvie NC

PROVIDER: S-EPMC8787811 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination.

Dalvie Neil C NC Lorgeree Timothy T Biedermann Andrew M AM Love Kerry R KR Love J Christopher JC

ACS synthetic biology 20211209 1

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 stabilit ...[more]

PMID: 34882409

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Project description:Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3'-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Project description:Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the organism's genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as C. albicans) and polyploid organisms. The effects of unleashing this nuclease within the genome of C. albicans are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a C. albicans strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in C. albicans, regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in C. albicans appears to limit transformation-associated genome changes.IMPORTANCE Genome editing is essential to nearly all research studies aimed at gaining insight into the molecular mechanisms underlying various biological processes, including those in the opportunistic pathogen Candida albicans The adaptation of the CRISPR-Cas9 system greatly facilitates genome engineering in many organisms. However, our understanding of the effects of CRISPR-Cas9 technology on the biology of C. albicans is limited. In this study, we sought to compare the extents of transformation-induced genomic changes within strains engineered using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation methods. CRISPR-Cas9-dependent transformation allows one to simultaneously target both homologs and, importantly, appears less mutagenic in C. albicans, since strains engineered using CRISPR-Cas9 display an overall decrease in concomitant genomic changes.

Dataset Information

Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination.

Publications

Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination.

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