RESUMEN
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RESUMEN
Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Edición Génica , Ribonucleoproteínas/genética , Acidaminococcus/enzimología , Epigénesis Genética/genética , Células HEK293 , Humanos , Mutación , Linfocitos T/metabolismoRESUMEN
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edición Génica/métodos , Mutagénesis , Streptococcus pyogenes/enzimología , Biotecnología/métodos , Proteínas Asociadas a CRISPR/genética , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Activación Enzimática , Variación Genética , Humanos , Dominios Proteicos , Streptococcus pyogenes/genética , Especificidad por SustratoRESUMEN
Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.
Asunto(s)
Camptotecina/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Resistencia a Antineoplásicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Ubiquitina/metabolismo , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Edición Génica , Humanos , Autoantígeno Ku/metabolismo , Complejos Multiproteicos/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Proteolisis , Interferencia de ARNRESUMEN
The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9). We also report that four to six bases at the 3' end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.
