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Abstract

Genetics

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

Published: December 26th, 2018

DOI:

10.3791/58556

1MCN Junior Research Group, Munich Center for Neurosciences, Ludwig Maximilian Universitat, BioMedical Center, 2Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, 3Institute of Developmental Genetics, Helmholtz Zentrum, German Research Center for Environmental Health, 4Physiological Genomics, Ludwig Maximilian Universitat, BioMedical Center

Abstract

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

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Keywords CRISPR Cas9

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