Reprogramming Microbial CO2-Metabolizing Chassis With CRISPR-Cas Systems

Abstract

Global warming is approaching an alarming level due to the anthropogenic emission of carbon dioxide (CO₂). To overcome the challenge, the reliance on fossil fuels needs to be alleviated, and a significant amount of CO₂ needs to be sequestrated from the atmosphere. In this endeavor, carbon-neutral and carbon-negative biotechnologies are promising ways. Especially, carbon-negative bioprocesses, based on the microbial CO₂-metabolizing chassis, possess unique advantages in fixing CO₂ directly for the production of fuels and value-added chemicals. In order to fully uncover the potential of CO₂-metabolizing chassis, synthetic biology tools, such as CRISPR-Cas systems, have been developed and applied to engineer these microorganisms, revolutionizing carbon-negative biotechnology. Herein, we review the recent advances in the adaption of CRISPR-Cas systems, including CRISPR-Cas based genome editing and CRISPR interference/activation, in cyanobacteria, acetogens, and methanogens. We also envision future innovations via the implementation of rising CRISPR-Cas systems, such as base editing, prime editing, and transposon-mediated genome editing.

Keywords: CRISPR; acetogen; carbon dioxide; cyanobacteria; genome editing; methanogen.

FIGURE 1

(A) CRISPR-Cas-based genome editing. Under the guidance of a gRNA containing a spacer complementary to the protospacer and a scaffold consisting of crRNA and tracer RNA, the Cas9 and gRNA complex finds and binds to the protospacer with a PAM, forming an R-loop. Then, Cas9 cuts the DNA in each strand, leaving a double-strand break (DSB) in the target. Finally, the target DNA will be modified with the repair of DSB. (B) Nuclease deactivated Cas9 protein (dCas9). When the nuclease activity of Cas protein was deactivated, the dCas9 still binds to the target sequence but it will not cleave the DNA anymore. (C) CRISPRi and CRISPRa. For CRISPRi, dCas9 binds to the promoter or coding region of a gene of interest and prevents the binding of RNAP, resulting in the repression of transcription. For CRISPRa, the dCas9-transcription factor (TF) fusion binds to the up region of the promoter. The TF helps recruit RNAP and allows the activation of transcription. We employed Cas9 from S. pyogenes as a representative in the figure, while a great variety of CRISPR systems can be used for genome editing, CRISPRi, and CRISPRa. TF stands for transcription factor, RNAP stands for RNA polymerase, CDS stands for coding sequence, and PAM stands for protospacer adjacent motif.

FIGURE 2

(A) Base editing. The cytosine base editor with a combination of dCas9 cytosine deaminase is shown as an example to illustrate the working mechanism. When dCas9 binds to its target sequence and an R-loop is formed, the cytosine deaminase (CDA) mutates a cytosine (C) to uracil (U), generating a uracil-guanine (U–G) mismatch. The C will then be replaced by thymine (T) along with the reparation of the mismatch upon DNA repair or replication, resulting in a C-to-T substitution at a one-nucleotide resolution. For adenine base editors, an A-to-G substitution will occur as a result of base editing. Notably, nCas9 can also be used as the effector for base editors. (B) Prime editing. The prime editing takes advantage of nCas9 and a fused reverse-transcription (RT). By designing a pegRNA consisting of the spacer for targeting, RT template with designed edits, and primer binding site (PBS), deletions, insertions and point mutations can be achieved. (C) Transposon-mediated CRISPR-Cas system for DNA integration. As a paradigm, dCas9 was fused to a transposase (Tns), and when the transposase was led to the target sequence, it recognizes the motif for transposition and integrates the donor DNA into the upstream of the protospacer, enabling large fragment insertions. Cas9 was used as the Cas effector for illustration of the basic working principles of base editing, prime editing and transposon-mediated editing.