CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening

Abstract

BackgroundThe bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches.ResultsHere we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs.ConclusionsThe simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3268-z) contains supplementary material, which is available to authorized users.

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

“…Here we present a detailed protocol for STAgR allowing the fast and simple generation of gRNA expression plasmids of varying sizes. This protocol can be used for one-step generation of gRNA plasmids with 2-8 gRNA expression cassettes (with or without two MS2 RNA aptamers) into the gRNA expression vector STAgR_Neo, and the use of human U6, mouse U6, human 7sK and human H1 promoters 28,32,33 . If more than four cassettes are desired, it is recommended to use at least two different promoter/scaffold types to increase efficiency.…”

SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries

“…Because they are based on a reference sequence, chemically synthesized libraries can also differ significantly from the actual subject of the study, limiting their application to a few model organisms with well-assembled genomes and low rates of DNA sequence polymorphism. To address these issues, several enzymatic approaches have been developed to create libraries from various DNA inputs 10,[13][14][15] .…”

“…and Köferle, et al15 did not select for PAMs but produced libraries from completely random fragments of a DNA sample. As a result, Cheng et al produced dense libraries targeting practically all valid PAMs and Köferle, et al produced libraries where 23-30% of the spacers were adjacent to PAMs and the spacer lengths distributed at about 27 bp.However, both libraries contained many spacers that did not target PAM sequences.…”

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

“…Both are based on controlled enzymatic digestion of target DNA rather than relying on custom oligonucleotide synthesis. While CORALINA 9 employs micrococcal nuclease, the only currently available alternative method, CRISPR-EATING 10 , makes use of restriction enzymes ( Hpa II, ScrF I, Bfa I and Mme I). Importantly, both techniques can be applied to any input DNA, which serves as the source of gRNA protospacer sequences.…”

“…Moreover, CORALINA is adaptable to customization, as appropriate input and vector choices define the library type, size and content. Here, a detailed protocol is presented that can be used for the generation of comprehensive gRNA libraries from diverse sources of DNA ( Figure 1 ), including bacterial artificial chromosomes (BACs) or genomic DNA 9 . The representative results accompanying this protocol were derived by applying the CORALINA protocol to BAC DNA.…”