Advances in CRISPR/Cas9 technology: shaping the future of photosynthetic microorganisms for biofuel production

Samreen Arshad; Muhammad Luqman Qadir; Nazim Hussain; Qurban Ali; Shiming Han; Daoud Ali

doi:10.1071/FP24255

REVIEW (Open Access)

Next Contents Vol 52(2)

Advances in CRISPR/Cas9 technology: shaping the future of photosynthetic microorganisms for biofuel production

Samreen Arshad ^A , Muhammad Luqman Qadir ^A , Nazim Hussain ^A ^* , Qurban Ali

^B ^* , Shiming Han ^C ^* and Daoud Ali ^D

+ Author Affiliations

- Author Affiliations

^A Center for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan. Email: arshadsamreen10@gmail.com, luqmanqadir0809@gmail.com

^B Department of Plant Breeding and Genetics, Faculty of Agricultural Sciences, University of the Punjab, Lahore, Pakistan.

^C School of Biological Sciences and Technology, Liupanshui Normal University, Liupanshui 553004, China.

^D Department of Zoology, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia. Email: Aalidaoud@ksu.edu.sa

^* Correspondence to: nazim.camb@pu.edu.pk, saim1692@gmail.com, hanshiliang888@163.com

Handling Editor: Suleyman Allakhverdiev

Functional Plant Biology 52, FP24255 https://doi.org/10.1071/FP24255

Submitted: 14 October 2024 Accepted: 15 January 2025 Published: 11 February 2025

© 2025 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND)

Abstract

Use of fossil fuels causes environmental issues due to its inefficiency and and imminent depletion. This has led to interest in identifying alternative and renewable energy sources such as biofuel generation from photosynthetic organisms. A wide variety of prokaryotic and eukaryotic microorganisms, known as microalgae, have the potential to be economical and ecologically sustainable in the manufacture of biofuels such as bio-hydrogen, biodiesel, bio-oils, and bio-syngas. By using contemporary bioengineering techniques, the innate potential of algae to produce biomass of superior quality may be enhanced. In algal biotechnology, directed genome modification via RNA-guided endonucleases is a new approach. CRISPR/Cas systems have recently been frequently used to modify the genetic makeup of several aquatic and freshwater microalgae. The majority of research has used the Cas9-driven Type II system, one of two classes and six unique kinds of CRISPR systems, to specifically target desired genes in algae, and knock them out and down, or both. Using CRISPR technology to modify its genetic makeup, microalgae has produced more biomass and increased in lipid content. This review highlights the attempts made so far to target microalgae genome modification, discusses the prospects for developing the CRISPR platform for large-scale genome modification of microalgae, and identifies the opportunities and challenges in the development and distribution of CRISPR/Cas9 components.

Keywords: biodiesel, biofuel, bio-oil, CRISPR–Cas9, cyanobacteria, genome-editing, microalgae, photosynthetic microorganisms.

References

Abbas A, Rehman A, Javed M (2021) Exploring the potential of in vitro tissue culture in breeding programs of legume and pulse crops: utilization and present condition. Bulletin of Biological and Allied Sciences Research 2021, 36.
| Crossref | Google Scholar |

Abbas A, Arshad A, Rehman A, Bukhari M, Zaman S (2024) Revolutionizing plant breeding programs with advancements in molecular marker-assisted selection. Bulletin of Biological and Allied Sciences Research 2024, 57.
| Crossref | Google Scholar |

Agarwal AK, Agarwal RA, Gupta T, Gurjar BR (2017) ‘Introduction to biofuels.’ (Springer)

Ajjawi I, Verruto J, Aqui M, Soriaga LB, Coppersmith J, Kwok K, Peach L, Orchard E, Kalb R, Xu W, Carlson TJ, Francis K, Konigsfeld K, Bartalis J, Schultz A, Lambert W, Schwartz AS, Brown R, Moellering ER (2017) Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator. Nature Biotechnology 35, 647-652.
| Crossref | Google Scholar | PubMed |

Alalwan HA, Alminshid AH, Aljaafari HAS (2019) Promising evolution of biofuel generations. Subject review. Renewable Energy Focus 28, 127-139.
| Crossref | Google Scholar |

Angermayr SA, Hellingwerf KJ, Lindblad P, Teixeira de Mattos MJ (2009) Energy biotechnology with cyanobacteria. Current Opinion in Biotechnology 20, 257-263.
| Crossref | Google Scholar | PubMed |

Azad AK, Rasul MG, Khan MMK, Sharma SC, Hazrat MA (2015) Prospect of biofuels as an alternative transport fuel in Australia. Renewable and Sustainable Energy Reviews 43, 331-351.
| Crossref | Google Scholar |

Baek K, Kim DH, Jeong J, Sim SJ, Melis A, Kim J-S, Jin E, Bae S (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Scientific Reports 6, 30620.
| Crossref | Google Scholar |

Baek K, Yu J, Jeong J, Sim SJ, Bae S, Jin E (2018) Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated mutagenesis. Biotechnology and Bioengineering 115, 719-728.
| Crossref | Google Scholar | PubMed |

Barrangou R, van der Oost J (Eds)(2013) ‘CRISPR-Cas systems: RNA-mediated adaptive immunity in bacteria and archea.’ (Springer: Berlin)

Barsanti L, Gualtieri P (2006) General overview. In ‘Algae: anatomy, biochemistry, and biotechnology’. (Eds L Barsanti, P Gualtieri) pp. 1–44. (CRC Press)

Behera S, Singh R, Arora R, Sharma NK, Shukla M, Kumar S (2015) Scope of algae as third generation biofuels. Frontiers in Bioengineering and Biotechnology 2, 90.
| Crossref | Google Scholar |

Bernheim A, Calvo-Villamañán A, Basier C, Cui L, Rocha EPC, Touchon M, Bikard D (2017) Inhibition of NHEJ repair by type II-A CRISPR-Cas systems in bacteria. Nature Communications 8, 2094.
| Crossref | Google Scholar |

Bhatia SK, Kim S-H, Yoon J-J, Yang Y-H (2017) Current status and strategies for second generation biofuel production using microbial systems. Energy Conversion and Management 148, 1142-1156.
| Crossref | Google Scholar |

Bhujade R, Chidambaram M, Kumar A, Sapre A (2017) Algae to economically viable low-carbon-footprint oil. Annual Review of Chemical and Biomolecular Engineering 8, 335-357.
| Crossref | Google Scholar | PubMed |

Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques 48, 463-465.
| Crossref | Google Scholar | PubMed |

Callegari A, Bolognesi S, Cecconet D, Capodaglio AG (2020) Production technologies, current role, and future prospects of biofuels feedstocks: a state-of-the-art review. Critical Reviews in Environmental Science and Technology 50, 384-436.
| Crossref | Google Scholar |

Carriquiry MA, Du X, Timilsina GR (2014) Production costs of biofuels. In ‘The impacts of biofuels on the economy, environment, and poverty: a global perspective’. (Eds G Timilsina, D Zilberman) pp. 33–46. (Springer)

Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA (2017) Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature 550, 407-410.
| Crossref | Google Scholar | PubMed |

Cheng JJ, Timilsina GR (2011) Status and barriers of advanced biofuel technologies: a review. Renewable Energy 36, 3541-3549.
| Crossref | Google Scholar |

Chisti Y (2007) Biodiesel from microalgae. Biotechnology Advances 25, 294-306.
| Crossref | Google Scholar | PubMed |

Codd GA, Meriluoto J, Metcalf JS (2016) Introduction: cyanobacteria, cyanotoxins, their human impact, and risk management. In ‘Handbook of cyanobacterial monitoring and cyanotoxin analysis’. (Eds J Meriluoto, L Spoof, GA Codd) pp. 1–8. (John Wiley & Sons, Ltd.)

Correa DF, Beyer HL, Fargione JE, Hill JD, Possingham HP, Thomas-Hall SR, Schenk PM (2019) Towards the implementation of sustainable biofuel production systems. Renewable and Sustainable Energy Reviews 107, 250-263.
| Crossref | Google Scholar |

Dejtisakdi W, Miller SM (2016) Overexpression of Calvin cycle enzyme fructose 1,6-bisphosphatase in Chlamydomonas reinhardtii has a detrimental effect on growth. Algal Research 14, 116-126.
| Crossref | Google Scholar |

Demirbaş A (2006) Oily products from mosses and algae via pyrolysis. Energy Sources, Part A: Recovery, Utilization, and Environmental Effects 28, 933-940.
| Crossref | Google Scholar |

Demirbas A (2007) Recent developments in biodiesel fuels. International Journal of Green Energy 4, 15-26.
| Crossref | Google Scholar |

Demirbas A (2009) Political, economic and environmental impacts of biofuels: a review. Applied Energy 86, S108-S117.
| Crossref | Google Scholar |

Dhokane D, Shaikh A, Yadav A, Giri N, Bandyopadhyay A, Dasgupta S, Bhadra B (2023) CRISPR-based bioengineering in microalgae for production of industrially important biomolecules. Frontiers in Bioengineering and Biotechnology 11, 1267826.
| Crossref | Google Scholar |

Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096.
| Crossref | Google Scholar |

Dunahay TG, Jarvis EE, Dais SS, Roessler PG (1996) Manipulation of microalgal lipid production using genetic engineering. Applied Biochemistry and Biotechnology 57, 223-231.
| Crossref | Google Scholar |

Falfushynska H (2024) Advancements and prospects in algal biofuel production: a comprehensive review. Phycology 4, 548-575.
| Crossref | Google Scholar |

Fang L, Lin HX, Low CS, Wu MH, Chow Y, Lee YK (2012) Expression of the Chlamydomonas reinhardtii Sedoheptulose-1,7-bisphosphatase in Dunaliella bardawil leads to enhanced photosynthesis and increased glycerol production. Plant Biotechnology Journal 10, 1129-1135.
| Crossref | Google Scholar | PubMed |

Fatima S, Cheema K, Shafiq M, Manzoor M, Ali Q, Haider M, Shahid M (2023) The genome-wide bioinformatics analysis of 1-aminocyclopropane-1-carboxylate synthase (acs), 1-aminocyclopropane-1-carboxylate oxidase (aco) and ethylene overproducer 1 (eto1) gene family of fragaria vesca (woodland strawberry). Bulletin of Biological and Allied Sciences Research 2023, 38.
| Crossref | Google Scholar |

Feng S, Xie X, Liu J, Li A, Wang Q, Guo D, Li S, Li Y, Wang Z, Guo T, Zhou J, Tang DYY, Show PL (2023) A potential paradigm in CRISPR/Cas systems delivery: at the crossroad of microalgal gene editing and algal-mediated nanoparticles. Journal of Nanobiotechnology 21, 370.
| Crossref | Google Scholar |

Fernandes BD, Mota A, Teixeira JA, Vicente AA (2015) Continuous cultivation of photosynthetic microorganisms: approaches, applications and future trends. Biotechnology Advances 33, 1228-1245.
| Crossref | Google Scholar | PubMed |

Freudenberg RA, Wittemeier L, Einhaus A, Baier T, Kruse O (2022) The spermidine synthase gene SPD1: a novel auxotrophic marker for Chlamydomonas reinhardtii designed by enhanced CRISPR/Cas9 gene editing. Cells 11, 837.
| Crossref | Google Scholar |

Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology 32, 279-284.
| Crossref | Google Scholar | PubMed |

Fu W, Chaiboonchoe A, Khraiwesh B, Sultana M, Jaiswal A, Jijakli K, Nelson DR, Al-Hrout A, Baig B, Amin A, Salehi-Ashtiani K (2017) Intracellular spectral recompositioning of light enhances algal photosynthetic efficiency. Science Advances 3, e1603096.
| Crossref | Google Scholar |

Gelfand I, Sahajpal R, Zhang X, Izaurralde RC, Gross KL, Robertson GP (2013) Sustainable bioenergy production from marginal lands in the US Midwest. Nature 493, 514-517.
| Crossref | Google Scholar | PubMed |

Genkov T, Meyer M, Griffiths H, Spreitzer RJ (2010) Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in chlamydomonas. Journal of Biological Chemistry 285, 19833-19841.
| Crossref | Google Scholar | PubMed |

Gordon GC, Korosh TC, Cameron JC, Markley AL, Begemann MB, Pfleger BF (2016) CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002. Metabolic Engineering 38, 170-179.
| Crossref | Google Scholar | PubMed |

Greiner A, Kelterborn S, Evers H, Kreimer G, Sizova I, Hegemann P (2017) Targeting of photoreceptor genes in Chlamydomonas reinhardtii via zinc-finger nucleases and CRISPR/Cas9. The Plant Cell 29, 2498-2518.
| Crossref | Google Scholar | PubMed |

Gupta D, Bhattacharjee O, Mandal D, Sen MK, Dey D, Dasgupta A, Kazi TA, Gupta R, Sinharoy S, Acharya K, Chattopadhyay D, Ravichandiran V, Roy S, Ghosh D (2019) CRISPR-Cas9 system: a new-fangled dawn in gene editing. Life Sciences 232, 116636.
| Crossref | Google Scholar |

Hassanien A, Saadaoui I, Schipper K, Al-Marri S, Dalgamouni T, Aouida M, Saeed S, Al-Jabri HM (2023) Genetic engineering to enhance microalgal-based produced water treatment with emphasis on CRISPR/Cas9: a review. Frontiers in Bioengineering and Biotechnology 10, 1104914.
| Crossref | Google Scholar |

Heimann K (2016) Novel approaches to microalgal and cyanobacterial cultivation for bioenergy and biofuel production. Current Opinion in Biotechnology 38, 183-189.
| Crossref | Google Scholar | PubMed |

Henley WJ, Litaker RW, Novoveská L, Duke CS, Quemada HD, Sayre RT (2013) Initial risk assessment of genetically modified (GM) microalgae for commodity-scale biofuel cultivation. Algal Research 2, 66-77.
| Crossref | Google Scholar |

Higo A, Isu A, Fukaya Y, Ehira S, Hisabori T (2018) Application of CRISPR interference for metabolic engineering of the heterocyst-forming multicellular cyanobacterium Anabaena sp. PCC 7120. Plant and Cell Physiology 59, 119-127.
| Crossref | Google Scholar | PubMed |

Ho DP, Ngo HH, Guo W (2014) A mini review on renewable sources for biofuel. Bioresource Technology 169, 742-749.
| Crossref | Google Scholar | PubMed |

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology 31, 827-832.
| Crossref | Google Scholar | PubMed |

Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262-1278.
| Crossref | Google Scholar | PubMed |

Hu J, Meng W, Su Y, Qian C, Fu W (2023) Emerging technologies for advancing microalgal photosynthesis and metabolism toward sustainable production. Frontiers in Marine Science 10, 1260709.
| Crossref | Google Scholar |

Huang C-H, Shen CR, Li H, Sung L-Y, Wu M-Y, Hu Y-C (2016) CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942. Microbial Cell Factories 15, 196.
| Crossref | Google Scholar |

IEA (2022) World energy outlook 2022. International Energy Agency (IEA), Paris, France.

Inam S, Muhammad A, Irum S, Rehman N, Riaz A, Uzair M, Khan MR (2024) Genome editing for improvement of biotic and abiotic stress tolerance in cereals. Functional Plant Biology 51, FP24092.
| Crossref | Google Scholar |

Irfan MF, Mirara F (2024) Biochar application in improving soil health and sustainability. Bulletin of Biological and Allied Sciences Research 2024, 81.
| Crossref | Google Scholar |

Irfan U, Haider M, Shafiq M, Sami A, Ali Q (2023) Genome editing for early and late flowering in plants. Bulletin of Biological and Allied Sciences Research 2023, 45.
| Crossref | Google Scholar |

Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A (1987) Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology 169, 5429-5433.
| Crossref | Google Scholar | PubMed |

Ishino Y, Krupovic M, Forterre P (2018) History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology. Journal of Bacteriology 200, e00580-17.
| Crossref | Google Scholar |

Javed M, Sami A, Haider M, Abbas A, Ali M, Naeem S, Amjad M, Ahmad A, Bostani R (2024) The contribution of transgenic rice to enhance grain yield. Bulletin of Biological and Allied Sciences Research 2024, 65.
| Crossref | Google Scholar |

Jeon S, Lim J-M, Lee H-G, Shin S-E, Kang NK, Park Y-I, Oh H-M, Jeong W-J, Jeong B-R, Chang YK (2017) Current status and perspectives of genome editing technology for microalgae. Biotechnology for Biofuels 10, 267.
| Crossref | Google Scholar |

Jiang WZ, Weeks DP (2017) A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii. Algal Research 26, 474-480.
| Crossref | Google Scholar |

Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP (2014) Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryotic Cell 13, 1465-1469.
| Crossref | Google Scholar | PubMed |

Jin C, Yao M, Liu H, Lee C-FF, Ji J (2011) Progress in the production and application of n-butanol as a biofuel. Renewable and Sustainable Energy Reviews 15, 4080-4106.
| Crossref | Google Scholar |

Jing Y (2023) Applications and prospects of CRISPR-Cas system in cyanobacteria. In ‘Bio web of conferences, 01009’. (EDP Sciences)

Junaid M, Gokce A (2024) Global agricultural losses and their causes. Bulletin of Biological and Allied Sciences Research 2024, 66.
| Crossref | Google Scholar |

Kanno M, Carroll AL, Atsumi S (2017) Global metabolic rewiring for improved CO₂ fixation and chemical production in cyanobacteria. Nature Communications 8, 14724.
| Crossref | Google Scholar |

Kao P-H, Ng I-S (2017) CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii. Bioresource Technology 245, 1527-1537.
| Crossref | Google Scholar | PubMed |

Kasai Y, Takagi S, Ota S, Ishii K, Takeshita T, Kawano S, Harayama S (2024) Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity. Biotechnology for Biofuels and Bioproducts 17, 36.
| Crossref | Google Scholar |

Kayani REZ, Rustam A, Saleem U, Bilal M, Bakhtiyar M, Jamshaid S, Sohail M, Farooq K, Khan S, Rehman B (2025) Chlorophytum comosum-mediated iron nanoparticles: an eco-friendly approach for antimicrobial and dye degradation applications. Bulletin of Biological and Allied Sciences Research 2025, 94.
| Crossref | Google Scholar |

Khanna M, Wang W, Hudiburg TW, Delucia EH (2017) The social inefficiency of regulating indirect land use change due to biofuels. Nature Communications 8, 15513.
| Crossref | Google Scholar |

Koonin EV, Makarova KS, Zhang F (2017) Diversity, classification and evolution of CRISPR-Cas systems. Current Opinion in Microbiology 37, 67-78.
| Crossref | Google Scholar | PubMed |

Krishnan A, Cano M, Burch TA, Weissman JC, Posewitz MC (2020) Genome editing using Cas9-RNA ribonucleoprotein complexes in the high-productivity marine alga Picochlorum celeri. Algal Research 49, 101944.
| Crossref | Google Scholar |

Kumar S (2015) GM algae for biofuel production: biosafety and risk assessment. Collection of Biosafety Reviews 9, 52-75.
| Google Scholar |

Lakhawat SS, Malik N, Kumar V, Kumar S, Sharma PK (2022) Implications of CRISPR-Cas9 in developing next generation biofuel: a mini-review. Current Protein & Peptide Science 23, 574-584.
| Crossref | Google Scholar | PubMed |

Li Y, Horsman M, Wu N, Lan CQ, Dubois-Calero N (2008) Biofuels from microalgae. Biotechnology Progress 24, 815-820.
| Crossref | Google Scholar | PubMed |

Li H, Shen CR, Huang C-H, Sung L-Y, Wu M-Y, Hu Y-C (2016) CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production. Metabolic Engineering 38, 293-302.
| Crossref | Google Scholar | PubMed |

Li L, Jiang W, Lu Y (2018) A modified gibson assembly method for cloning large DNA fragments with high GC contents. In ‘Synthetic metabolic pathways: methods and protocols’. (Eds MK Jensen, JD Keasling) pp. 203–209. (Humana Press)

Li-Beisson Y, Peltier G (2013) Third-generation biofuels: current and future research on microalgal lipid biotechnology. OCL 20, D606.
| Crossref | Google Scholar |

Lin W-R, Ng I-S (2020) Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation. Enzyme and Microbial Technology 133, 109458.
| Crossref | Google Scholar |

Lin T, Rodríguez LF, Shastri YN, Hansen AC, Ting KC (2014) Integrated strategic and tactical biomass–biofuel supply chain optimization. Bioresource Technology 156, 256-266.
| Crossref | Google Scholar | PubMed |

Lin P-C, Saha R, Zhang F, Pakrasi HB (2017) Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocysti s sp. PCC 6803. Scientific Reports 7, 17503.
| Crossref | Google Scholar |

Lin W-R, Tan S-I, Hsiang C-C, Sung P-K, Ng I-S (2019) Challenges and opportunity of recent genome editing and multi-omics in cyanobacteria and microalgae for biorefinery. Bioresource Technology 291, 121932.
| Crossref | Google Scholar |

Lin J-Y, Lin W-R, Ng I-S (2022) CRISPRa/i with Adaptive Single Guide Assisted Regulation DNA (ASGARD) mediated control of Chlorella sorokiniana to enhance lipid and protein production. Biotechnology Journal 17, 2100514.
| Crossref | Google Scholar |

Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJM, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV (2015) An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology 13, 722-736.
| Crossref | Google Scholar | PubMed |

Mallick N, Bagchi SK, Koley S, Singh AK (2016) Progress and challenges in microalgal biodiesel production. Frontiers in Microbiology 7, 1019.
| Crossref | Google Scholar |

Mazard S, Penesyan A, Ostrowski M, Paulsen IT, Egan S (2016) Tiny microbes with a big impact: the role of cyanobacteria and their metabolites in shaping our future. Marine Drugs 14, 97.
| Crossref | Google Scholar |

Mehdizadeh Allaf M, Peerhossaini H (2022) Cyanobacteria: model microorganisms and beyond. Microorganisms 10, 696.
| Crossref | Google Scholar |

Muñoz CF, de Jaeger L, Sturme MHJ, Lip KYF, Olijslager JWJ, Springer J, Wolbert EJH, Martens DE, Eggink G, Weusthuis RA, Wijffels RH (2018) Improved DNA/protein delivery in microalgae – a simple and reliable method for the prediction of optimal electroporation settings. Algal Research 33, 448-455.
| Crossref | Google Scholar |

Naduthodi MIS, Barbosa MJ, van der Oost J (2018) Progress of CRISPR-Cas based genome editing in photosynthetic microbes. Biotechnology Journal 13, 1700591.
| Crossref | Google Scholar |

Naeem S, Sami A, Haider M, Ali M, Khaliq A, Akram M, Mudasar M, Ali Q, Junaid M (2024) Heat stress in citrus: a molecular functional and biochemical perception. Bulletin of Biological and Allied Sciences Research 2024, 69.
| Crossref | Google Scholar |

Naik SN, Goud VV, Rout PK, Dalai AK (2010) Production of first and second generation biofuels: a comprehensive review. Renewable and Sustainable Energy Reviews 14, 578-597.
| Crossref | Google Scholar |

Ni E, Deng L, Chen H, Lin J, Ruan J, Liu Z, Zhuang C, Zhou H (2021) OsCER1 regulates humidity-sensitive genic male sterility through very-long-chain (VLC) alkane metabolism of tryphine in rice. Functional Plant Biology 48, 461-468.
| Crossref | Google Scholar | PubMed |

Niu F, Jiang Q, Sun X, Hu Z, Wang L, Zhang H, Bozhkov P (2021) Large DNA fragment deletion in lncRNA77580 regulates neighboring gene expression in soybean (Glycine max). Functional Plant Biology 48, 1139-1147.
| Crossref | Google Scholar | PubMed |

Nowak CM, Lawson S, Zerez M, Bleris L (2016) Guide RNA engineering for versatile Cas9 functionality. Nucleic Acids Research 44, 9555-9564.
| Crossref | Google Scholar | PubMed |

Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen KM, Gregg A, Noggle S, Tessier-Lavigne M (2016) Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125-129.
| Crossref | Google Scholar | PubMed |

Parsaeimehr A, Ebirim RI, Ozbay G (2022) CRISPR-Cas technology a new era in genomic engineering. Biotechnology Reports 34, e00731.
| Crossref | Google Scholar |

Patel VK, Soni N, Prasad V, Sapre A, Dasgupta S, Bhadra B (2019) CRISPR-Cas9 system for genome engineering of photosynthetic microalgae. Molecular Biotechnology 61, 541-561.
| Crossref | Google Scholar | PubMed |

Perrine Z, Negi S, Sayre RT (2012) Optimization of photosynthetic light energy utilization by microalgae. Algal Research 1, 134-142.
| Crossref | Google Scholar |

Plagens A, Richter H, Charpentier E, Randau L (2015) DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes. FEMS Microbiology Reviews 39, 442-463.
| Crossref | Google Scholar | PubMed |

Poliner E, Pulman JA, Zienkiewicz K, Childs K, Benning C, Farré EM (2018) A toolkit for Nannochloropsis oceanica CCMP 1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production. Plant Biotechnology Journal 16, 298-309.
| Crossref | Google Scholar | PubMed |

Quintana N, van der Kooy F, van de Rhee MD, Voshol GP, Verpoorte R (2011) Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering. Applied Microbiology and Biotechnology 91, 471-490.
| Crossref | Google Scholar | PubMed |

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281-2308.
| Crossref | Google Scholar | PubMed |

Rasala BA, Muto M, Lee PA, Jager M, Cardoso RMF, Behnke CA, Kirk P, Hokanson CA, Crea R, Mendez M, Mayfield SP (2010) Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnology Journal 8, 719-733.
| Crossref | Google Scholar | PubMed |

Razzak SA, Hossain MM, Lucky RA, Bassi AS, de Lasa H (2013) Integrated CO₂ capture, wastewater treatment and biofuel production by microalgae culturing – a review. Renewable and Sustainable Energy Reviews 27, 622-653.
| Crossref | Google Scholar |

Renzaho AMN, Kamara JK, Toole M (2017) Biofuel production and its impact on food security in low and middle income countries: implications for the post-2015 sustainable development goals. Renewable and Sustainable Energy Reviews 78, 503-516.
| Crossref | Google Scholar |

Riaz A, Thomas J, Ali HH, Zaheer MS, Ahmad N, Pereira A (2024) High night temperature stress on rice (Oryza sativa)–insights from phenomics to physiology. A review. Functional Plant Biology 51, FP24057.
| Crossref | Google Scholar |

Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347-355.
| Crossref | Google Scholar | PubMed |

Saravanan AP, Mathimani T, Deviram G, Rajendran K, Pugazhendhi A (2018) Biofuel policy in India: a review of policy barriers in sustainable marketing of biofuel. Journal of Cleaner Production 193, 734-747.
| Crossref | Google Scholar |

Sarsekeyeva F, Zayadan BK, Usserbaeva A, Bedbenov VS, Sinetova MA, Los DA (2015) Cyanofuels: biofuels from cyanobacteria. Reality and perspectives. Photosynthesis Research 125, 329-340.
| Crossref | Google Scholar | PubMed |

Schuergers N, Wilde A (2015) Appendages of the cyanobacterial cell. Life 5, 700-715.
| Crossref | Google Scholar | PubMed |

Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84-87.
| Crossref | Google Scholar | PubMed |

Shao Y, Wang L, Guo N, Wang S, Yang L, Li Y, Wang M, Yin S, Han H, Zeng L, Zhang L, Hui L, Ding Q, Zhang J, Geng H, Liu M, Li D (2018) Cas9-nickase–mediated genome editing corrects hereditary tyrosinemia in rats. Journal of Biological Chemistry 293, 6883-6892.
| Crossref | Google Scholar | PubMed |

Sharma AK, Nymark M, Sparstad T, Bones AM, Winge P (2018) Transgene-free genome editing in marine algae by bacterial conjugation–comparison with biolistic CRISPR/Cas9 transformation. Scientific Reports 8, 14401.
| Crossref | Google Scholar |

Shekh AY, Shrivastava P, Gupta A, Krishnamurthi K, Devi SS, Mudliar SN (2016) Biomass and lipid enhancement in Chlorella sp. with emphasis on biodiesel quality assessment through detailed FAME signature. Bioresource Technology 201, 276-286.
| Crossref | Google Scholar | PubMed |

Shin S-E, Lim J-M, Koh HG, Kim EK, Kang NK, Jeon S, Kwon S, Shin W-S, Lee B, Hwangbo K, Kim J, Ye SH, Yun J-Y, Seo H, Oh H-M, Kim K-J, Kim J-S, Jeong W-J, Chang YK, Jeong B-R (2016) CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii. Scientific Reports 6, 27810.
| Crossref | Google Scholar |

Shin W-S, Lee B, Kang NK, Kim Y-U, Jeong W-J, Kwon J-H, Jeong B-R, Chang YK (2017) Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris. Scientific Reports 7, 17929.
| Crossref | Google Scholar |

Shin YS, Jeong J, Nguyen THT, Kim JYH, Jin E, Sim SJ (2019) Targeted knockout of phospholipase A₂ to increase lipid productivity in Chlamydomonas reinhardtii for biodiesel production. Bioresource Technology 271, 368-374.
| Crossref | Google Scholar | PubMed |

Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harbor Perspectives in Biology 2, a000414.
| Crossref | Google Scholar |

Singh G, Sharma S, Rawat S, Sharma RK (2022) Plant Specialised Glycosides (PSGs): their biosynthetic enzymatic machinery, physiological functions and commercial potential. Functional Plant Biology 49, 1009-1028.
| Crossref | Google Scholar | PubMed |

Smith EG, D’Angelo C, Sharon Y, Tchernov D, Wiedenmann J (2017) Acclimatization of symbiotic corals to mesophotic light environments through wavelength transformation by fluorescent protein pigments. Proceedings of the Royal Society B: Biological Sciences 284, 20170320.
| Crossref | Google Scholar |

Song I, Kim J, Baek K, Choi Y, Shin B, Jin E (2020) The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii. Microbial Cell Factories 19, 220.
| Crossref | Google Scholar |

Spreitzer RJ, Peddi SR, Satagopan S (2005) Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco. Proceedings of the National Academy of Sciences 102, 17225-17230.
| Crossref | Google Scholar |

Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Biophysical Journal 106, 695A.
| Crossref | Google Scholar |

Talebi AF, Tohidfar M, Bagheri A, Lyon SR, Salehi-Ashtiani K, Tabatabaei M (2014) Manipulation of carbon flux into fatty acid biosynthesis pathway in Dunaliella salina using AccD and ME genes to enhance lipid content and to improve produced biodiesel quality. Biofuel Research Journal 1, 91-97.
| Crossref | Google Scholar |

Tanveer M, Abidin ZU, Alawadi HFN, Shahzad AN, Mahmood A, Khan BA, Qari S, Oraby HF (2024) Recent advances in genome editing strategies for balancing growth and defence in sugarcane (Saccharum officinarum). Functional Plant Biology 51, FP24036.
| Crossref | Google Scholar |

Tanwar A, Sharma S, Kumar S (2018) Targeted genome editing in algae using CRISPR/Cas9. Indian Journal of Plant Physiology 23, 653-669.
| Crossref | Google Scholar |

Tsai C-H, Warakanont J, Takeuchi T, Sears BB, Moellering ER, Benning C (2014) The protein compromised hydrolysis of triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in chlamydomonas. Proceedings of the National Academy of Sciences 111, 15833-15838.
| Crossref | Google Scholar |

Urtubia HO, Betanzo LB, Vásquez M (2016) Microalgae and cyanobacteria as green molecular factories: tools and perspectives. In ‘Algae–organisms for imminent biotechnology’. (Eds N Thajuddin, D Dhanasekaran) pp. 1–27. (IntechOpen)

Vassilev SV, Vassileva CG (2016) Composition, properties and challenges of algae biomass for biofuel application: an overview. Fuel 181, 1-33.
| Crossref | Google Scholar |

Voloshin RA, Kreslavski VD, Zharmukhamedov SK, Bedbenov VS, Ramakrishna S, Allakhverdiev SI (2015) Photoelectrochemical cells based on photosynthetic systems: a review. Biofuel Research Journal 2, 227-235.
| Crossref | Google Scholar |

Wang B, Wang J, Zhang W, Meldrum DR (2012) Application of synthetic biology in cyanobacteria and algae. Frontiers in Microbiology 3, 344.
| Crossref | Google Scholar |

Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80-84.
| Crossref | Google Scholar | PubMed |

Wang Q, Lu Y, Xin Y, Wei L, Huang S, Xu J (2016) Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9. The Plant Journal 88, 1071-1081.
| Crossref | Google Scholar | PubMed |

Wang N, Qian Z, Luo M, Fan S, Zhang X, Zhang L (2018) Identification of salt stress responding genes using transcriptome analysis in green alga Chlamydomonas reinhardtii. International Journal of Molecular Sciences 19, 3359.
| Crossref | Google Scholar |

Wang M, Ye X, Bi H, Shen Z (2024) Microalgae biofuels: illuminating the path to a sustainable future amidst challenges and opportunities. Biotechnology for Biofuels and Bioproducts 17, 10.
| Crossref | Google Scholar |

Wei L, Wang Q, Xin Y, Lu Y, Xu J (2017) Enhancing photosynthetic biomass productivity of industrial oleaginous microalgae by overexpression of RuBisCO activase. Algal Research 27, 366-375.
| Crossref | Google Scholar |

Wendt KE, Ungerer J, Cobb RE, Zhao H, Pakrasi HB (2016) CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microbial Cell Factories 15, 115.
| Crossref | Google Scholar |

Wu X, Kriz AJ, Sharp PA (2014) Target specificity of the CRISPR-Cas9 system. Quantitative Biology 2, 59-70.
| Crossref | Google Scholar | PubMed |

Yamano T, Zetsche B, Ishitani R, Zhang F, Nishimasu H, Nureki O (2017) Structural basis for the canonical and non-canonical PAM recognition by CRISPR-Cpf1. Molecular Cell 67, 633-645.e3.
| Crossref | Google Scholar | PubMed |

Yang B, Liu J, Jiang Y, Chen F (2016) Chlorella species as hosts for genetic engineering and expression of heterologous proteins: progress, challenge and perspective. Biotechnology Journal 11, 1244-1261.
| Crossref | Google Scholar | PubMed |

Yang B, Liu J, Ma X, Guo B, Liu B, Wu T, Jiang Y, Chen F (2017) Genetic engineering of the Calvin cycle toward enhanced photosynthetic CO₂ fixation in microalgae. Biotechnology for Biofuels 10, 229.
| Crossref | Google Scholar |

Yao L, Cengic I, Anfelt J, Hudson EP (2016) Multiple gene repression in cyanobacteria using CRISPRi. ACS Synthetic Biology 5, 207-212.
| Crossref | Google Scholar | PubMed |

Yoshimitsu Y, Abe J, Harayama S (2018) Cas9-guide RNA ribonucleoprotein-induced genome editing in the industrial green alga Coccomyxa sp. strain KJ. Biotechnology for Biofuels 11, 326.
| Crossref | Google Scholar |

Zaboikin M, Zaboikina T, Freter C, Srinivasakumar N (2017) Non-homologous end joining and homology directed DNA repair frequency of double-stranded breaks introduced by genome editing reagents. PLoS ONE 12, e0169931.
| Crossref | Google Scholar |

Zakar T, Laczko-Dobos H, Toth TN, Gombos Z (2016) Carotenoids assist in cyanobacterial photosystem II assembly and function. Frontiers in Plant Science 7, 173308.
| Crossref | Google Scholar |

Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759-771.
| Crossref | Google Scholar | PubMed |

Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiology 161, 20-27.
| Crossref | Google Scholar | PubMed |

Zhu Z, Sun J, Fa Y, Liu X, Lindblad P (2022) Enhancing microalgal lipid accumulation for biofuel production. Frontiers in Microbiology 13, 1024441.
| Crossref | Google Scholar |