Comparison of CRISPR-Cpf1 with Cre/loxP for gene knockout in Corynebacterium glutamicum

Abstract:

[Background] Corynebacterium glutamicum is an important industrial platform strain. Efficient and convenient genetic manipulation tools is in urgent need for improving C. glutamicum strains in commercial applications. [Objective] Gene argR and argF of C. glutamicum ATCC 13032 was deleted by CRISPR-Cpf1-assisted-homologous recombination and Cre/loxP-assisted-homologous recombination knockout system respectively. To provide guidance for reasonable selection of gene knockout systems, the advantages and disadvantages of these two methods are investigated in detail. [Methods] Firstly, Cre/loxP-assisted-homologous recombination replaced the targeted gene in genome with kanR fragment containing loxP sites at 5′ and 3′ ends. Then recombinase Cre expressed by pDTW109 recognized and bound on the loxP sites, and catalyzed the homologous recombination between two loxP sites to remove the kanR fragment. Eventually, the pDTW109 was eliminated by elevating the temperature to 37 °C based on its temperature-sensitive characteristic. In the knockout of targeted gene by CRISPR-Cpf1-assisted genome editing, the pre-crRNA was processed by Cpf1 and the resultant crRNA guided Cpf1 to bind to the specific sequence and cleave the target DNA. The targeted gene was removed by homologous recombination. The recombinant plasmid was also eliminated by elevating the cultivation temperature. [Results] In the genes knockout of C. glutamicum, Cre/loxP-assisted system allowed a complete N rounds of iterative gene knockout in 8N+2 d, while CRISPR-Cpf1-assisted system only need 5N+2 d. Theoretically, the latter could achieve the simultaneous knockout of multiple genes, however suffers from disadvantage of low homologous recombination efficiency and high false-positive rates. [Conclusion] In comparison with Cre/loxP system, CRISPR-Cpf1 assisted genome editing is time-saving and labor-saving in gene knockout of C. glutamicum, and theoretically it can be employed in knockout of multiple genes at a time. Consequently, CRISPR-Cpf1-assisted system has higher overall efficiency. However, its editing efficiency still has great potentials for improvement.

Key words:Corynebacterium glutamicum, CRISPR-Cpf1, Cre/loxP, Homologous recombination, Gene knockout