Development and Application of CRISPR/Cas in Microbial Biotechnology

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has been rapidly developed as versatile genomic engineering tools with high efficiency, accuracy and flexibility, and has revolutionized traditional methods for applications in microbial biotechnology. Here, key points of building reliable CRISPR/Cas system for genome engineering are discussed, including the Cas protein, the guide RNA and the donor DNA. Following an overview of various CRISPR/Cas tools for genome engineering, including gene activation, gene interference, orthogonal CRISPR systems and precise single base editing, we highlighted the application of CRISPR/Cas toolbox for multiplexed engineering and high throughput screening. We then summarize recent applications of CRISPR/Cas systems in metabolic engineering toward production of chemicals and natural compounds, and end with perspectives of future advancements.

Keywords: CRISPR/Cas; gene regulation; genome editing; guide RNA; microbial biotechnology.

FIGURE 1

Guidelines for expression of Cas protein and sgRNA in CRISPR/Cas system. (A) Scheme of CRISPR/Cas9 system. The Cas9-sgRNA (or Cas9-crRNA-tracrRNA) complex binds to DNA target arising from Watson-Crick base pairing of spacer sequence, and triggers double strand break (DSB) when next to a short protospacer adjacent motif (PAM, ‘NGG’ for Cas9 from S. pyogenes). (B) Expression cassette for Cas9. For efficient targeting to nucleus in eukaryotes, the Cas9 should be fused to NLS (nuclear localization sequence) at one end or both ends. (C) Scheme of CRISPR/Cas12a (Cpf1) system. Cas12a triggers DSB through a similar scheme of Cas9, but depends on different PAM (‘NTTT’) and less folded crRNA, and creates a sticky end at 18–23 bases away from the PAM. (D) Expression cassette for sgRNA. A promoter of RNA polymerase III (RNAP III) is usually required for directing sgRNA in nucleus and with less modification. A 20 bp spacer should be well designed according to target DNA sequence for efficient editing rates and avoiding off-target effects. (E) Multi-sgRNA expression through multi-cassettes. Repeated elements, such as promoters, gRNA scaffold and terminators are repeated for different spacer sequences. (F) Multi-sgRNA expression through crRNA array and tracrRNA (HI-CRISPR system). Different spacers are separated with direct repeats (DRs) and expressed by one promoter of RNAP III. The pre-crRNA is transcribed and processed into mature crRNA by RNase III and unknown nuclease(s). The tracrRNA and Cas9 protein are complexed with mature crRNA to form the dual-RNA-guided nuclease. (G) gRNA multiplexing strategies. Both RNAP II and RNAP III promoter can be used for expression the sgRNA array, where sgRNAs are separated by features for RNA cleavage. RNA endonuclease Csy4 recognizes a 28 nucleotide sequence flanking the sgRNA sequence and cleaves after the 20th nucleotide. The hammerhead ribozyme and HDV ribozyme flanked the 5′ and 3′ of the sgRNA, respectively, allowing for self-cleaving production of sgRNAs, which are not dependent on the presence of an exogenous protein. Polycistronic tRNA-gRNA architecture allows the production of multiple sgRNAs by endogenous RNase P and RNase Z.

FIGURE 2

DNA repair and donor design for DNA deletion, insert and mutation. (A) The Cas9-sgRNA complex binds to DNA target and triggers a double strand break (DSB), which is subsequently repaired generally through non-homologous end joining (NHEJ) or homology-directed (HDR) pathway. NHEJ directs ligation of two break ends with little or no sequence homology required, resulting in small insertions or deletions (indels); while HDR repairs DSB according to a DNA template with homology sequence, resulting in precise editing when supplemented with ds- or ss-DNA donors. (B) A Cas9 nickase mutant with HNH or RuvC inactive domain introduces a single strand break (SSB), which can be repaired by HDR rather than NHEJ pathway. (C) Donor designs for gene interruption, deletion and mutation. Gene interruption: Small deletion (e.g., 8 bp) or insertion is integrated to shift reading frame, or stop codon is introduced to interrupt gene translation. Gene deletion: A donor fused with sequence upstream and downstream ORF is sued for gene deletion (‘*’ indicate the deleted gene). Gene mutation: Sequence mutations can be introduced by a donor, where seed sequence and PAM should be destroyed to avoid cutting again by Cas9-sgRNA complex. Chr, chromosome. (D) Donor design for sequence insertion. A donor contain long sequence is integrated through HDR, and longer homology arms are required when inserting long sequence. (E) Another strategy employing CRISPR I-F or V-K (e.g., Cas12k) mediates DNA integration with Tn7-like transposons (e.g., tnsB/tnsC/tniQ).

FIGURE 3

The nuclease-deactivated Cas9 (dCas9) mediated CRISPRi and CRISPRa system. (A) dCas9 blocks recruiting of RNA polymerase (RNAP). (B) dCas9 can sterically block the transcriptional elongation of RNAP. (C) dCas9 fuses repressors (e.g., KRAB, Mxi1) to repress gene transcription. (D) KRAB is fused to RNA-binding domains (e.g., COM-KRAB) and achieves gene repression when targeting DNA sites overlaps the TSS using an scaffold RNA. (E) Fusion of dCas9 with the histone demethylase LSD1 suppresses gene expression. (F) Fusion of dCas9 with activators (e.g., VP64, ω-subuint of RNAP) activates gene transcription. (G) The VPR strategy for gene activation. The dCas9 has been fused to the combinatory transcriptional activator VP64-p65-Rta (VPR) to amplify the activation effects. (H) The SAM system. The dCas9 is fused to VP64 and the sgRNA has been modified to contain two MS2 RNA aptamers to recruit the MS2 bacteriophage coat protein (MCP), which was fused to the transcriptional activators p65 and heat shock factor 1 (HSF1). (I) The SunTag system. The tandem repeats of a small peptide GCN4 are utilized to recruit multiple copies of scFv (single-chain variable fragment) in fusion with the transcriptional activator VP64. (J) dCas9 is fused with the catalytic core of the human acetyltransferase p300, which catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation.

FIGURE 4

Orthogonal CRISPR systems. (A) The orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID). This orthogonal tri-functional CRISPR system employed dLbCpf1-VP for CRISPRa, dSpCas9-RD1152 for CRISPRi, and SaCas9 for CRISPRd (gene deletion), which recognized different type of sgRNA and PAMs. dLbCpf1, dCpf1 from Lachnospiraceae bacterium ND2006; dSpCas9, dCas9 from S. pyogenes; SaCas9, Cas9 from S. aureus. (B) The orthogonal CRISPR system with different RNA scaffolds. The gRNA fused with MS2 is used for activation through binding of MCP-VP64 or MCP-SoxS^R93A. The gRNA fused with com is used for repression through binding of Com-KRAB. Alternatively, a sgRNA without MS2 or com scaffold can hinder gene expression either. (C) CRISPR and CRISPRi via different crRNA length. Cas12a triggers DSB and genome editing with 20 bp-spacer in crRNA, while it blocks transcription with a short crRNA (16 bp-spacer).

FIGURE 5

Strategies for precise single base editing. (A) Two-step stuffer-assisted point mutation. In the first step, a 20-nucleotide target genome sequence close to the target is replaced by a heterologous stuffer fragment via homologous recombination. In the second step, the stuffer fragment acts as the target sequence, recognized by a second gRNA, and the original sequence with arbitrary mutation is inserted back. Chr, chromosome. (B) ‘C→T’ mutation through DSB independent pathway. The dCas9 or Cas9 nickase (nCas9, D10A) is fused with cytidine deaminase and uracil DNA glycosylase inhibitor (UGI), and binds to a DNA target. Cytidine deaminase converts the cytidine (‘C’) to uracil (‘U’) in the non-targeted strand, which is protected by UGI from the nucleotide excision repair (NER) pathway. And in the next replication cycle, the ‘G:C’ base pair is repaired to ‘T:A’. (C) ‘A→G’ mutation through DSB independent pathway. The dCas9 or nCas9 is fused with adenosine deaminase and binds to a DNA target. Adenosine deaminase converts the adenosine (‘A’) to hypoxanthine (‘I’) in the non-targeted strand. And in the next replication cycle, the ‘T:A’ base pair is repaired to ‘C:G’.