CRISPR-based gene editing technology and its application in microbial engineering

Abstract

Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-mediated technologies have provided an efficient tool for genetic engineering of cells and organisms. Here, we review the three emerging gene editing tools (ZFNs, TALENs, and CRISPR-Cas) and briefly introduce the principle, classification, and mechanisms of the CRISPR-Cas systems. Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems, as well as the novel base editor (BE), prime editor (PE), and CRISPR-associated transposase (CAST) technologies, are described in detail. In addition, we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications. Finally, the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.

Keywords: CRISPR-Cas system; Gene editing; Industrial microorganisms; Probiotics.

Graphical abstract

Fig. 1

Comparison of gene editing methods based on ZFNs, TALENs, and CRISPR-Cas9 technologies. Both ZFNs and TALENs are engineered nucleases, and their Fok I nucleases are fused with codon-specific and nucleotide-specific recognition modules, respectively. After the recognition module binds to the target site, Fok I nucleases are activated after dimerization. CRISPR-Cas9, however, is an RNA-guided nuclease that is activated after the guide RNA is base-paired with target sites. These systems all cause DNA double-strand breaks (DSBs) near the target site, introducing gene editing through subsequent repair pathways such as NHEJ, MMEJ, or HDR. The latest CRISPR-based gene editing tools that do not rely on DSBs are described in a later section.

Fig. 2

The three stages (adaptation, processing, and interference) of CRISPR-Cas immunity against invading genetic elements. During the adaptation stage, short fragments of foreign nucleic acids are integrated into the CRISPR array to form an immune memory. The CRISPR array is then transcribed into pre-crRNA, which is processed into mature crRNA by a related ribonuclease. In the final stage of target interference, mature crRNAs guide the CRISPR-Cas effector complex to cleave foreign nucleic acids through complementary base-pairing between the crRNA and target nucleic acid.

Fig. 3

Schematic diagram of the classification and architecture of different types of CRISPR-Cas systems. The CRISPR-Cas system is highly diverse and is currently divided into two broad categories: class 1 and class 2. Class 1 systems encode multi-subunit effector complexes, while class 2 systems encode single-subunit effectors. Different types of systems encode specific Cas proteins involved in adaptation, processing, and interference. Genes that may be missing in some subtypes are indicated by dashed outlines. Genes encoding adaptation proteins are shown in brown. Genes encoding proteins involved in pre-crRNA processing are shown in purple. Genes encoding proteins involved in target interference are shown in brick red. The staggered diamonds and rectangles represent repeats and spacers, respectively, which make up the CRISPR array.

Fig. 4

Strategies for gene editing based on exogenous and endogenous CRISPR-Cas systems. For gene editing based on exogenous CRISPR-Cas systems, Cas nuclease (usually Cas9 or Cas12) and short artificial mini-CRISPR arrays are provided to create double-stranded breaks at specific sites. Donor DNA is also needed for the introduction of desired mutations via the HDR pathway. For gene editing based on endogenous CRISPR-Cas systems, only artificial mini-CRISPR arrays and donor DNA are needed. The crRNA generated by artificial mini-CRISPR arrays forms effector complexes with endogenous Cas proteins, resulting in cleavage of target sites. Subsequently, the target mutation is achieved by homologous recombination between the donor DNA and host genome.

Fig. 5

CRISPR-based gene editing tools without DSBs: base editors (BEs), prime editors (PEs), and CRISPR-associated transposases. For BEs, dCas9 or nCas9 is fused with deaminase. When the sgRNA guides Cas9 to bind to the target, deaminase deamidates the unwound single-stranded DNA, ultimately resulting in base conversion. For PEs, nCas9 is fused with reverse transcriptase and the sgRNA was engineered to a more complex pegRNA. Following the actions of nCas9, the 3′ end of the pegRNA can pair with the broken single-stranded DNA and designed mutations are then introduced into the target site under the action of reverse transcriptase. For CRISPR-related transposase (CAST)-mediated gene integration, transposon donor DNA is first captured and processed by TnsA and TnsB. Then, TnsA, TnsB, and the processed substrate form an integrated complex. The CRISPR elements of CAST systems bind to a specific site under the guidance of crRNA and recruit TniQ, TnsC, and TnsA/B complexes to integrate the donor DNA fragment downstream of the target site.