Determination of Cas9/dCas9 associated toxicity in microbes

Abstract

23The CRISPR-Cas9 system has been used extensively in eukaryotic and prokaryotic systems for 24 various applications. In case of the latter, a couple of previous studies had shown Cas9 protein 25 expression associated toxicity. We studied the same in five microbes, viz Escherichia coli, 26 Salmonella typhimurium, Mycobacterium smegmatis, Xanthomonas campestris and 27 Deinococcus radiodurans. Transformation efficiency of plasmids carrying genes coding for 28 Cas9 or dCas9 was used to gauge toxicity associated with Cas9 protein expression. Results 29 showed differential levels of Cas9 toxicity among the bacteria and lower transformation 30 efficiency for cas9/dcas9 bearing plasmids compared to controls in general. This indicated 31 lethal effect of Cas9/dCas9 expression. While E. coli and S. typhimurium seemed to tolerate 32 Cas9/dCas9 fairly well, in GC rich microbes, M. smegmatis, X. campestris and D. radiodurans, 33 Cas9/dCas9 associated toxicity was acute. 34 35 36 37 38 39 40 41 42 43 44 3 | P a g e Introduction: 45 Recombinant DNA technology together with High Throughput Sequencing in recent 46times, has allowed us to harvest a large amount of genetic information from the microbial 47 world. The technologies have been used extensively to find out which genes determine how 48 microbes, grow, travel, starve, cause diseases, ward of predators and even die. This information 49 is especially important for studying pathogenic bacteria, bacteria of industrial importance and 50 ones with special stress tolerance abilities. Perturbing the normal functioning of the genome 51 has emerged as the best method to probe function and dynamics of individual genes. 52 Discovery of the CRISPR -Cas viral defence systems opened up another novel and 53 efficient tool box for genome editing, gene silencing, targeted gene methylation, etc. in all 54 kinds of organisms from bacteria to humans [1][2][3][4]. The ease and efficiency of the system 55 has made it an extremely popular go-to system for various applications. The system has further 56 had widespread applications in metabolic engineering of bacteria as it allows easy 57 programming and multiplexing [5][6]. Among the CRISPR-Cas systems, the Cas9 system from 58 Streptomyces pyogens has gained popularity on account of being one of the earliest systems to 59 be discovered and its simplicity of usage [7][8][9]. The system comprises a single protein, Cas9 60 and the sgRNA , which together can be easily employed to bring about a host of desired changes 61 inside the cell of virtually any living being [10]. While the nucleoprotein, Cas9 itself has been 62 extensively used for genome editing [7][8][11][9], its nuclease deficient variant, dCas9 has 63 been useful for regulation of gene expression [3][1]. Systems employing the Cas9 variants have 64 shown great promise for use in eukaryotic, particularly mammalian systems [11]. Attempts to 65 use them in microbes have met with mixed success. 66 The Cas9/dCas9 and also Cas9 nickase systems were used successfully to probe gene 67 function...

Targeted-Antibacterial-Plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity

“…By contrast, the non-specific TAP-dCas9-nsp add no effect on curli formation or aggregation in the OmpR234 strain. Besides, we confirmed that the constitutive production of the Cas9 or dCas9 from the TAPs do not cause growth defects (Supplementary Fig.1d) or elongated cell morphology (Supplementary Fig.1e), contrasting with the toxic effects reported in some systems 15–18 . These results demonstrate that TAPs ability to induce Cas9-mediated killing or dCas9-mediated gene expression inhibition is efficient and depends on the accurate targeting by the spacer sequence.…”

Genome Editing in Bacteria: CRISPR-Cas and Beyond

“…In any case, they may have common drawbacks related to the continuous expression of a foreign Cas9 protein. SpCas9 overexpression can be highly cytotoxic in E. coli and many other bacteria (it will be explained within the next section) leading to little or no colonies, even when devoid of its nuclease activity [ 50 , 51 ].…”

Exploring nano-enabled CRISPR-Cas-powered strategies for efficient diagnostics and treatment of infectious diseases

“…Early findings have shown the potential for non-specific nuclease activity toxicity of the Cas9 protein. However, the idea must be amended in comparable toxicities with dCas9 [257]. On the other hand, a low dose of NCs including metals, oxides of metals, polymers, and composites is usually non-toxic, prolonged delivery of such agents can lead to long-term aggregation and potential toxicity.…”