The CRISPR tool kit for genome editing and beyond

Abstract

CRISPR is becoming an indispensable tool in biological research. Once known as the bacterial immune system against invading viruses, the programmable capacity of the Cas9 enzyme is now revolutionizing diverse fields of medical research, biotechnology, and agriculture. CRISPR-Cas9 is no longer just a gene-editing tool; the application areas of catalytically impaired inactive Cas9, including gene regulation, epigenetic editing, chromatin engineering, and imaging, now exceed the gene-editing functionality of WT Cas9. Here, we will present a brief history of gene-editing tools and describe the wide range of CRISPR-based genome-targeting tools. We will conclude with future directions and the broader impact of CRISPR technologies.

Fig. 1

The basic working principle of major genome-editing technologies. Meganucleases are engineered restriction enzymes that recognize long stretches of DNA sequences. Each zinc finger nuclease recognizes triple DNA code whereas each TALE recognizes an individual base. Unlike protein–DNA recognition in ZFNs and TALENs, simple RNA–DNA base pairing and the PAM sequence determine CRISPR targeting specificity. All these tools result in DNA double-strand breaks, which are repaired either by error-prone non-homology end joining or homology-directed repair. While NHEJ results in random indels and gene disruption at the target site, HDR can be harnessed to insert a specific DNA template (single stranded or double stranded) at the target site for precise gene editing

Fig. 2

CRISPR-based genome-targeting tools are widely used. Number of PubMed publications over the last 12 years that had the word “CRISPR” or “Cas9” in the abstract or title. **Number of publications in 2018 is projected to be more than 5000

Fig. 3

Major application areas of CRISPR-Cas-based technologies beyond genome editing. While WT Cas9 enables genome editing through its guidable DNA cleavage activity, catalytically impaired Cas9 enzymes have been repurposed to achieve targeted gene regulation, epigenome editing, chromatin imaging, and chromatin topology manipulations. Furthermore, the catalytically impaired nickase Cas9 enzyme has been used as a platform for base editing without double strand breaks. In addition to DNA-targeting Cas proteins, novel RNA-targeting CRISPR/Cas systems have been described as well

Fig. 4

Major strategies to recruit DNA- and chromatin-targeting and modifying enzymes via the CRISPR-Cas system. The schematics show various strategies of recruiting effector proteins to a target site using RNA guidable DNA binding capacity of Cas9-sgRNA complex. Effector proteins can be directly fused to active Cas9 or catalytically inactive dCas9 through a linker peptide. Additionally, the sgRNA scaffold can be engineered to contain multiple RNA aptamers that specifically bind to a known RNA binding proteins (RBP) such as MCP or PCP. Effector proteins than can be guided to a target locus by fusing them to the RBPs. In the Tripartite strategy, multiple different effectors are being recruited through dCas9 as well as engineered sgRNA scaffold. The SunTag approach utilizes a repeating peptide array of protein scaffold to recruit multiple copies of an antibody-fused effector protein. Chemically inducible strategies enable temporal control over the activity of Cas9 or Cas9 fused effector proteins. In split Cas9, each halves of Cas9 protein can be induced to form functional complex. In the intein-Cas9 approach, the intein protein segment can be chemically induced to excise from Cas9 and result in its activation