Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

Li Yu Sung, Meng Ying Wu, Mei Wei Lin, Mu Nung Hsu, Vu Anh Truong, Chih Che Shen, Yi Tu, Kuen Yuan Hwang, An Pang Tu, Yu Han Chang*, Yu Chen Hu

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

31 Scopus citations

Abstract

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.

Original languageEnglish
Pages (from-to)1066-1079
Number of pages14
JournalBiotechnology and Bioengineering
Volume116
Issue number5
DOIs
StatePublished - 05 2019
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2019 Wiley Periodicals, Inc.

Keywords

  • CRISPR
  • CRISPRi
  • Cas9 ortholog
  • SaCas9
  • St1Cas9
  • metabolic engineering

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