Applied Microbiology: Open Access
Open Access

A Short Note on CRISPR Gene Design

Andrezza Nascimento^{* *}Correspondence: Andrezza Nascimento, Department of Medical Investigation, University of São Paulo, São Paulo, Brazil, Email:

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About the Study

Controlling biological populations, such as pests, parasites, viruses, and invasive species, remains a crucial priority for addressing the issues those ecosystems and agriculture face, as well as those posed by human disease. A gene drive, one of the architectures of CRISPR/Cas biotechnology, offers the capacity to modify or eradicate populations on a large scale. Super- Mendelian inheritance has now been found in fungi and metazoans, including disease vectors like mosquitos. Titration of nuclease activity, anti-CRISPR-dependent inhibition, and the use of non-native DNA target sites are among the molecular safeguards discovered in yeast and fly model systems to boost biosafety and control over drive systems in vivo [1-3].

In Saccharomyces cerevisiae, we designed a CRISPR/Cas9 gene drive that enables for the safe and quick testing of several drive designs and control mechanisms. Investigations have done whether Non-Homologous End-Joining (NHEJ) had happened within diploid cells that had lost the target allele after drive activation, but found no evidence of NHEJ in any of the analysed populations and were also able to successfully multiplex two more non-native target sequences [3]. Focusing at 'resistant' clones that still had both the drive and target selection markers after Streptococcus pyogenes Cas9 expression; de novo mutation or NHEJ-based repair could not account for the bulk of these heterozygous clones.

Finally, a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus was produced, and resistant clones in this system could be reactivated during a second round of Cas9 induction [4].

CRISPR/Cas genome editing has enabled recent breakthroughs in a variety of disciplines, including agriculture, biotechnology, and basic laboratory research. The introduction of specific chromosomal breaks inside a genome of interest, together with DNA repair, allows practically any genetic change to be generated. A Gene Drive (GD) system is a strong arrangement that uses CRISPR/Cas to swiftly 'push' a genetic element of choice through a native population. This biotechnology might theoretically be used to impart a desirable trait to a population or to eradicate local populations (through extreme bias of sex determination is taken as an example) [5,6].

Controlling specific biological populations is a major difficulty for many enterprises, and it is exacerbated by global health crises such as animal and plant pests and parasites, pathogen spread (through insect vectors), and invasive organisms' change of natural habitats. Given the potential for CRISPR GDs to be widely used in the future, more laboratory research into biosafety, control, and reversal mechanisms is essential. Current CRISPR-based GDs have been produced and tested in fungi, insects, and even vertebrates in the lab, with various degrees of effectiveness. Previously, we designed a highly tractable drive system in budding yeast that can be used to evaluate novel drive arrangements, CRISPR components, DNA repair, and inhibition or control modes.

The integration of the basic CRISPR system (nuclease and related guide RNA expression cassettes) at a specific genomic locus is the basic mechanism of a GD. The induction of a Double-Strand Break (DSB) in the target allele inside the same locus where the GD was positioned on the homologous chromosome is possible when Cas9/sgRNA is expressed within a diploid genome. The DSB will be repaired via Homology- Directed Repair (HDR), with donor DNA coming from the drive-containing chromosome; the GD cassette will then be replicated to replace the entire target locus. All homozygous diploid progeny for the GD are produced by heterozygous pairing of a drive-containing individual with a Wild-Type (WT) individual (heterozygote); this allows for super-Mendelian inheritance of the drive locus through a population [6].

Some of our findings in Saccharomyces cerevisiae have been tested in higher eukaryotes, such as the targeting of non-native DNA sequences within target loci (such as eGFP or other programmed artificial sequences), the titration of Cas9 activity within a GD, and split drives that separate guide RNAs from nucleases across multiple loci.

The occurrence of drive resistance; methods to slow, interrupt, or reverse active drives; and how population management may affect larger ecosystems during possible field deployment of drive systems are all key continuing concerns for GD research.

We use budding yeast to test (i) the possibility of DNA repair by the non-homologous end-joining (NHEJ) system; (ii) additional controls for our examination of the drive and target loci following drive action; (iii) multiplexing to independent target sequences; (iv) examination of'resistant' clone formation; and (v) a modified second-generation drive harbouring an integrated guide RNA cassette.

References

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