Multiplex genome editing of microorganisms using CRISPR-Cas

Abstract

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.

Keywords: CRISPR-Cas; Cas12a; Cas9; cell factories; genome editing; multiplex.

Figure 1.

(A) Cas9 and Cas12a expression and cleavage schemes. Left panel: Cas9 requires tracrRNA transcription and RNase III expression for CRISPR array transcript processing. Cas9 forms a complex with crRNA and tracrRNA and cleaves target DNA generating blunt ends. Right panel: Cas12a processes its own CRISPR array transcript to obtain individual crRNAs without the requirement of any tracrRNA or RNAse III co-expression. Cas12a stays in complex with crRNA and cleaves target DNA generating staggered ends. (B) Double strand break (DSB) repair mechanisms. DSBs can be repaired via non-homologous end joining (NHEJ), alternative non-homologous end joining repair pathways such as microhomology-mediated end joining (MMEJ), or via homologous direct recombination. NHEJ and MMEJ repair pathways can lead to the incorporation of deletions or insertions (only in case of NHEJ) in the targeted region. HDR is combined with the supplementation of donor DNA (dDNA), which can be double stranded or single stranded. dDNA can be used for insertion of long DNA sequences, deletion of genomic fragments, or introduction of single point mutations (SNPs).

Figure 2.

Multiplexing using single gRNA expression cassettes. (A) Expression of several sgRNA cassettes from a single expression vector. (B) Expression of several sgRNA cassettes from multiple expression vectors (each harboring a different marker). (C) Expression of several sgRNA cassettes from multiple expression vectors (all harboring the same marker). (D) Transient supplementation with in vitro assembled RNPs or in vitro transcribed gRNAs.

Figure 3.

Multiplexing using gRNA polycistronic cassettes. (A) Expression of gRNAs from synthetic array dependent on Csy4 processing. In this case, Csy4 has to be co-expressed. (B) Expression of gRNAs from synthetic array dependent on endoribonuclease splicing. In most of the reviewed examples, these synthetic arrays are expressed using tRNAs as RNA pol III promoters. (C) Expression of gRNAs from native-like CRISPR array dependent on Cas9, tracrRNA and RNAse III processing. (D) Expression of gRNAs from native-like CRISPR array dependent on Cas12a processing.