Synbio Technologies has recently made a break through in synthetic biology through use of sgRNA libraries.
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sgRNA Libraries Authored by Sybio Technologies
Synthetic biology has one main goal: retooling and repurposing biological systems for new and novel applications. One of the premier examples of this revamping of biological systems is the CRISPR-Cas9 defense mechanism. Originally found in bacterial genomes, CRISPR was used as a defense mechanism against viral DNA. What happened in this system was when a virus inserted its DNA into the cells of the bacteria, the CRISPR system would activate and causes a double strand break within the viral sequence, rendering it inactive. A section of the viral DNA would then be stored within the genome of the bacterial. This was to make sure that if the virus every did come back and try to harm the bacteria again, the CRISPR system would be recognized the sequence, and clip the viral DNA. Multiple scientists, in multiple different locations, discovered this defense mechanism and saw the opportunity for it to be repurposed. The idea was to use CRISPR as a new mechanism of achieving genome modifications. Scientists figured out that sequences could be design and loaded into the CRISPR-Cas9 system. The loaded sequences, otherwise known as sgRNA sequences, were designed to correspond to a sequence of interest of a particular gene. When the sgRNA sequenced match up with the foreign DNA the CRISPR-Cas9 defense mechanism would be activated, and the foreign DNA would be cut. This prompted a need for large scale design and construction of the sgRNA sequences.
One of the most important factors that comes into play is the sgRNA that corresponds to the foreign sequence the researchers want to study. Since multiple genes are normally implicated in any given research topic, many sgRNA sequences may be needed. A common method of generating many sgRNA sequences is through an sgRNA library. An sgRNA library harbors many sgRNA sequences for various model organisms. Although a large number, each sequence is still designed to accurately corresponding to the genetic sequence of interest. When using an sgRNA library, a specific sgRNA sequence can easily be obtained and utilized. The sgRNA then matches up with the corresponding gene of interest. Once matched up, the Cas9 nuclease kicks into action and cuts the genetic sequence. The now cut DNA can be left cut, rending it inactive, or an additional sequence can be inserted into the break and ligated. This allows for the genetic modification sought by scientists all over the world. In order to achieve these genome modifications, a specific sgRNAs must be designed and constructed.
sgRNA sequences act as a guide to recognize and combine with the target DNA sequence. The sgRNA lengths vary, depending on the overall genetic sequence length. To date, an array of sgRNA sequences have been designed and constructed, varying in length, GC content, and overall structure. This includes many different model organisms ranging from e. coli to mice, with every organism in between. The sgRNA sequence will be slightly different for each of these organisms, but one common factor remains: the sgRNA sequence must be precisely and accurate designed and constructed. Without the correct sgRNA sequence the Cas9 nuclease will not be able to recognize the gene’s sequence, resulting in no double stranded break. There are, however, instances in which a large variety of variant sgRNA sequences are required to conduct the research of interest. For these cases, it is best to generate an sgRNA library to have a large number of sequences.
Generating an sgRNA library is a synthetic biology application that can be easily achieved by a skilled and experienced biotechnology company, such as Synbio Technologies. The sgRNA libraries contain a large number of different sgRNA sequences to be used later for various aspects of research across many topics. sgRNA libraries start with the construction of the oligo sequence, and culminate in the packaging of the sgRNA sequence into a lentivirus to be stored and called upon at a later date. High throughput screen must be conducted to assure the overall quality of the sgRNA library remains intact. Without this screening, the sgRNA library may be rendered useless. For this reason, biotech companies like Synbio Technologies are sure to screen their sgRNA libraries before shipping to their customers.
sgRNA Library Construction Steps:
1. Oligo Synthesis
a. Oligo synthesis allows for the manufacturing of the corresponding sgRNA sequence of interest.
b. Synbio Technologies relies upon its Syno® Synthesis Platform to synthesize oligo sequences with 100% accuracy.
i. This accuracy guarantee is value for all sequences no matter the length, GC content, or structure.
a. The newly synthesized oligo sequences are then run through PCR to generate a sufficient amount of product.
i. In order to generate a sufficient sgRNA library, a large amount of PCR copies must be present.
3. Ligation in gRNA Library
a. The sgRNA sequences are then ligated into lentivirus vectors.
b. At this point, the plasmid sgRNA library is constructed.
c. The following steps are taken to ensure the overall quality of the plasmid sgRNA library and to construct the pooled sgRNA library.
4. Lentivirus Library
a. This allows for the recombinant viruses to be selected in the next phase of the quality control.
b. Once packaged into the lentivirus, the sgnRNA library is now considered to be a packed pooled sgRNA library.
sgRNA Library Applications:
The designed and constructed sgRNA libraries have been utilized by labs all over the world for years, and they don’t show any signs of leaving. sgRNA libraries allow researchers to knockout multiple genes within a model organism with relative ease and accuracy. This is done by utilizing the large pool of different sgRNA sequences within the library itself. The knockout of multiple genes at once is the basis for many different aspects of biological research. sgRNA libraries, in turn, play a large role in the drug discovery, phenotypic changes, and report assays. All of which are a major part of many biological research pipelines. It is these sgRNA libraries that are utilized to generate the genetic modifications critical to these research topics.
CRISPR-Cas9 has revolutionized the fields of genetics and biology in general. The bacterial defense mechanism is a perfect example of how synthetic biology repurposes biological systems for new and novel applications. To make expedite the overall process and generate multiple genetic knockouts at once, an sgRNA library can be constructed. This sgRNA library contains a large number of various sgRNA sequences corresponding to many different genes in an organism’s genome. These sgRNA sequences can be used to knockout each and every one of the corresponding genes. For this reason, generating an sgRNA library is a critical aspect of many research pipelines. The vast number of sequences that the sgRNA library can harbor offers many advantages to traditional methods of generating genetic knockouts.