CRISPR‐Cas9‐Edited Site Sequencing (CRES‐Seq): An Efficient and High‐Throughput Method for the Selection of CRISPR‐Cas9‐Edited Clones

Abstract

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc.

CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis: pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency

“…Percentage frequency of HDR to create the EHMT1 _SNV were determined by Amplicon sequencing. Transfected cells were then single cell cloned by limiting dilution in 96 well plates, replica plated, and DNA lysate prepared [ 11 ]. Further rounds of amplicon sequencing on DNA lysate determined clonal cell lines.…”

CRISPR‐Cas9–Mediated Genome Editing Confirms EPDR1 as an Effector Gene at the BMD GWAS‐Implicated ‘STARD3NL’ Locus

“…The WT product was 2370 bp in size, whereas deletions in the CRISPR‐edited pooled cells ranged from 595 to 1739 bp (PCR band size 631–1775 bp). To further verify each CRISPR deletion, all PCR products were sequenced using a multiplexed next‐generation sequencing adapter primer strategy modified from previously published studies ( 19 , 20 ) (see Supplementary Fig. S2 ).…”

“…To further verify each CRISPR deletion, all PCR products were sequenced using a multiplexed next generation sequencing adapter primer strategy modified from previously published studies (19) (20) (see Supplemental Figure 2). While there is some variability among replicates, it is apparent that the upstream (US)-3, downstream (DS)-3 sgRNA pair were the most efficient at CRISPR-Cas9 mediated deletion of the proxy SNP region, followed closely by the US-3 and DS-4 pair (Figure 2).…”

Base Editing in Human Cells to Produce Single‐Nucleotide‐Variant Clonal Cell Lines

“…In these cases, the harvested genomic DNA samples from Basic Protocol 2 or Basic Protocol 5 should be amplified and prepared for Illumina NGS. Targeted amplicon sequencing for genome editing experiments has been previously described in depth by Veeranagouda and coworkers, as well as by Yang and coworkers (Veeranagouda, Debono-Lagneaux, Fournet, Thill, & Didier, 2018;Yang, Yang, Byrne, Pan, & Church, 2014). We recommend quantifying base editing using a 300-cycle, paired-end NGS run with a 200-to 250-bp amplicon.…”

“…Genomic DNA samples can also be prepared for Illumina NGS to robustly quantitate editing efficiencies. Targeted amplicon sequencing is the most common method with which to do this, and has been previously described (Gaudelli et al., 2017; Veeranagouda et al., 2018; Yang et al., 2014). We recommend the use of the CRISPResso2 open‐access software to quantify base‐editing efficiencies from fastq files (Clement et al., 2019).…”