Chemically modified siRNA: tools and applications. Optimizing sgRNA structure to improve CRISPR–Cas9 knockout efficiency. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.
Synthetic CRISPR RNA-Cas9-guided genome editing in human cells. Chemically modified guide RNAs enhance CRISPR–Cas genome editing in human primary cells. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Rationally engineered Cas9 nucleases with improved specificity.
Type V CRISPR–Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition. Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Genome-wide specificities of CRISPR–Cas Cpf1 nucleases in human cells. Generation of knockout mice by Cpf1-mediated gene targeting. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. The crystal structure of Cpf1 in complex with CRISPR RNA. Crystal structure of Cpf1 in complex with guide RNA and target DNA. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Cpf1 is a Single RNA-guided endonuclease of a class 2 CRISPR–Cas system. RNA-guided human genome engineering via Cas9. Multiplex genome engineering using CRISPR/Cas systems.
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Evolution and classification of the CRISPR–Cas systems. CRISPR germline engineering-the community speaks. Our findings could facilitate a wide range of genome editing applications.īosley, K. We also show that the applicability of crRNAs from Cpf1 orthologues is different for AsCpf1 and LbCpf1, as LbCpf1 is more conservative in the recognition of the loop structure at the 5′ handle. This combination induced a more pronounced improvement in the gene-cutting efficiency when using LbCpf1 than when using AsCpf1. Notably, we demonstrate that the combination of lead crRNA and ψ-mRNA substantially increased the gene-cutting efficiency by over 300% compared to the control group. We also show that a ψ-modification is a suitable chemical alteration for AsCpf1 mRNA. Here, we report the systematic investigation of 42 chemically or structurally engineered crRNAs, and establish comprehensive structure–activity (genome editing efficiency) relationships. Yet, to the best of our knowledge, the genome editing efficiency and off-target effects of engineered crRNAs and Cpf1 mRNAs have not been explored. Furthermore, the structure of guide RNAs also plays a notable role in gene cutting for the CRISPR–Cas9 system 20, 21. And the incorporation of chemically modified nucleotides in guide RNAs has been shown to retain insertion and deletion (indel) percentages in the CRISPR–Cas9 nuclease system 19. Also, a chimaeric single-guide RNA with three chemically modified nucleotides at both the 5′ and 3′ ends strongly improved Cas9-mediated genome editing in human primary T cells 18. For example, chemical modifications of CRISPR–Cas9 led to enhanced activity in a number of human cells 18, 19. To increase the genome editing efficiency of CRISPR–Cas systems, previous studies have explored a variety of approaches 16– 19.
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