CRISPR/Cas, comprised of Clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas), provides adaptive immunity against foreign DNA in bacteria and archaea. Type II CRISPR/Cas9 has attracted considerable interest as a tool to probe and manipulate biological systems. It has been engineered to introduce genome editing in a simple, flexible, and efficient mann...

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novici...

UNLABELLED The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (...

The relative ease of using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)associated proteins (Cas-proteins) in genome editing has rapidly made this RNA-guided endonuclease system the method of choice in almost all current applications involving genetic manipulations, with Cas9 from S. pyogenes (SpCas9) being the most well characterized and broadly used [1]. Compared to the e...

UNLABELLED Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced...

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a...

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defec...

It has nowbeen two years sincewe first wrote about the therapeutic potential of CRISPR-Cas9 in ourMay 2015 Editorial “CRISPR–Cas9 Based Therapeutics: Not So Fast”. At the time, we shared serious concerns resonating within the community that the potential for undesirable off-target mutations was significant, and that the technology was not ready for introduction into humans as a gene modificatio...

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas...

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 prot...