Synthesis of an arrayed sgRNA library targeting the human genome.

Tobias Schmidt,Jonathan L Schmid-Burgk,Veit Hornung

doi:10.1038/srep14987

Tobias Schmidt, Jonathan L Schmid-Burgk + Show 1 more

Open Access

https://doi.org/10.1038/srep14987

Copy DOI

Journal: Scientific Reports	Publication Date: Oct 8, 2015
Citations: 53	License type: CC BY 4.0

Affiliation: University Hospital Bonn, University of Bonn

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) provide an adaptive immune system to bacteria and archaea targeting foreign genetic material[1,2]
We developed an amplification-free ligation-independent cloning (LIC)-based library assembly method combined with an orthogonal deep sequencing validation strategy to facilitate the synthesis of large, arrayed single guide RNA (sgRNA) libraries
Based on LIC, we developed a cloning strategy that allows determining the spacer sequence of an sgRNA expression plasmid only by a single ssDNA oligonucleotide, enabling low-quantity chip-synthesized oligo pools to be used as input DNA without prior PCR amplification (Fig. 1)

Summary

Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) provide an adaptive immune system to bacteria and archaea targeting foreign genetic material[1,2]. Deep sequencing allowed the identification of sgRNA sequences that were enriched in selected populations In these studies PCR- and Gibson-assembly-based approaches were employed to create large polyclonal pools of sgRNA vectors from chip-synthesized oligonucleotides covering the entire human or mouse transcriptome. CIRPSR/Cas[9] constitutes a promising tool for the fast and efficient generation of large numbers of knockout cells, e.g. comprising targets from entire pathways To this end, arrayed sgRNA libraries encompassing the entire protein coding genome will constitute a powerful resource. Since the synthesis of sgRNAs using individual oligonucleotides is laborious and costly, the approach of generating arrayed libraries from chip-synthesized oligo pools is currently the most attractive choice To this end, a method would be desirable that allows singling out clonal sgRNA constructs from polyclonal source material with minimal sequence bias. In four iterative rounds of shotgun assembly and sequencing we were able to generate an arrayed genome-spanning library comprising clonal sgRNAs for targeting most genes in the human genome

Methods

Results

Conclusion