CADS: CRISPR/Cas12a-Assisted DNA Steganography for Securing the Storage and Transfer of DNA-Encoded Information

Because DNA has the merit of high capacity and complexity, DNA steganography, which conceals DNA-encoded messages, is very promising in information storage. The classical DNA steganography method hides DNA with a “secret message” in a mount of junk DNA, and the message can be extracted by polymerase chain reaction (PCR) using specific primers (key), followed by DNA sequencing and sequence decoding. As leakage of the primer information may result in message insecurity, new methods are needed to better secure the DNA information. Here, we develop a pre-key by either mixing specific primers (real key) with nonspecific primers (fake key) or linking a real key with 3′-end redundant sequences. Then, the single-stranded DNA (ssDNA) <i>trans</i> cleavage activity of CRISPR/Cas12a is employed to cut a fake key or remove the 3′-end redundant sequences, generating a real key for further information extraction. Therefore, with the Cas12a-assisted DNA steganography method, both storage and transfer of DNA-encoding data can be better protected

iBrick: A New Standard for Iterative Assembly of Biological Parts with Homing Endonucleases

<div><p>The BioBricks standard has made the construction of DNA modules easier, quicker and cheaper. So far, over 100 BioBricks assembly schemes have been developed and many of them, including the original standard of BBF RFC 10, are now widely used. However, because the restriction endonucleases employed by these standards usually recognize short DNA sequences that are widely spread among natural DNA sequences, and these recognition sites must be removed before the parts construction, there is much inconvenience in dealing with large-size DNA parts (<i>e.g</i>., more than couple kilobases in length) with the present standards. Here, we introduce a new standard, namely iBrick, which uses two homing endonucleases of I-SceI and PI-PspI. Because both enzymes recognize long DNA sequences (>18 bps), their sites are extremely rare in natural DNA sources, thus providing additional convenience, especially in handling large pieces of DNA fragments. Using the iBrick standard, the carotenoid biosynthetic cluster (>4 kb) was successfully assembled and the actinorhodin biosynthetic cluster (>20 kb) was easily cloned and heterologously expressed. In addition, a corresponding nomenclature system has been established for the iBrick standard.</p></div

Ligation efficiency of iBrick assembly.

<p>* Clones on plates were evenly divided into 4 even sections with only one counted and the total number was then roughly calculated as four times of the number.</p><p>Ligation efficiency of iBrick assembly.</p

iBrick elements used in this study.

<p>iBrick elements used in this study.</p

Letter allocations for parts and elements in iBrick standard.

<p>Letter allocations for parts and elements in iBrick standard.</p

Heterologous expression of the <i>act</i> cluster in <i>Streptomyces</i> 4F.

<p>(A) Constructed expression plasmid pIB2Am1_0-X000005. (B&C) Verification of pIB2Am1_0-X000005 with both the experimental digestion (B) and the electronic restriction (C) with XcmI. M, 1 kb DNA ladder with sizes labeled; -, pIB2Am1_0-X000004; +, pIB2Am1_0-X000005. For “−”, theoretical sizes are 8.32 and 1.88 kb; for “+”, sizes are 9.93, 5.28, 4.18, 3.38, 2.67, 2.08, 1.88, 1.08 and 0.78 kb. Same patterns were obtained for the experimental group and the electronic analysis group. (D) Heterologous expression of <i>act</i> cluster in <i>Streptomyces</i> 4F, employing pIB2Am1_0-X000004 as a negative control. Strains were cultured on R2YE plate at 30°C for 2 days.</p

Schematic diagram of iBrick forward assembly.

<p>(A) Construction of iBrick parts, which should be prefixed and suffixed in iBrick standard. (B) Forward assembly procedure. Part B is firstly released with I-SceI and PI-PspI digestion, and is then inserted into the PI-PspI digested vector, obtaining an assembled part AB. Alternatively, the vector can also be cut with I-SceI followed by insertion of part B to obtain part BA, which is designated the reverse assembly.</p

Letter abbreviations for antibiotic resistance markers in iBrick standard^*.

<p>* Most of the letter abbreviations are the same as those described by Shetty <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110852#pone.0110852-Shetty1" target="_blank">[8]</a>.</p><p>Letter abbreviations for antibiotic resistance markers in iBrick standard^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110852#nt102" target="_blank">*</a>}.</p

DNA sequences of the prefix, suffix and scar in iBrick assembly.

<p>Parts A and B are prefixed with I-SceI (shown in blue) and suffixed with PI-PspI (shown in red). After cleavage, compatible cohesive end (“TTAT”) can be ligated together to produce a 21-bp scar between parts. When translated in frame, this 21-bp DNA sequence encodes 7 amino acids.</p

iBrick parts used in this study.

<p>iBrick parts used in this study.</p