CRISPR-Cas Systems: A Functional Perspective and Innovations

Abstract

Adaptation is a fundamental tenet of evolutionary biology and is essential for the survival of all organisms, including prokaryotes. The evolution of clustered regularity exemplifies this principle of interspaced short palindromic repeats (CRISPR) and associated proteins (Cas), an adaptive immune system that confers resistance to viral infections. By integrating short segments of viral genomes into their own, bacteria and archaea develop a molecular memory that enables them to mount a rapid and targeted response upon subsequent viral challenges. The fortuitous discovery of this immune mechanism prompted many studies and introduced researchers to novel tools that could potentially be developed from CRISPR-Cas and become clinically relevant as biotechnology rapidly advances in this area. Thus, a deeper understanding of the underpinnings of CRISPR-Cas and its possible therapeutic applications is required. This review analyses the mechanism of action of the CRISPR-Cas systems in detail and summarises the advances in developing biotechnological tools based on CRISPR, opening the field for further research.

Keywords: CRISPR-Cas; gene editing; gene therapy.

Figure 1

Mechanism of adaptation in CRISPR-Cas systems. After including foreign genetic material inside the bacterium, Cas1 and Cas2 proteins are responsible for recognising and excising a specific portion of it: the protospacer. Then, this is included within the CRISPR sequence to carry out the subsequent steps of bacterial adaptive immunity. Some CRISPR subtypes use other Cas proteins to acquire said protospacer. Abbreviation: PAM: Protospacer adjacent motive.

Figure 2

Mechanism of crRNA biogenesis and interference in CRISPR-Cas systems. The pre-crRNA transcription occurs from the CRISPR array. Subsequently, different Cas and non-Cas proteins (RNAase III) are responsible for the crRNA maturation. Then, the crRNA and certain specialised complexes and proteins are responsible for recognising and destroying foreign DNA and/or RNA. Abbreviations: DNA: deoxyribonucleic acid; RNA: ribonucleic acid.

Figure 3

CRISPR-Cas systems recent discoveries. In recent years, the scientific community has been searching for new tools to improve the properties of CRISPR-Cas systems. These include (A) crRNA chemical modifications; (B) base editing; (C) base prime editors; (D) gene regulation; (E) ChyMErA. Abbreviations: DNA: deoxyribonucleic acid; RNA: ribonucleic acid; CHyMErA: Cas Hybrid for Multiplexed Editing and Screening Applications; pegRNA: prime editing guide RNA; ABE: adenine base editor; CBE: cytidine base editor.