CRISPR/Cas9 in Genome Editing and Beyond.

  title={CRISPR/Cas9 in Genome Editing and Beyond.},
  author={Haifeng Wang and Marie La Russa and Lei S. Qi},
  journal={Annual review of biochemistry},
The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9… 

Figures from this paper

Precision Control of CRISPR-Cas9 Using Small Molecules and Light.

A perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas 9 is provided.

A Review of CRISPR-Based Genome Editing: Survival, Evolution and Challenges.

This review focuses on the recent advances of CRISPR/Cas system by outlining the evolutionary and biotechnological implications of current strategies for improving the specificity and accuracy of these genome-editing technologies.

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Detailed protocols for implementing the RNP strategy in a range of contexts are outlined, highlighting its distinct benefits and diverse applications and showing how Cas9 RNP editing enables the facile genetic manipulation of entire organisms.

Repurposing CRISPR-Cas12b for mammalian genome engineering

The identification of a Cas12b system from the Alicyclobacillus acidiphilus (AaCas12b), which maintains optimal nuclease activity over a wide temperature range (31 °C–59
°C), establishes CRISPR-Cas 12b as a versatile tool for mammalian genome engineering.

CRISPR/Cas9 as a tool for Genome Editing: A Mini Review on Development and Approaches

The basis of this technique and its application in the treatment of genetic diseases are explored while highlighting challenges as well as future directions.

A glance at genome editing with CRISPR–Cas9 technology

It is reviewed that CRISPR–Cas organization is restricted to two classes and possesses different protein effectors, and the strategies to increase the Cas9 specificity and reduce off-target activity to achieve accurate genome editing are presented.

CRISPR/Cas9: Nature's gift to prokaryotes and an auspicious tool in genome editing

The basic mechanisms underlying CRISPR/Cas9 working principles are explored along with some of its current applications in a number of diverse fields.

Genome Editing for Crop Improvement: Status and Prospects

This chapter reviews the CRISPR-Cas strategy by focusing on components of the tool kit and available variants, delivery into plant cells, and gene modification detection assays.

CRISPR-Cas9 Structures and Mechanisms.

This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage and provides a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas 9-based therapies against genetic diseases.



Potential pitfalls of CRISPR/Cas9‐mediated genome editing

Although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off‐target effects and the incidence of homology‐directed repair.

RNA-guided genome editing in plants using a CRISPR-Cas system.

The results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants and suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity.

CRISPR-Cas systems for editing, regulating and targeting genomes

A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells, which will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

Development and Applications of CRISPR-Cas9 for Genome Engineering

RNA-Guided Human Genome Engineering via Cas9

The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

Multiplex Genome Engineering Using CRISPR/Cas Systems

Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Rational design of a split-Cas9 enzyme complex

This study explores the structural features that enable Cas9 to bind and cleave target DNAs, and the results suggest a way of regulating Cas9 by physical separation of the catalytic domains from the rest of the protein scaffold, and proposes that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

CRISPR-Cas9: a new and promising player in gene therapy

The research on CRISPR-mediated gene therapy is reviewed, its advantages, limitations and possible solutions are discussed, and directions for future research are proposed, with an emphasis on the opportunities and challenges of CRISpr-Cas9 in cancer gene therapy.

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

The use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

Genome editing using Cas9 nickases.