利用CRISPR技术对莱茵衣藻进行DNA-free的基因组编并对产生突变进行分析

Jihyeon  Yu; Kwangryul  Baek; EonSeon  Jin; Sangsu  Bae

doi:10.21769/BioProtoc.2352

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Peer-reviewed

DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis

利用CRISPR技术对莱茵衣藻进行DNA-free的基因组编并对产生突变进行分析

JY Jihyeon Yu ^*

KB Kwangryul Baek ^*

EJ EonSeon Jin email

Sangsu Bae email

(*contributed equally to this work) 发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2352 浏览次数: 14642

评审: Vinay PanwarKaisa KajalaAnonymous reviewer(s)

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Abstract

We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.

Keywords: Genome editing (基因组编辑)

CRISPR-Cas9 ( CRISPR-Cas9)

Microalgae ( 微藻)

Ribonucleoproteins ( 核糖核蛋白)

Chlamydomonas reinhardtii ( 莱茵衣藻)

DNA-free transformation (DNA-free转化)

Background

We recently reported (Baek et al., 2016) a one-step transformation of the model green microalga Chlamydomonas reinhardtii (Harris, 2001) using preassembled Cas9 protein-guide RNA ribonucleoproteins (RNPs). The manner of DNA-free CRISPR-Cas9 delivery has several advantages such as no need for codon optimization and specific promoters in different species of microalgae. Furthermore, it reduces off-target effects and may also be less cytotoxic in cells because the Cas9 protein is transiently active and then degraded by endogenous proteases in cells (Kim et al., 2014). In addition, the resulting gene-edited microalgae could be exempt from genetically modified organism (GMO) regulations due to the absence of foreign DNA sequences. In this protocol, the detailed procedures of an entire workflow are contained from the initial target selection of CRISPR to the mutant analysis using NGS technology (Bae et al., 2014a and 2014b; Park et al., 2015 and 2017).

Materials and Reagents

Cas9 protein purification
1. 1.5 ml Eppendorf tubes
2. Sterile 50 ml conical tubes
3. 10 ml syringe
4. 0.45 μm syringe filter
5. Protein concentrator–100K MWCO (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 88533 )
6. Poly-Prep Chromatography columns (Bio-Rad Laboratories, catalog number: 7311550 )
7. BL21-Pro cells
8. pET28 plasmid containing 6xHis-SpCas9 (Addgene)
9. Kanamycin
10. Lysozyme
11. PMSF
12. Ni-NTA agarose beads (QIAGEN, catalog number: 30210 )
13. Bradford reagent (Bio-Rad Laboratories, catalog number: 5000205 )
14. Bovine serum albumin (BSA)
15. Sodium chloride (NaCl)
16. Tryptone
17. Yeast extract
18. IPTG
19. Sodium phosphate monobasic (NaH₂PO₄)
20. Imidazole
21. Sodium hydroxide (NaOH)
22. HEPES
23. Ethylenediaminetetraacetic acid (EDTA)
24. DL-dithiothreitol (DTT)
25. Sucrose
26. Glycerol
27. LB medium (see Recipes)
28. 1 M IPTG stock (see Recipes)
29. Lysis buffer (see Recipes)
30. Wash buffer (see Recipes)
31. Elution buffer (see Recipes)
32. Cas9 storage buffer (see Recipes)

In vitro transcription and library PCR

1.5 ml Eppendorf tubes

Forward and reverse oligos (see Tables 1 and 2)

Table 1. Pre-index primers

Pre-index forward primer	5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT gDNA target-3’
Pre-index reverse primer	5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT gDNA target-3’

Table 2. Index primer sequences

	index sequence [i5]	Index forward primer sequence
D501	tatagcct	AATGATACGGCGACCACCGAGATCTACACtatagcctACACTCTTTCCCTACACGAC
D502	atagaggc	AATGATACGGCGACCACCGAGATCTACACatagaggcACACTCTTTCCCTACACGAC
D503	cctatcct	AATGATACGGCGACCACCGAGATCTACACcctatcctACACTCTTTCCCTACACGAC
D504	ggctctga	AATGATACGGCGACCACCGAGATCTACACggctctgaACACTCTTTCCCTACACGAC
D505	aggcgaag	AATGATACGGCGACCACCGAGATCTACACaggcgaagACACTCTTTCCCTACACGAC
D506	taatctta	AATGATACGGCGACCACCGAGATCTACACtaatcttaACACTCTTTCCCTACACGAC
D507	caggacgt	AATGATACGGCGACCACCGAGATCTACACcaggacgtACACTCTTTCCCTACACGAC
D508	gtactgac	AATGATACGGCGACCACCGAGATCTACACgtactgacACACTCTTTCCCTACACGAC
	index sequence [i7]	Index reverse primer sequence
D701	cgagtaat	CAAGCAGAAGACGGCATACGAGATcgagtaatGTGACTGGAGTTCAGACGTGT
D702	tctccgga	CAAGCAGAAGACGGCATACGAGATtctccggaGTGACTGGAGTTCAGACGTGT
D703	aatgagcg	CAAGCAGAAGACGGCATACGAGATaatgagcgGTGACTGGAGTTCAGACGTGT
D704	ggaatctc	CAAGCAGAAGACGGCATACGAGATggaatctcGTGACTGGAGTTCAGACGTGT
D705	ttctgaat	CAAGCAGAAGACGGCATACGAGATttctgaatGTGACTGGAGTTCAGACGTGT
D706	acgaattc	CAAGCAGAAGACGGCATACGAGATacgaattcGTGACTGGAGTTCAGACGTGT
D707	agcttcag	CAAGCAGAAGACGGCATACGAGATagcttcagGTGACTGGAGTTCAGACGTGT
D708	gcgcatta	CAAGCAGAAGACGGCATACGAGATgcgcattaGTGACTGGAGTTCAGACGTGT
D709	catagccg	CAAGCAGAAGACGGCATACGAGATcatagccgGTGACTGGAGTTCAGACGTGT
D710	ttcgcgga	CAAGCAGAAGACGGCATACGAGATttcgcggaGTGACTGGAGTTCAGACGTGT
D711	gcgcgaga	CAAGCAGAAGACGGCATACGAGATgcgcgagaGTGACTGGAGTTCAGACGTGT
D712	ctatcgct	CAAGCAGAAGACGGCATACGAGATctatcgctGTGACTGGAGTTCAGACGTGT

dNTP mix
Phusion DNA polymerase (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: F530L )
PCR purification kit
ATP, CTP, GTP, UTP, 100 mM MgCl₂, DEPC-treated water
T7 RNA polymerase (New England Biolabs, catalog number: M0251L )
DNase I (RNase-free) (New England Biolabs, catalog number: M0303L )
RNase inhibitor murine (New England Biolabs, catalog number: M0314L )
RNeasy MinElute Cleanup Kit (QIAGEN, catalog number: 74204 )
Illumina Miseq Reagent Kit (v2)

Transfection
1. 1.5 ml Eppendorf tubes
2. Sterile 15 ml and 50 ml conical tubes
3. Purified Cas9 protein and sgRNA
4. Chlamydomonas reinhardtii strains CC-4349 cw15 mt-(Chlamydomonas Resource Center)
5. PCR DNA purification kit (MG Med, catalog number: MD008 )
6. TAP medium (Harris, 1989 or Thermo Fisher Scientific, Gibco^TM, catalog number: A1379801 )
7. TAP sucrose solution (see Recipes)
8. TAP agar plates (see Recipes)
9. Top agar (see Recipes)

Equipment

Cas9 protein purification
1. Centrifuge: swing rotor (LaboGene, model: 1580R )
2. Sonicators (Qsonica, model: Q125 )
3. Pipettes
4. Shaking incubator (JS Research, model: JSSI-300C )
In vitro transcription and library PCR
1. Incubator (JS Research, model: JSGI-050T )
2. Thermal cycler (Bio-Rad Laboratories, model: C1000 Touch^TM Thermal Cycler )
3. Micro centrifuge (LaboGene, model: 1730R )
4. Spectrophotometer
Transfection
1. 100 ml flask
2. Spectrophotometer (GE Healthcare, Amersham Biosciences, model: Ultrospec 2100 pro )
3. Hemocytometer (Marienfeld-Superior, catalog number : 0650030 )
4. Microscope (Olympus, model: CH30 )
5. Micro high speed centrifuge (Hanil, model: MICRO 17TR )
6. Orbital shaker (N-biotek, model: NB-101M )
7. Clean bench (BioFree, model: BF-150BSC )
8. Electroporation device (Bio-Rad Laboratories, model: Gene Pulser Xcell^TM Electroporation Systems )
9. Electroporation cuvettes (Bio-Rad Laboratories, 0.4 cm gap)

Procedure

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Yu, J., Baek, K., Jin, E. and Bae, S. (2017). DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis. Bio-protocol 7(11): e2352. DOI: 10.21769/BioProtoc.2352.