Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing

Yuyi Tang,Yan Fu

doi:10.1186/s13578-018-0255-x

Abstract

Artificial nuclease-dependent DNA cleavage systems (zinc-finger nuclease, ZFN; transcription activator like effectors, TALENs) and exogenous nucleic acid defense systems (CRISPR/Cas) have been used in the new era for genome modification. The most widely used toolbox for genome editing, modulation and detection contains Types II, V and VI of CRISPR/Cas Class 2 systems, categorized and characterized by Cas9, Cas12a and Cas13 respectively. In this review, we (1) elaborate on the definition, classification, structures of CRISPR/Cas Class 2 systems; (2) advance our understanding of new molecular mechanisms and recent progress in their applications, especially beyond genome-editing applications; (3) provide the insights on the specificity, efficiency and versatility of each tool; (4) elaborate the enhancement on specificity and efficiency of the CRISPR/Cas toolbox. The expanding and concerted usage of the CRISPR/Cas tools is making them more powerful in genome editing and other biotechnology applications.

Highlights

Introduction of CRISPR/Cas Clustered regularly interspaced short palindromic repeats (CRISPRs), formerly known as short regularly spaced repeats (SRSRs) [1], are a family of repetitive DNA sequences
The results showed that Cas12a was sensitive to single mismatch at positions 1–18 in the 5′-protospacer adjacent motif (PAM) proximal region where double mismatches could even induce a nearly complete loss of Cas12a activity but tolerated single or double mismatches in the 3′-PAM-distal region [82, 102]
Discovery of the well-known CRISPR/CRISPR associated protein 9 (Cas9) system is a historical leap in modern biology (Fig. 4), especially for genome editing

Summary

Introduction

Introduction of CRISPR/Cas Clustered regularly interspaced short palindromic repeats (CRISPRs), formerly known as short regularly spaced repeats (SRSRs) [1], are a family of repetitive DNA sequences. Cas9-tracrRNA-crRNA complex plays an important role in recognition and binding of Cas9 on target sites and specific cleavage. Applications of CRISPR/Cas9 on genome‐editing To simplify the protocol of CRISPR/Cas9 genome-editing, a single guide RNA (sgRNA) is designed to function as the crRNA-tracrRNA complex.

Results

Conclusion