The COVID-19 pandemic has brought great challenges to traditional nucleic acid detection technology. Thus, it is urgent to develop a more simple and efficient nucleic acid detection technology. CRISPR-Cas12 has signal amplification ability, high sensitivity and high nucleic acid recognition specificity, so it is considered as a nucleic acid detection tool with broad development prospects and high application value. This review paper discusses recent advances in CRISPR-Cas12-based nucleic acid detection, with an emphasis on the new research methods and means to improve the nucleic acid detection capability of CRISPR-Cas12. Strategies for improving sensitivity, optimization of integrated detection, development of simplified detection mode and improvement of quantitative detection capabilities are included. Finally, the future development of CRISPR-Cas12-based nucleic acids detection is prospected.
Myxococcus xanthus DK1622 has two RecA genes, recA1 (MXAN_1441) and recA2 (MXAN_1388), with unknown functional differentiation. Herein, we showed that both recA genes were induced by ultraviolet (UV) irradiation but that the induction of recA1 was more delayed than that of recA2. Deletion of recA1 did not affect the growth but significantly decreased the UV-radiation survival, homologous recombination (HR) ability, and induction of LexA-dependent SOS genes. In contrast, the deletion of recA2 markedly prolonged the lag phase of bacterial growth and increased the sensitivity to DNA damage caused by hydrogen peroxide but did not change the UVradiation resistance or SOS gene inducibility. Protein activity analysis demonstrated that RecA1, but not RecA2, catalyzed DNA strand exchange (DSE) and LexA autocleavage in vitro. Transcriptomic analysis indicated that RecA2 has evolved mainly to regulate gene expression for cellular transportation and antioxidation. This is the first report of functional divergence of duplicated bacterial recA genes. The results highlight the evolutionary strategy of M. xanthus cells for DNA HR and genome sophistication.
2 RecA is a ubiquitous multifunctional protein for bacterial homologous recombination 3 and SOS response activation. Myxococcus xanthus DK1622 possesses two recA genes, 4 and their functions and mechanisms are almost unclear. Here, we showed that the 5 transcription of recA1 (MXAN_1441) was less than one-tenth of recA2 (MXAN_1388).6 Expressions of the two recA genes were both induced by ultraviolet (UV) irradiation, 7 but in different periods. Deletion of recA1 did not affect the growth, but significantly 8 decreased the UV-irradiation survival, the homologous recombination ability, and the 9 induction of the LexA-dependent SOS genes. Comparably, the deletion of recA2 10 markedly prolonged the lag phase for cellular growth and antioxidation of hydrogen 11 peroxide, but did not change the UV-irradiation resistance and the SOS-gene 12 inducibility. The two RecA proteins are both DNA-dependent ATPase enzymes. We 13 demonstrated that RecA1, but not RecA2, had in vitro DNA recombination capacity 14 and LexA-autolysis promotion activity. Transcriptomic analysis indicated that the 15 duplicate RecA2 has evolved to mainly regulate the gene expressions for cellular 16 transportation and antioxidation. We discuss the potential mechanisms for the 17 functional divergence. This is the first time to clearly determine the divergent functions 18 of duplicated recA genes in bacterial cells. The present results highlight that the 19 functional divergence of RecA duplicates facilitates the exertion of multiple RecA 20 functions. 21 22 Author summary 23 Myxobacteria has a large-size genome, contains many DNA repeats that are rare in 24 the prokaryotic genome. It encodes bacterial RecA that could promote recombination 25 between homologous DNA sequences. How myxobacteria avoid the undesired 26 recombination between DNA repeats in its genome is an interesting question. M. 27 xanthus encodes two RecA proteins, RecA1 (MXAN_1441) and RecA2 28 (MXAN_1388). Both RecA1 and RecA2 shows more than 60% sequence consistency 29 with E. coli RecA (EcRecA) and can partly restore the UV resistance of E. coli recA 30 mutant. Here, our results proved their divergent functions of the two RecAs. RecA1 31 retains the ability to catalyze DNA recombination, but its basal expression level is 32 very low. RecA2 basal expression level is high, but no recombination activity is 33 detected in vitro. This may be a strategy for M. xanthus to adapt to more repetitive 34 sequences in its genome and avoid incorrect recombination. 35 36 Highlights: 37 1. M. xanthus has two recAs, which are expressed with significantly different levels. Both 38 recAs are inducible by UV irradiation, but in different stages. 39 2. The absence of recA1 reduces bacterial UV-irradiation resistance, while the absence of 40 recA2 delays bacterial growth and antioxidant capacity. 41 3. RecA1 retains the DNA recombination and SOS induction abilities, while RecA2 has 42 evolved to regulate the expression of genes for cellular transport and antioxidation. 43 44 RecA, an ATPase recombinase, is ...
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