In nature, the CRISPR-Cas system is a prokaryotic (bacteria and archaea) adaptive immune mechanism used to cut invading nucleic acids. The CRISPR-Cas9 system has the advantages of simplicity, convenience, and quickness, ultimately leading to its widespread and rapid use while promoting the change of research technology. In the past decade, CRISPR-Cas9 has changed genomic engineering by relying on base pairing of nucleic acids rather than traditional protein-DNA recognition.

Common gene editing tools include CRISPR-Cas9, CRISPR-Cas12a, Cascade, and Cas3. The CRISPR-Cas9 system is the most widely used genome editing tool. Streptococcus pyogenes Cas9 (SpCas9) is the first one used outside prokaryotic cells and reprogrammed for mammalian cell genome editing. It is the most commonly used Cas9. After DNA target recognition, SpCas9 usually produces a blunt double strand break (DSB). Cas12a generates a staggered cut with a 5’ overhang at DNA target sites without a tracrRNA. Before recruiting Cas3 targeted DNA sequences, Cascade first binds to DNA through PAM and spacer recognition. Due to its promiscuous recognition of PAM sequences, Cascade provides greater target site flexibility.

Fig 1 Overview of the main CRISPR–Cas gene editing tools.

Gene Regulation by CRISPR-Cas
In addition to gene editing by forming DNA breaks, the catalytic activity of Cas9 can be destroyed by mutating RuvC (D10A) and HNH (H840A) sites of Cas9, while maintaining its RNA-guided DNA targeting ability to form dCas9. dCas9 is combined with a variety of effectors, such as transcription inhibitors or activators, epigenetic modifiers, and fluorophores, so as to expand the application range of CRISPR-Cas system.

Fig 2 Targeted gene regulation and other applications

Future
The application of the CRISPR-Cas system to eukaryotic cells has completely changed the field of genome engineering. The progress of CRISPR gRNA libraries and next-generation sequencing makes genome-wide genetic and epigenetic screening easy, which is helpful to find new therapeutic targets.

As CRISPR-Cas-based therapies enter clinical trials, although there are some application limitations, such as small adeno-associated virus vector, off-target effect, immunogenicity of Cas9 protein, etc., the use of CRISPR-Cas shows great potential in correcting genetic diseases and enhancing cell therapy.

Synbio Technologies’s CRISPR-Cas9 Gene Editing Services
Synbio Technologies provides one-stop CRISPR-Cas9 gene editing services, including sgRNA design, chip synthesis, sgRNA library construction, NGS verification, virus packaging, and bioinformatics analysis. At present, Synbio Technologies has delivered hundreds of high-quality CRISPR-Cas9 sgRNA libraries covering more than 20 species including animals, plants, and microorganisms. Our offered libraries include various genomic knockout libraries, interference libraries, and activation libraries, which can meet the customized needs of many different customers. We always strive to provide our customers around the globe with accurate, fast, and convenient gene editing services.

Reference[1] Adrian Pickar-Oliver and Charles A.Gersbach. The next generation of CRISPR Cas technologies and applications. NatRevMolCellBiol. 2019Aug; 20(8):490–507. doi:10.1038/s41580-019-0131-5