The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at ...single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87-100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115-245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications.
Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by ...clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control.
Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPRs). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control.
Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for ...therapeutic genome editing
. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in the loading of the cargo. Here, we report the design of customizable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA) that enables the controlled stoichiometry of CRISPR components and limits the possible safety concerns in vivo. We describe the synthesis of a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating, called a nanocapsule (NC), around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. The NC is synthesized by in situ polymerization, has a hydrodynamic diameter of 25 nm and can be customized via facile surface modification. NCs efficiently generate targeted gene edits in vitro without any apparent cytotoxicity. Furthermore, NCs produce robust gene editing in vivo in murine retinal pigment epithelium (RPE) tissue and skeletal muscle after local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing.
RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be ...challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation.
Display omitted
•Chemical modification of Cas13 guide RNAs improves RNA targeting in human cells•Modifications at the guide RNA 3′ end increase Cas13 activity and prolong knockdown•Cas13 ribonucleoproteins yield high knockdown in human primary immune cells
By screening multiple chemical modifications of the CRISPR RNA (crRNA), Méndez-Mancilla et al. identify specific chemical modifications and placement of modified bases that improve CRISPR-Cas13 transcript knockdown in human cells. The authors demonstrate that Cas13 ribonucleoproteins complexed with chemically modified crRNAs can modify the transcriptome of human primary T cells.
Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence ...is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation—a late transition state—and defies common descriptions of a “seed” region. Our results provide a quantitative basis for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease.
Display omitted
•DNA target binding by CRISPR-Cas12a is rate limiting for DNA cleavage•Multiple cleavage sites are present on each DNA strand•Specificity against mismatches suggests a late transition state for R-loop formation•Data explain in vivo cleavage patterns by Cas12a and differences from Cas9
Strohkendl et al. dissect DNA binding and cleavage by CRISPR-Cas12a. They show that binding is functionally irreversible, yet Cas12a discriminates against mismatches with the target DNA extending beyond a seed region. These results suggest that R-loop propagation is readily reversible, enabling Cas12a to select DNA sequences more precisely than Cas9.
Display omitted
•Focused on the structural biology of core effector complexes in type III CRISPR-Cas systems.•Summarize the distinct molecular mechanisms of type III CRISPR-Cas systems.•Provide an ...in-depth discussion of structural basis of type III CRISPR-Cas interference, enhancing the understanding of CRISPR-Cas mediated bacterial immunity.
CRISPR-Cas system is an RNA-guided adaptive immune system widespread in bacteria and archaea. Among them, type III CRISPR-Cas systems are the most ancient throughout the CRISPR-Cas family, proving anti-phage defense through a crRNA-guided RNA targeting manner and possessing multiple enzymatic activities. Type III CRISPR-Cas systems comprise four typical members (type III-A to III-D) and two atypical members (type III-E and type III-F), providing immune defense through distinct mechanisms. Here, we delve into structural studies conducted on three well-characterized members: the type III-A, III-B, and III-E systems, provide an overview of the structural insights into the crRNA-guided target RNA cleavage, self/non-self discrimination, and the target RNA-dependent regulation of enzymatic subunits in the effector complex.
Display omitted
•Efficient genome editing using crRNA, tracrRNA and Cas9 ribonucleoproteins (ctRNPs).•Delivery of CRISPR ctRNPs into mammalian cells via lipofection and electroporation.•Delivery of ...CRISPR ctRNPs into mouse zygotes via microinjection.•Efficient HDR using CRISPR RNPs and with ssODNs or in vitro synthesized long ssDNAs.
Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA.
Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves ...otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications.
The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption ...of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using “error-prone” DNA-repair machinery to generate precise edits.
Display omitted
•DNA repair profiles of 223 sites in the human genome reveal nonrandom outcomes•The protospacer sequence, not genomic context, determines Cas9 DSB repair outcomes•Each DNA repair profile is composed of specific contributions from c-NHEJ and MMEJ•DNA repair profiling can be used to anticipate repair outcomes in primary cells
van Overbeek, Capurso et al. demonstrate that repair outcomes are nonrandom at S. pyogenes Cas9-mediated DSBs and are determined by the protospacer sequence rather than genomic context. DNA repair profiling reveals specific contributions of c-NHEJ and MMEJ at each site and an approach to generate precise edits without exogenous template.
CRISPR/Cas is a revolutionary gene editing technology with wide‐ranging utility. The safe, non‐viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the ...synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg−1). Intravenous co‐delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.
Special delivery: The synthesis and development of zwitterionic amino lipids (ZALs) is reported. ZALs are uniquely able to deliver long RNAs (Cas9 mRNA and targeted sgRNA) from a single ZAL nanoparticle (ZNP) to enable CRISPR/Cas gene editing.
PDF
