Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing

CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction

Resultados de seis años de estudios citogenéticos en líquido amniótico

Fundamento: la pesquisa de diversas enfermedades genéticas es uno de los programas preventivos con actual importancia a nivel de la salud pública. La detección precoz de cromosomopatías y la toma de conducta oportuna disminuyen la morbimortalidad y mejora la calidad de vida de los pacientes.Objetivo: describir el comportamiento de los resultados de estudios citogéneticos en líquido amniótico realizados a gestantes de Las Tunas durante un periodo de seis años, de 2008 a 2014.Metodología: se realizó un estudio descriptivo retrospectivo para evaluar los resultados de estudios citogenéticos en líquido amniótico realizados durante seis años, de 2008 al 2014. Se revisaron los registros estadísticos, se evaluaron los resultados, los criterios de indicación, el comportamiento de los grupos de edades en las mujeres de edad avanzada y las cromosomopatías diagnosticadas.Resultados: predominaron las muestras con resultados, que superaron a las no concluyentes y a las positivas, por cada 100 mujeres estudiadas con riesgo, se diagnostica 2,3 casos positivos de cromosomopatías; como criterio de indicación prevalecieron las embarazadas con edad gestacional avanzada, siendo el grupo etario de 37 a 40 el más representado; en los casos positivos predominó las cromosomopatías numéricas, del tipo trisomía 21 o Síndrome de Down, con una frecuencia de 1,2 por cada 100 embarazada de riesgo estudiada.Conclusiones: el programa de diagnóstico citogenético en líquido amniótico ha sido un instrumento eficaz de detección prenatal de defectos congénitos por cromosomopatías, de gran utilidad en el proceso de asesoramiento genético. </p

Naturally Occurring Off-Switches for CRISPR-Cas9

CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These anti-CRISPRs were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable off-switches for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT

Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly

Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly