CRISPR/Cas9系统的PCR介导的一日合成向导RNA

Naim Hassan; Farhana    Easmin; Keisuke    Ekino; Satoshi Harashima

doi:10.21769/BioProtoc.4082

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Peer-reviewed

PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System

CRISPR/Cas9系统的PCR介导的一日合成向导RNA

NH Naim Hassan ^*

FE Farhana Easmin ^*

KE Keisuke Ekino

Satoshi Harashima email

(*contributed equally to this work) 发布: 2021年07月05日第11卷第13期 DOI: 10.21769/BioProtoc.4082 浏览次数: 3697

评审: Priyanka DasLifeng LiuAnonymous reviewer(s)

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Abstract

Nowadays, CRISPR (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated protein (Cas9) system play a major role in genome editing. To target the desired sequence of the genome successfully, guide RNA (gRNA) is indispensable for the CRISPR/Cas9 system. To express gRNA, a plasmid expressing the gRNA sequence is typically constructed; however, construction of plasmids involves much time and labor. In this study, we propose a novel procedure to express gRNA via a much simpler method that we call gRNA-TES (gRNA-transient expression system). This method employs only PCR, and all the steps including PCR and yeast transformation can be completed within 1 day. In comparison with the plasmid-based gRNA delivery system, the performance of gRNA-TES is more effective, and its total time and cost are significantly reduced.

Keywords: CRISPR/Cas9 (CRISPR/Cas9)

Genome editing (基因组编辑)

Guide RNA (向导RNA)

PCR-based (基于PCR的)

Yeast (酵母)

Background

Developing genome editing techniques is one of the central issues of genome science. For the past decade, the CRISPR/Cas9 system has contributed to easier and more precise genome editing as compared with previously developed techniques such as ZFN (zinc finger nuclease) and TALEN (transcription activator-like effector nuclease). For successful CRISPR/Cas9 engineering, design, expression, and delivery of the guide RNA (gRNA) components are the key factors (Stovicek et al., 2017). For prokaryotes like Escherichia coli and eukaryotes such as Saccharomyces cerevisiae, the most commonly employed method for expressing gRNA is to use a plasmid (Jiang et al., 2013; Li et al., 2015; DiCarlo et al., 2013; Bao et al., 2015; Jakočiunas et al., 2015a and 2015b); however, plasmid construction, including cloning steps for necessary components, is laborious, costly, and time-consuming. To express gRNA more simply, in this study we developed a method that we call gRNA-transient expression system (gRNA-TES), where gRNA is expressed from the PCR product. gRNA-TES is very fast and effective: it takes only 5-6 h to complete the whole process, including preparation of PCR products for expression of gRNA in yeast cells and yeast transformation. By contrast, it takes at least 3-4 days to construct a plasmid expressing gRNA including verification. As expected, when applied to replacement of desired chromosome regions in yeast, gRNA-TES effectively replaces single and multiple chromosomal regions (Easmin et al., 2019 and 2020). Therefore, we believe that gRNA-TES will be useful for other types of genome editing including segmental deletion, duplication, and splitting of chromosomes. Lastly, gRNA-TES is effective in yeast; therefore, it should be emphasized that gRNA-TES may also be efficacious in other organisms if suitable gene promoters are incorporated.

Materials and Reagents

0.1-10 μl pipette tips (BMBio, catalog number: BMT-10GXLR)
20-200 μl pipette tips (BMBio, catalog number: BMT-200R)
PCR tubes (Axygen, catalog number: SKPCRF)
p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene, catalog number: 43803)
p414-TEF1p-Cas9-CYC1t (Addgene, catalog number: 43802)
Escherichia coli DH5α competent cells (NIPPON GENE, catalog number: 316-06233)
DNA, MB-grade from fish sperm (Roche Diagnostics, catalog number: 11467140001)
KOD plus neo (TOYOBO, catalog number: KOD-401)
2 mM dNTP solution (included with KOD plus neo)
25 mM magnesium sulfate (MgSO₄) (included with KOD plus neo)
Oligonucleotide Primer Fw A for construction of fragment A (5’-GTTCGAAACTTCTCCGCAGT GAAAGATAAATGATCN₂₀GTTTTAGAGCTAGAAATAGCAAG-3’) (synthesized by Hokkaido System Science, Japan) (N₂₀ represents the 20-nt upstream sequence of the target PAM sequence)
Oligonucleotide primer Rv A for construction of fragment A (5’- ACTCACAAATTAGAGCTTCA -3’) (synthesized by Hokkaido System Science, Japan)
Oligonucleotide primer Fw B for construction of fragment B (5’- CGAACGACCGAGCGCAGCGA-3’) (synthesized by Hokkaido System Science, Japan)
Oligonucleotide primer Rv B for construction of fragment B (5’- TTTATCTTTCACTGCGGAGAAGTTTCGAAC-3’) (synthesized by Hokkaido System Science, Japan)
Ex Taq^® DNA polymerase (TaKaRa, catalog number: RR001A)
10× Ex Taq buffer (Mg²⁺ plus) (included with Ex Taq^® DNA Polymerase)
2.5 mM each dNTP mix (included with Ex Taq^® DNA Polymerase)
Ampicillin (Nacalai tesque, catalog number: 02739-74)
Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883-250G)
Polyethylene glycol 3,350 (Sigma-Aldrich, catalog number: P4338-500G)
Sodium hydroxide (NaOH) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 192-15985)
Control primer 1 for amplifying the CNE1 region (5’-TCACAGGGTCGATTGCAAGG-3’) (synthesized in Hokkaido System Science, Japan)
Control primer 2 for amplifying the CNE1 region (5’-CTGGTGGTTCAGTGCCATCT-3’) (synthesized in Hokkaido System Science, Japan)
Oligonucleotide primer 1 for checking replacement (Use the 200-176 nt upstream sequence of the target region)
Oligonucleotide primer 2 for checking replacement (Use the 66-90 nt downstream reverse sequence of the target region)
Prime Star Max Premix 2× (TaKaRa, catalog number: R045A)
Agar (FUJIFILM Wako Pure Chemical Corporation, catalog number: 010-08725)
Gene Ladder Wide 2 (Nippon Gene, catalog number: 310-06971)
Glucose (FUJIFILM Wako Pure Chemical Corporation, catalog number: 043-31163)
Yeast nitrogen base without amino acids (BD, Difco, catalog number: DF0919-15-3)
Peptone (BD, Bacto^TM, catalog number: 211677)
Yeast extract (BD, Bacto^TM, catalog number: 288620)
Adenine HCL (FUJIFILM Wako Pure Chemical Corporation, product code: 016-00802)
Synthetic Complete (SC) medium (see Recipes)
YPDA medium (see Recipes)

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Hassan, N., Easmin, F., Ekino, K. and Harashima, S. (2021). PCR-mediated One-day Synthesis of Guide RNA for the CRISPR/Cas9 System. Bio-protocol 11(13): e4082. DOI: 10.21769/BioProtoc.4082.