CRISPR Gene Editing

The CRISPR-Cas system is a novel targeted gene-editing technique from bacterial immune system. CRISPR, relies on the ability of CRISPR single guide RNAs (sgRNAs) to target Cas9 endonuclease to precise genomic locations, where Cas9 introduces double-strand breaks (DSBs). CRISPR-Cas9 system is one of the easiest and most rapidly adapted genome editing tool, which is transforming the molecular biology research nowadays. CRISPR-Cas systems have been widely used in genome editing, transcriptional and epigenetic regulation and so on. The previous gene editing tools MNs (meganucleases), ZFNs, and TALENs achieve sequence-specific DNA-binding via protein-DNA interactions, whereas CRISPR and targetrons are RNA-guided systems.

The benefit of the CRISPR-Cas system over already existing gene editing technologies is the easiness of design and ultimately the low cost. Comparing with ZFNs and TALENs, CRISPR-Cas technology does not require elaborate design and time-consuming assembly of individual DNA-binding proteins. The CRISPR-Cas system is versatile and only requires a single Cas9 nuclease that can be programmed by engineering the sgRNA. The relatively easy and cost effective assembly of the targeting construct will allow this technology to become a routine method of application for molecular biology research. CRISPR-Cas systems have become one of the most popular tools due to their simplicity, high efficiency and versatility.

CRISPR Gene Editing Contains The Following Section

Discovery of CRISPR as A Gene Editing Technology

The nuclease system CRISPR-Cas provides an alternative gene editing platform. The CRISPR gene editing sysytem is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA. Notably, CRISPR had been simply known as a peculiar prokaryotic DNA repeat element for several decades before it was recognized as the bacterial immune system and subsequently harnessed as a powerful reprogrammable gene-targeting tool. There have been several critical findings that paved the way for CRISPR systems to become the CRISPR gene-editing technology. The most important work, which arguably marked the beginning of CRISPR as a biotechnology tool, has been the demonstration that Cas9 enzymes can be reprogrammed to target a desired DNA sequence in bacteria.

The basic working principle of major gene-editing technologies

Fig 1. The basic working principle of major gene-editing technologies.

The endogenous CRISPR system requires two short RNAs: the mature crRNA and a trans-activating crRNA (tracrRNA). The crRNA is composed of the part that serves as guiding sequence and another part base pairs with the tracrRNA. Both crRNA and tracrRNAs are required to form the Cas9 protein-RNA complex that cleaves DNA with DSBs at target sites. Notably, Jinek et al. showed that CRISPR-Cas9 can also be guided by a single chimeric RNA formed by the fusion of tracrRNA and crRNA, called single guide RNA (sgRNA). The studies subsequently shown that CRISPR can be adapted for in vivo genome editing in eukaryotic cells. Due to high editing efficiency and ease of use, researchers from diverse fields quickly adopted CRISPR technology as a method of choice for various genome-targeting purposes.

Different CRISPR Systems for Gene Editing

Although researchers repurposed many different CRISPR-Cas systems for gene targeting, the most widely used one is the type II CRISPR-Cas9 system from Streptococcus pyogenes. However, researchers are still actively exploring other CRISPR systems to identify Cas9-like effector proteins that may have differences in their sizes, PAM requirements, and substrate preferences. In the last few years, Dozens of different CRISPR-Cas proteins have been repurposed for gene editing. Some of the proteins that have been discovered, such as Cpf1 proteins are particularly interesting. In contrast to the native Cas9, which requires two separate short RNAs, Cpf1 naturally requires one sgRNA. Furthermore, it cuts DNA at target sites 3′ downstream of the PAM sequence in a staggering fashion, generating a 5′ overhang rather than producing blunt ends like Cas9.

In the near future, We believe that many more other CRISPR-Cas systems for gene editing will be discovered. The rapid development of gene editing with the endonuclease systems has dramatically changed the biomedical research.These CRISPR-Cas systems are not only great platforms for investigating the genes functions, but also provide a valuable means to treat many diseases from Mendelian disorders to cancers. The researches on nucleases for genome manipulation will revolutionize medical cares for many complex genetic diseases in the future.

Evolution of Second-Generation CRISPR Gene-editing Tools

One of the key progresses in the field of CRISPR technology has been the development of base-editing technology. Unlike WT Cas9, which results in DSBs and random indels at the target sites, these so-called second-generation gene-editing tools are able to precisely convert a single base into another without causing DNA DSBs. The nickase Cas9 is the foundational platform for the base editor tools that enables direct C to T or A to G conversion at the target site without DSBs. Komor et al. demonstrated that a fusion complex composed of nickase Cas9 fused to an APOBEC1 deaminase and Uracyl Glycosylase inhibitor (UGI) protein effectively converts Cytosine (C) into Thymine (T) at the target site without causing DSBs.

The activation-induced adenosine deaminase (AID) has also been fused to dCas9 to achieve direct A-G conversion at the target sites. Notably, in the absence of UGI in the complex, the dCas9-AID complex becomes a powerful local mutagenic agent that acts as a gain of function screening tool. These novel base-editing approaches significantly expand the scope of gene targeting. Researchers harnessed the efficiency of this CRISPR base editor to alter genetic code and introduce early STOP codons in genes. Studies showed that by editing C into T at CGA (Arg), CAG (Gln), and CAA (Gln) codons, we can create TGA (opal), TAG (amber), or TAA (ochre) STOP codons, respectively. The CRISPR-STOP approach is an efficient and less deleterious alternative to WT-Cas9-mediated gene knockout (KO) studies.

CRISPR Gene Editing Related References

1. Mei et al. Modified Cas (CRISPR-associated proteins) for Genome Editing and Beyond. Advanced Techniques in Biology & Medicine. 2016; 4:3.
2. Mazhar Adli. The CRISPR tool kit for genome editing and beyond. NATURE COMMUNICATIONS. May 15 2018; 9:1911.
3. Ianis G. Matsoukas. Commentary: RNA editing with CRISPR-Cas13. Front Genet. Apr 18 2018; 9:134.
4. Zhang. Genome Editing with ZFN, TALEN and CRISPR-Cas Systems:The Applications and Future Prospects. Adv Genet Eng. Feb 1 2014; 3:1.
5. Khatodia and Khurana. Trending: the Cas nuclease mediated genome editing technique. Biotech Today. December 2014; Vol. 4, No. 1:46-49.