Can SpRY recognize any PAM in human cells? / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.

Sistema CRISPR/Cas: Edición genómica de precisión / CRISPR/Cas system: precision genome editing

La función original de los sistemas CRISPR/Cas es destruir el DNA de virus bacterianos. Este sistema ha evolucionado para identificar y cortar secuencias de diferentes DNA de virus de DNA evitando la infección. En la célula, está compuesto de genes Cas que producen nucleasas guiadas por RNA capaces de cortar el DNA. Si el RNA guía encuentra DNA de un virus con el que se puede emparejar, recluta a la nucleasa Cas9 que lo corta. Este sistema es utilizado in vitro para editar genes basándose en la producción de rupturas de doble cadena y su posterior reparación. Actualmente existen varias plataformas para el diseño de RNAs guía, aunque también es posible realizarlo de forma manual. Los componentes del sistema son entregados a la célula mediante un plásmido o una ribonucleoproteína. En esta revisión nos centraremos en la función original de CRISPR/Cas en procariotas y en cómo los investigadores la han modificado para proporcionar nuevas técnicas de edición de genomas. Discutiremos sobre las ventajas de esta nueva técnica, las formas en que podemos utilizarla y algunas de las limitaciones que aún encontramos en su aplicación

The original function of CRISPR/Cas systems is to destroy the DNA of bacterial viruses. This system has evolved to identify sequences of different DNA viruses and cut them in order to avoid infection. In the cell, the system is made up of Cas genes which produce RNA-guided nucleases capable of cutting DNA. If the guide RNA finds viral DNA with which it can pair up, it recruits the Cas9 nuclease to cut it. This system is used in vitro for gene edition, relying on the production of double-strand breaks and their subsequent repair. Currently, there are several platforms for the design of the guide RNA, and it is also possible to design it manually. The components of the system can be delivered to the cell through a plasmid or through a ribonucleoprotein. In this review we will focus on the original function of CRISPR/Cas in prokaryotes, and in how researchers have modified it in order to provide new genome editing techniques. We will discuss the advantages of this new technique, the ways in which it can be used, and some of the limitations found in its application