Novel Microbial Modification Tools to Convert Lipids into Other Value-Added Products

  • Priya Kumari
  • Farnaz Yusuf
  • Naseem A. GaurEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1995)


CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) is a microbial adaptive immune system that has revolutionized the field of molecular biology and genome engineering. The Type II CRISPR system consists of Cas9 nuclease of Streptococcus pyogenes and the RNA complex that guides Cas9 nuclease to a specific sequence of DNA in the genome. The CRISPR-Cas9 technology has reformed our ability to edit DNA and to regulate expression levels of genes of interest to high precision and accuracy. It is a powerful technology, which is used for genome engineering of a wide range of organisms for various applications. Here, we describe a method involving CRISPR-Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms for biotechnological applications in yeast. The complete procedure of genome editing including target sequence selection, cloning gRNA with a target sequence, transformation, and verification of the desired mutation/deletion or insertion can be achieved within 2–3 weeks in yeast.

Key words

CRISPR-Cas9 gRNA CRISPR RNA Trans-activating CRISPR RNA Genome editing Homology-directed repair Nonhomologous end joining 


  1. 1.
    Song J-W, Jeon E-Y, Song D-H, Jang H-Y, Bornscheuer UT, Oh D-K, Park J-B (2013) Multistep enzymatic synthesis of long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils. Angew Chem Int Ed 52(9):2534–2537CrossRefGoogle Scholar
  2. 2.
    Keasling JD, Chou H (2008) Metabolic engineering delivers next-generation biofuels. Nat Biotech 26(3):298–299CrossRefGoogle Scholar
  3. 3.
    Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB (2010) Microbial biosynthesis of alkanes. Science 329(5991):559CrossRefGoogle Scholar
  4. 4.
    Lee JW, Na D, Park JM, Lee J, Choi S, Lee SY (2012) Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat Chem Biol 8(6):536–546CrossRefGoogle Scholar
  5. 5.
    Ostergaard S, Olsson L, Nielsen J (2000) Metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 64(1):34–50CrossRefGoogle Scholar
  6. 6.
    Lartigue C, Vashee S, Algire MA, Chuang RY, Benders GA, Ma L, Noskov VN, Denisova EA, Gibson DG, Assad-Garcia N, Alperovich N, Thomas DW, Merryman C, Hutchison CA 3rd, Smith HO, Venter JC, Glass JI (2009) Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 325(5948):1693–1696CrossRefGoogle Scholar
  7. 7.
    Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345CrossRefGoogle Scholar
  8. 8.
    Zhu B, Cai G, Hall EO, Freeman GJ (2007) In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques 43(3):354–359CrossRefGoogle Scholar
  9. 9.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826CrossRefGoogle Scholar
  10. 10.
    Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotech 32(4):347–355CrossRefGoogle Scholar
  11. 11.
    La Russa MF, Qi LS (2015) The new state of the art: Cas9 for gene activation and repression. Mol Cell Biol 35(22):3800–3809CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Yeast Biofuel Group, DBT-ICGEB Centre for Advanced Bioenergy ResearchInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia

Personalised recommendations