Table 4.

Summary of the Main Articles Related to CRISPR-Cas Transformation in C. reinhardtii and Valuable Information Regarding the Technical Aspects.

Author and Year	System	Targeted Gene	Codon Optimization	Cas	Proof of Concept	C. reinhardtii Strain	Efficiency of Mutations	Transformation Method	Off Target	Antibiotics
Jiang et al., 2014 [18]	knock-out to disrupt FKB12 gene	FKB12: C. reinhardtii cell lacking FKB12 are fully resistant to rapamycin antibiotics	yes	Cas9 and dCas9 as control of toxicity	Cells becoming resistant to rapamycin	CC-503	Only one colony and failure to recover more transformants	electroporation	not studied	rapamycin
Baek et al., 2016 [15]	knock-out using DNA-free CRISPR-Cas9 method, disrupting CpFTSY and ZEP and generating a strain producing zeaxanthin	The knockout of the CpFTSY gene confers smaller, or truncated, chlorophyll (Chl) antenna size of the photosystems. The knockout of zeaxanthin epoxidase (ZEP) leads to constitutive accumulation of zeaxanthin	yes	Cas9	CpFTSY: pale green color ZEP: zeaxanthin accumulation CpFTSY/ZEP: zeaxanthin and golden color	CC-4349 cw15 mt-	CpFTSY:0.56% ZEP: 0.46%	electroporation	CpFTSY: far up to 4 nucleotides from the on-target ZEP: no off-target	no antibiotics used
Sung shin et al., 2016 [22]	Complex RNP for knock-out, plasmids CpSRP43 and pCr202	MAA7 encodes for the beta subunit of tryptophan synthase	yes	Cas9	5-fluoro-indole	CC-124	40%	electroporation	no off-target events	no antibiotics used
		CpSRP43: antenna assembly genes encode for chloroplast			Light colony color and low chlorophyll dosage		1.4%
		ChLM: when disrupted affects chlorophyll biosynthesis and reduces the accumulation of major thylakoid-associated proteins					1.7%
Greiner et al., 2017 [14]	RNP and plasmid knock-out of PSY1	PSY1: Phytoene synthase 1 catalyze carotenoids synthesis and photosynthesis	yes	Cas9	white colony	CC-125	not mentioned	electroporation	no off-target events	paromomycin antibiotics
Baek et al., 2017 [23]	Knock-out of zeaxanthin epoxidase with RNP CRISPR	ZEP: zeaxanthin epoxidase, when disrupted the microalgae accumulates zeaxanthin	-	Cas9	zeaxanthin dosage	CC-4349, CC-124, CC-620, CC-4051, CC-4533	not mentioned	glass beads	no off-target (Cas-OFFinder, reported in [15])	no antibiotics used
Ferenczi et al., 2017 [21]	RNP CRISPR for the gene Fkb12 with a repair plasmid ssODN	Fkb12: mediates the interaction between the antibiotic rapamycin and the cell cycle regulator target of rapamycin, which leads to cell death.	yes	LbCPpf1	high tolerance to rapamycin	CC-1883, CC-2931	29%	electroporation	no predicted off-target sites with Cas-ORFinder	Rapamycin
Findinier et al., 2019 [83]	RNP CRISPR for the gene CrFzI	CrFzi: phenotypic analyses revealed a specific requirement of CrFzl for survival upon light stress. Consistent with this, strong irradiance leads to increased photoinhibition of photosynthesis in mutant cells	not mentioned	Cas9	when Fzi is disrupted, it promotes thylakoid fusion and resistance to light stress	cc-4533, cw15.J3	not mentioned	electroporation	not mentioned	Hygromycin resistant gene
Guzmán-Zapata et al., 2019 [20]	plasmid CRISPR	Apt: sensitivity to a toxin 2-fluoroadenine (2-FA)	Yes	Cas9	growth on the 2-FA toxin	CC-125, CC-3403, SAG73.72, CC-3403-uvr8-2	not mentioned	glass beads/particle bombardment	not mentioned	no antibiotic used
Kim et al., 2020 [84]	knock- out RNP CRISPR Cas9 and knock-in antibiotics resistance gene and YFP	CrFTSY: confers smaller, or truncated, chlorophyll (Chl) antenna size of the photosystems ([15])	not mentioned	Cas9	pale green color and measurement of luciferase activity	CC-4349, CC-124, and CC-503	up to 30%	electroporation	not mentioned	hygromycin, paromomycin