A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing

Abstract

Background: CRISPR-Cas9 is a revolutionary genome editing technique that allows for efficient and directed alterations of the eukaryotic genome. This relatively new technology has already been used in a large number of 'loss of function' experiments in cultured cells. Despite its simplicity and efficiency, screening for mutated clones remains time-consuming, laborious and/or expensive.

Results: Here we report a high-throughput screening strategy that allows parallel screening of up to 96 clones, using next-generation sequencing. As a proof of principle, we used CRISPR-Cas9 to disrupt the coding sequence of the homeobox gene, Evx1 in mouse embryonic stem cells. We screened 67 CRISPR-Cas9 transfected clones simultaneously by next-generation sequencing on the Ion Torrent PGM. We were able to identify both homozygous and heterozygous Evx1 mutants, as well as mixed clones, which must be identified to maintain the integrity of subsequent experiments.

Conclusions: Our CRISPR-Cas9 screening strategy could be widely applied to screen for CRISPR-Cas9 mutants in a variety of contexts including the generation of mutant cell lines for in vitro research, the generation of transgenic organisms and for assessing the veracity of CRISPR-Cas9 homology directed repair. This technique is cost and time-effective, provides information on clonal heterogeneity and is adaptable for use on various sequencing platforms.

Figure 1

Barcoded library preparation strategy. Forward and reverse PCR primers were designed with a unique ~10 nt barcode along with a ~20 nt site specific sequence, which will amplify around the CRISPR-Cas9 targeted site. In the first PCR cycle, either the forward or reverse barcode is added to end of the PCR product. In the second PCR cycle, the opposite barcode is added to each PCR product. In each subsequent cycle, both the forward and reverse barcodes are amplified along with the targeted region. After the PCR, sequencing platform specific adaptors are ligated to the pool of barcoded amplicons in a single reaction.

Figure 2

CRISPR-Cas9 screening workflow. Duplicate 96 well plates of cell clones are generated. One plate is frozen down or maintained for later use. From the other plate, genomic DNA is extracted, and the targeted region is amplified and uniquely barcoded in each well. The DNA is then pooled, adaptors ligated, and a fragment library is prepared. The sequencing library is then sequenced on the IonTorrent PGM. Reads are filtered and visualized on IGV, to identify the desired clones. The desired clones are then thawed and/or expanded for subsequent experiments.

Figure 3

Visualization of CRISPR-Cas9 mutant clones. A) Coverage across the barcoded amplicon in a variety of different ‘knockout’ CRISPR-Cas9 clones. The wildtype clone has full coverage over the whole amplicon, while the mutant clones have variable indels around the targeted site. Deletions are represented by a gap in the coverage, insertions are shown as black bars, while substitutions are shown in red. Blue bars represent mismatched mapped reads, due to a large deletion. Brown bars show presumed sequencing errors, which each have significant strand bias and commonly occur at the same position in multiple samples. B) The sequencing reads and coverage for clone A6, which is a compound heterozygote with a 1 bp deletion on one allele and a 13 bp deletion on the other allele. The deletions at the targeted site are present at equal proportion on both strands (blue and red), whereas the sequencing error upstream is present only on the red strand.