CRISPR-Cas9/Cas12a biotechnology and application in bacteria

Abstract

CRISPR-Cas technologies have greatly reshaped the biology field. In this review, we discuss the CRISPR-Cas with a particular focus on the associated technologies and applications of CRISPR-Cas9 and CRISPR-Cas12a, which have been most widely studied and used. We discuss the biological mechanisms of CRISPR-Cas as immune defense systems, recently-discovered anti-CRISPR-Cas systems, and the emerging Cas variants (such as xCas9 and Cas13) with unique characteristics. Then, we highlight various CRISPR-Cas biotechnologies, including nuclease-dependent genome editing, CRISPR gene regulation (including CRISPR interference/activation), DNA/RNA base editing, and nucleic acid detection. Last, we summarize up-to-date applications of the biotechnologies for synthetic biology and metabolic engineering in various bacterial species.

Keywords: Base editing; Cas12a (Cpf1); Cas13; Cas9; DNA/RNA detection.

Fig. 1

Numbers of NCBI PubMed publications containing Cas9 and Cas12a (Cpf1). The 2018 data are collected at the end of August.

Fig. 2

The biological mechanisms of type II CRISPR-Cas9 (a) and type V CRISPR-Cas12a (b). The immune defense presented on the upper contains three steps: spacer acquisition, crRNA biogenesis, and target interference. The Cas domain organizations are presented at the bottom. spCas9 has two nuclease domains HNH and RuvC which cleaves complementary and non-complementary DNA strands respectively, while fnCas12a uses the single nuclease domain RuvC for both DNA cleavage. Orange triangles, the cleavage site; PAM, protospacer adjacent motif; NUC, nuclease lobe; REC, recognition lobe; PI, PAM interaction domain; WED, wedge domain; BH, bridge helix; tracrRNA, trans-activating CRISPR RNA.

Fig. 3

CRISPR genome editing (a) and CRISPR gene regulation including CRISPR interference (b, CRISPRi) and CRISPR activation (c, CRISPRa). The genome editing starts from the introduction of DSBs (double-stranded breaks) followed by NHEJ and HDR DNA repair. CRISPRi uses dCas (dCas9 or dCas12a) to sterically block RNA polymerase (RNAP) to repress gene expression. CRISPRa is achieved by fusing the ω-subunit of the RNAP or the bacterial RNAP activator SoxS to dCas9, and activates transcription by recruitment of the RNAP assembly.

Fig. 4

The emerging CRISPR biotechnologies for in vivo manipulations. a) DNA base editing to switch C to T. the system contains a nuclease-defective Cas (dCas9/nCas9/dCas12a), a fused cytidine deaminase, and a fused uracil glycosylase inhibitor (UGI). b) DNA base editing to switch A to G. A nuclease-defective Cas (dCas9/nCas9) is fused to an evolved tRNA adenosine deaminase that converts A to G via I. c) RNA base editing to switch A to I. A catalytically-inactive dCas13 tethered to an adenosine deaminase, acts on RNA to convert A to I. d) RNA-guided RNase Cas13 mediates RNA cleavages. Asterisk, nucleotide change; triangle, cleavage.