Single-Nucleotide Microbial Genome Editing Using CRISPR-Cas12a.

Microbial genome editing can be achieved by donor DNA-directed mutagenesis and CRISPR-Cas12a-mediated negative selection. Single-nucleotide-level genome editing enables the manipulation of microbial cells exactly as designed. Here, we describe single-nucleotide substitutions/indels in the target DNA of E. coli genome using a mutagenic DNA oligonucleotide donor and truncated crRNA/Cas12a system. The maximal truncation of nucleotides at the 3'-end of the crRNA enables Cas12a-mediated single-nucleotide-level precise editing at galK targets in the genome of E. coli.

Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs.

Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for time and labor savings. However, achieving high accuracy remains a challenge. In an Escherichia coli model system, we achieved highly efficient single-nucleotide level simultaneous editing of the galK and xylB genes using the 5'-end-truncated single-molecular guide RNA (sgRNA) method. Furthermore, we successfully demonstrated the simultaneous editing of three genes (galK, xylB, and srlD) at single-nucleotide resolution. To showcase practical application, we targeted the cI857 and ilvG genes in the genome of E. coli. While untruncated sgRNAs failed to produce any edited cells, the use of truncated sgRNAs allowed us to achieve simultaneous and accurate editing of these two genes with an efficiency of 30%. This enabled the edited cells to retain their lysogenic state at 42 °C and effectively alleviated l-valine toxicity. These results suggest that our truncated sgRNA method holds significant potential for widespread and practical use in synthetic biology.

Recent Advances in CRISPR-Cas Technologies for Synthetic Biology.

With developments in synthetic biology, "engineering biology" has emerged through standardization and platformization based on hierarchical, orthogonal, and modularized biological systems. Genome engineering is necessary to manufacture and design synthetic cells with desired functions by using bioparts obtained from sequence databases. Among various tools, the CRISPR-Cas system is modularly composed of guide RNA and Cas nuclease; therefore, it is convenient for editing the genome freely. Recently, various strategies have been developed to accurately edit the genome at a single nucleotide level. Furthermore, CRISPR-Cas technology has been extended to molecular diagnostics for nucleic acids and detection of pathogens, including disease-causing viruses. Moreover, CRISPR technology, which can precisely control the expression of specific genes in cells, is evolving to find the target of metabolic biotechnology. In this review, we summarize the status of various CRISPR technologies that can be applied to synthetic biology and discuss the development of synthetic biology combined with CRISPR technology in microbiology.

Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3'-Truncated sgRNA.

The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia coli cells obtained by oligonucleotide-directed genome editing. Although double-, triple-, and quadruple-base substitutions were accurately and efficiently performed in the genome, the performance of single-base editing was poor. To resolve this limitation, we serially truncated the 3'-end of sgRNAs and determined the maximal truncation required to maintain the target DNA cleavage activity of Cas12f1. Negative selection of single-nucleotide-edited cells was efficiently performed with the maximally 3'-truncated sgRNA-Cas12f1 complex in vivo. Moreover, Sanger sequencing showed that the accuracy of single-nucleotide substitution, insertion, and deletion in the microbial genome was improved. These results demonstrated that a truncated sgRNA approach could be widely used for accurate CRISPR-mediated genome editing.

Control of λ Lysogenic Escherichia coli Cells by Synthetic λ Phage Carrying cIantisense.

Enterobacterial phage λ is a temperate phage that infects Escherichia coli and has a lytic-lysogenic life cycle. CI, a λ repressor, regulates the expression of lytic transcripts and acts as a major genetic switch that determines the lysogenic state. To manipulate the genome of phage λ, the CRISPR-Cas9 genome editing system was constructed in lysogenic E. coli MG1655 cells. For instance, we successfully changed cI857 to cIWT in the phage genome through Cas9-mediated single-nucleotide editing. A lytic phage was prepared by introducing an amber mutation in the middle of the cI gene, but it could not lyse lysogenic MG1655 cells. We prepared a phage expressing cI antisense mRNA by reverse substitution of the cI gene. Lysis of λ cI857 lysogenic cells occurred by the infection of the λ cIantisense. These results suggest an effective lysogenic cell control method by a synthetic phage expressing antisense mRNA of the genetic switch gene. It is expected to be applied as a tool to control harmful lysogenic microorganisms.

Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated crRNAs.

Mismatch tolerance, a cause of the off-target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3'-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 inEscherichia coli. Five nucleotides could be maximally truncated at the crRNA 3'-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3'-end-truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3'-end-truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB. Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution.

Advances in Accurate Microbial Genome-Editing CRISPR Technologies.

Previous studies have modified microbial genomes by introducing gene cassettes containing selectable markers and homologous DNA fragments. However, this requires several steps including homologous recombination and excision of unnecessary DNA regions, such as selectable markers from the modified genome. Further, genomic manipulation often leaves scars and traces that interfere with downstream iterative genome engineering. A decade ago, the CRISPR/Cas system (also known as the bacterial adaptive immune system) revolutionized genome editing technology. Among the various CRISPR nucleases of numerous bacteria and archaea, the Cas9 and Cas12a (Cpf1) systems have been largely adopted for genome editing in all living organisms due to their simplicity, as they consist of a single polypeptide nuclease with a target-recognizing RNA. However, accurate and fine-tuned genome editing remains challenging due to mismatch tolerance and protospacer adjacent motif (PAM)-dependent target recognition. Therefore, this review describes how to overcome the aforementioned hurdles, which especially affect genome editing in higher organisms. Additionally, the biological significance of CRISPR-mediated microbial genome editing is discussed, and future research and development directions are also proposed.

Mismatch Intolerance of 5'-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing.

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5'-truncated sgRNAs inhibited target recognition. These results suggest that a 5'-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5'-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5' end of truncated sgRNAs. Further, 5'-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5'-NG), which may enable whole-genome single-base editing.

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs.

Genome editing has been revolutionized by the CRISPR-Cas9 system. CRISPR-Cas9 is composed of single-molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a protospacer adjacent motif (PAM) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR-Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR-Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced two- to four-base mutations in galK in Escherichia coli with high editing efficiencies (81%-86%). However, single-point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed one- or two-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli A single-point nucleotide mutation (T504A or C578A in the galK gene) was successfully introduced in 36%-95% of negatively selected E. coli cells using single-base mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single-base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes.

Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture.

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.

Non-radioactive and colorimetric quantification of monocyte adhesion to endothelial cells in early atherogenesis.

Monocyte adhesion to vascular endothelium is an initial step in atherogenesis. To quantify this, we incubated monocytes with cultured endothelial cells, and quantified the adhered live monocytes using a colorimetric assay. Endothelium activated with lipopolysaccharide attracted monocytes in a dose-dependent manner and the adhesion was attenuated with post-treatments with L-ascorbic acid (53%), alpha- (40%) and gamma-tocopherol (39%), resveratrol (39%), and Lithospermum erythrorhizon root extract (45%). This non-radioactive, colorimetric assay may be useful for screening anti-atherogenic compounds in early atherogenesis.

Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans.

Methionine (Met) residues in proteins are susceptible to oxidation. The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA). The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli. The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT). However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH. The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer. K(m) and k(cat) values for the enzyme were approximately 1.18 mM and 3.64 min(-1), respectively. Other kinetics properties of the enzyme were also evaluated.