Jun 12, 2026
The application of a novel Cas12a nuclease aligned CRISPR system in Ulva prolifera
- Zheng Qin1,
- Toshiki Uji2
- 1Graduate School of Fisheries Sciences, Hokkaido University, Japan;
- 2Faculty of Fisheries Sciences, Hokkaido University, Japan
Protocol Citation: Zheng Qin, Toshiki Uji 2026. The application of a novel Cas12a nuclease aligned CRISPR system in Ulva prolifera. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj5rwxgk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 11, 2026
Last Modified: June 12, 2026
Protocol Integer ID: 318987
Keywords: Seaweed, Genome editing, CRISPR, Cas12a, Delivery system, Ulva prolifera, crispr system in ulva prolifera, editing system in the green seaweed ulva prolifera, novel crispr cas12a, green seaweed ulva prolifera, crispr genome, crispr system, novel cas12a nuclease, ulva prolifera, aligned gene, mutant screening
Abstract
The protocol describes a novel CRISPR Cas12a-aligned gene-editing system in the green seaweed Ulva prolifera. It has 3 main steps:
1. Gamete induction and collection by phototaxis.
2. CRISPR genome editing RNP assembly and delivery by modified Polyethene Glycol (PEG) 4000 system.
3. Mutant screening by 2-fluoroadenine (2-FA).
Materials
Sorbitol (TCI S0065)
0.5M Tris-HCl pH 7.5
1M CaCl2
PEG 4000 (Wako)
PEG 8000 (MPbio)
2-Fluoroadenine (TCI F0647)
EnGen sgRNA Synthesis Kit (NEB #E3322S),
Monarch RNA Clean up Kit (NEB #T2040)
Cas9 Nuclease (NEB #M0646T)
crRNA (IDT)
ST8
AsCas12a (IDT 1081068)
Distilled water
Gamete collection
Prepare the Low Salinity Buffer:
Sorbitol 1.822 g
Distilled water 8.7 mL
1x PES medium 1 mL
0.5M Tris-HCl pH 7.5 200 µL
1M CaCl2 100 µL
The gametes are collected by phototactic migration after cells are obtained by centrifugation at 5,000 g for 5 min at 20°C. Finally, resuspend thoroughly in Low Salinity Buffer at a density of 1.0×10^^4 per µL.
RNP preparations
For CRISPR/Cas9 Genome Editing:
Mix 1 µL crRNA+1 µL tracrRNA and incubate at 95°C for 5 min as the synthetic sgRNA. After cooling down, Cas9 proteins are added to the synthetic sgRNA and incubated at 37°C for 5-10 min to make the CRISPR RNP complexes.
For CRISPR/AsCas12a and ST8 Genome Editing:
Mix 1 µL dissolved crRNA with AsCas12a/ST8 (3.34 µmol/µL), then incubate at room temperature for 10-20 min as the CRISPR RNP complexes.
Transformation
Prepare the Transformation Buffers:
40% PEG 4000:
PEG 4000 4 g
Distilled water 10 mL
40% PEG 4000 with CaCl2:
PEG 4000 4 g
Distilled water 9.7 mL
0.5M Tris-HCl pH 7.5 200 µL
1M CaCl2 100 µL
Final concentration: 10mM Tris-HCl pH 7.5, 10mM CaCl2, 40% PEG 4000.
60% PEG 4000:
PEG 4000 6 g
Distilled water 10 mL
40% PEG 8000:
PEG 8000 4 g
Distilled water 10 mL
60% PEG 8000:
PEG 8000 6 g
Distilled water 10 mL
Prepare the reaction as following:
Gametes 100 µL
60% PEG or UTB2 (Prefer 60% PEG) 100 µL
CRISPR RNP complex 2.5 µL
Gently mix the reagents and incubate at room temperature for 30 min.
Centrifuge at 5,000 × g for 5 min and remove the supernatant.
Wash the gametes with filtered seawater and centrifuge at 5,000 g for 5 min to obtain the pellet.
Resuspend the pellet in 1/2 PES and add to a 60 mm dish.
Incubate the dish at 20°C in the dark for 36-48 hours before 2-FA treatment.
2-FA selection
The 1/2 PES medium was removed, and the selection medium (10 uM 2-FA) was added to the dish and cultured for 2-3 weeks; change the selection medium every week.
Transfer the 2-FA-resistant thalli to 1/2 PES medium for the following experiments and change the medium every week.
Protocol references
Badis Y, Scornet D, Harada M, Caillard C, Godfroy O, Raphalen M, Gachon C, Coelho S, Motomura T, Nagasato C, Cock J. 2021. Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus. New Phytologist 231, 2077-2091.
Ichihara K, Yamazaki T, Kawano S. 2022. Genome editing using a DNA-free clustered regularly interspaced short palindromic repeats-Cas9 system in green seaweed Ulva prolifera_. Phycological Research 70_, 50-56.
Qin Z, Surnido W, Mizuta H, and Uji T. 2025. Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera_. BMC Plant Biology 25_, 1323.