Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9

Abstract

The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology.

Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance

“…ZFNs and TALENs are effective genome engineering technologies but their major limitation is that tailoring the DNA binding proteins to target a sequence of interest can be costly and time-consuming (Ceasar et al, 2016). Furthermore, engineering TALENs to generate targeted DSBs requires two TALEN proteins capable of binding in a tail-to-tail orientation to facilitate the dimerization of FokI nuclease domain (Sun and Zhao, 2013).…”

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

“…This system used an engineered single guide RNA (sgRNA) in which the 20 bp comprising the CRISPR RNA (crRNA) upstream of a protospacer adjacent motif (PAM; NGG or NAG for Cas9) was fused to the trans -activating crRNA (tracrRNA) sequence of the Cas9 protein. In plants, CRISPR-Cas9 mediated genome editing of single and multiple gene targets has been shown for several vascular plants (Ceasar et al 2016; Ma et al 2016; Zhang et al 2016). In nonvascular plants, the CRISPR-Cas9 system has been used in Marchantia polymorpha to target the AUXIN RESPONSE FACTOR 1 (ARF1) gene following Agrobacterium -mediated transformation (Sugano et al 2014), and in P. patens to target the ADENINE PHOSPHORIBOSYL TRANSFERASE ( PpAPT ) gene by protoplast transformation (Collonnier et al 2016).…”

Detection of Sp110 by Flow Cytometry and Application to Screening Patients for Veno-occlusive Disease with Immunodeficiency

“…Of note, flow cytometry was sufficiently sensitive to detect a decrease in Sp110 protein associated with a 50% decrease in mRNA which mimics the condition in individuals who carry a heterozygous mutation in this gene. To further confirm the specificity of the Sp110 antiserum, we generated a Jurkat T cell clone that contained targeted loss-of-function mutations in Sp110 using the CRISPR/Cas9 system [11] (ΔSp110-Jurkat; the altered SP110 sequence of the targeted Jurkat clone is depicted in Supplemental Fig. 2).…”