RESUMO
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , DNA/genética , DNA/metabolismo , DNA Complementar , Humanos , Oligonucleotídeos/genéticaRESUMO
Genome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.
Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Edição de Genes , Proteína 9 Associada à CRISPR/genética , Escherichia coli/genética , Proteínas Luminescentes/genética , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Discovered as an adaptive immune system of prokaryotes, CRISPR-Cas provides many promising applications. DNA-cleaving Cas enzymes like Cas9 and Cas12a, are of great interest for genome editing. The specificity of these DNA nucleases is determined by RNA guides, providing great targeting adaptability. Besides this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics, that can be exploited for different applications. Although Cas nucleases are highly promising, some room for improvement remains. New developments and discoveries like base editing, prime editing, and CRISPR-associated transposons might address some of these challenges.
Assuntos
Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Clivagem do DNA , Desoxirribonucleases/genética , Endodesoxirribonucleases/genética , RNA Guia de Cinetoplastídeos/genética , Imunidade Adaptativa , Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a DNA , Desoxirribonucleases/química , Endodesoxirribonucleases/química , Edição de Genes/métodos , HumanosRESUMO
Quantifying DNA cleavage by CRISPR-Cas nucleases is usually done by separating the cleaved products from the non-cleaved target by agarose gel electrophoresis. We devised a method that eliminates the quantification from band intensity on agarose gel, and uses a target with a fluorescent dye on the one end and a biotin on the other. Cleavage of the target will separate the dye from the biotin, and cause the dye to stay in solution when streptavidin beads are introduced. All non-cleaved target will be eliminated from solution and no longer contribute to detectable fluorescence. Cleavage will therefore increase the fluorescent signal. A control, which has no streptavidin treatment, is taken along to correct for any errors that might have been introduced by pipetting, inactivation of the fluorescent dye or release of the biotin during several steps of the procedure. With this method we were able to quantify the fraction of active Cas12a in a purification sample and assess the cleavage rate.
