谷氨酸棒状杆菌CRISPR-Cpf1和Cre/loxP基因敲除技术的比较

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

摘要:

【背景】谷氨酸棒状杆菌的基因敲除系统较为匮乏且效率不高，难以对其进行代谢工程改造，不利于高性能工业菌株的构建及规模生产。【目的】分别采用CRISPR-Cpf1和Cre/loxP基因敲除系统对谷氨酸棒状杆菌ATCC 13032 (Corynebacterium glutamicum ATCC 13032)基因组上的argR和argF基因进行敲除，比较两种敲除方法的优缺点，为合理选择敲除系统提供依据。【方法】特异性重组的Cre/loxP敲除系统是首先利用同源重组将基因组上的靶基因替换为两端带有重组位点loxP的kanR片段，然后由重组酶Cre识别loxP位点并发生重组反应，从而去除替换到基因组上的kanR片段，进一步利用质粒的温敏特性将其消除，从而实现靶基因的敲除。CRISPR-Cpf1敲除系统是利用Cpf1对pre-crRNA进行加工，形成的成熟crRNA引导Cpf1识别和结合到靶DNA的特定序列上并切割双链DNA分子，通过同源重组作用去除靶基因，基于质粒自身的温敏特性将其消除，从而完成基因敲除的整个过程。【结果】Cre/loxP系统可在8N+2 d内完成N轮迭代基因敲除，而CRISPR-Cpf1系统可在5N+2 d内完成N轮迭代基因无痕敲除，理论上还可以一次对多个靶位点进行编辑，效率更高，但存在同源重组效率较低、假阳性率高等缺点。【结论】与Cre/loxP系统相比，CRISPR-Cpf1辅助的同源重组基因敲除方法可省时、省力地实现基因的无痕敲除，理论上还可实现多个基因的同时敲除、总体效率更高，然而编辑效率还有提高的空间。

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.