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.
Highlights
The bacterial Clustered regularly interspaced short palindromic repeats (CRISPR) system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability
While CORALINA guide RNA molecules (gRNAs) could be derived in principle from any source of DNA we used complete genomic DNA from two very large and well annotated genomes (Mus musculus and Homo sapiens) to test the optimal conditions, limits, and bottlenecks of our method
We independently generated and analyzed three pooled gRNA libraries from both species to assess the reproducibility of CORALINA
Summary
The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The CRISPR system has been adapted to many cell types and species, where, without exception and with a high. Genome editing with CRISPR has proven remarkably efficient [11], extending to cell types and species where this was so far not (or only insufficiently) applicable (e.g. plasmodium [12], Cryptosporidium [13], tunicates [14], wheat [15], rice [16], tomato [17], silk worms [18], C. elegans [19], beetles [20], sea lampreys [21], zebrafish [22], salmons [23], pigs [24], rats [25], goats [26], rabbits [27], and many more). For human and mouse, only gRNA libraries of limited complexity have been generated so far and none are available for other mammals
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