**Conflict of interest**

this strategy, the authors obtained 31% of mutation efficiency [74]. This team targeted two other genes of the diatom using this technology and obtained from 25 to 63% of mutation level [74]. Adaptability of the CRISPR/Cas9 system has been demonstrated in other diatoms like *Thalassiosira pseudonana* [75] as well as in the heterokont, *Nannochloropsis oceanica* in order to knock out the nitrate reductase activity [76]. In conclusion, CRISPR/cas9 system is a promising technology to generate genome-modified organisms in microalgae. **Table 1** compares this system with the other nuclease systems cited above in terms of their technical characteristics

The study of mutants impaired in a glycosidase or a glycosyltransferase implied in the *N*-glycan pathway is of great interest. Indeed, the synthesis of oligosaccharides is a sequential process. Inactivation of an enzyme usually results in the accumulation of its *N*-glycan substrate which enables the step-by-step dissection of the entire pathway. Moreover, mutant phenotyping of the glycosylation pathway allows to investigate to which extent the protein *N*-glycan processing is required for normal growth and development. An indexed and mapped mutant library has been created in *C. reinhardtii* by single random insertional mutagenesis of gene cassettes in 2016 [79]. This library already envisioned to study the function of genes encoding putative glycosyltransferases, glycosidases or even putative translocators in microalgae and to confirm

The production of biopharmaceuticals in microalgae currently requires a better understanding of the *N*-glycosylation pathway mechanism and regulation. Such information can be gained by the use of mutant libraries like the one recently developed for *C. reinhardtii*. Indeed, characterization of each individual mutants will allow an understanding of a specific step of the *N*-glycan processing, and mutant cells could represent interesting cell lines for the pro-

Once these pathways would be completely deciphered in the microalgae model intended to be used for the production of biopharmaceuticals, the humanization of the *N*-glycosylation pathway could be initiated using designed engineered nucleases strategies recently developed in microalgae. We can now consider that transformed microalgae by these innovative new genomic tools will constitute in a near future one of the most suitable green cell factories

The authors are indebted to all coworkers and students at the Glyco-MEV laboratory who are currently contributing to the microalgae research project or did so in the past. They are also

and highlights their advantages and disadvantages.

their physiological role from reverse genetic studies.

duction of biopharmaceuticals bearing a chosen *N*-glycan profiling.

for the production of humanized biopharmaceuticals.

**3.2. Mutant libraries**

186 Microalgal Biotechnology

**4. Conclusion**

**Acknowledgements**

The authors have declared no conflict of interest.
