Abstract
The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.
Highlights
The past two decades have seen the explosion of available genomic editing tools for eukaryotic systems including Zinc finger nucleases (ZFNs) [1], transcription activator-like effector (TALE) guided nucleases (TALENs) [2], CRISPR/Cas nucleases [3,4,5], and most recently the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, and Base-Editors [6]
These systems operate via endogenous DNA repair mechanisms to bring about nucleotide changes and have greatly increased the routine throughput of genomic editing experiments
Sequence changes arise during repair of double-strand DNA breaks, with repair primarily carried out by the non-homologous end joining (NHEJ) pathway, which generates small insertions or deletions, and less commonly large deletions [7,8]
Summary
The past two decades have seen the explosion of available genomic editing tools for eukaryotic systems including Zinc finger nucleases (ZFNs) [1], transcription activator-like effector (TALE) guided nucleases (TALENs) [2], CRISPR/Cas nucleases [3,4,5], and most recently the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, and Base-Editors [6]. The exact editing of single nucleotide polymorphisms (SNPs) within primary cells or cell lines is essential to functionally validate causative SNPs identified from genome-wide association studies [15] Methods for both editing cells and isolating clones of edited cells have become much more accessible, techniques for high-throughput screening of large numbers of clones are still required. The protocol provides exact allelic sequence for resolution of complex alleles in diploid or polyploid cells and provides sequence files that can be analyzed with provided plateScreen scripts, or input into other analysis tools (e.g., OutKnocker [21]) This protocol will be highly useful for studies where isolation of clones with exact nucleotide changes is necessitated; and may be incorporated into an automated robotics system for even higher throughput applications
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