CRISPR is a powerful, yet simple gene-editing technology that enables researchers to quickly and efficiently achieve highly targeted edits in genomes to disrupt defective genes and/or increase their function. The technology homes in on a specific locus, allowing users to add to, remove, or change the genomic sequence.
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In this podcast, Jason Liu, field application scientist at Roche, will talk to us about using next-generation sequencing to ensure gene-editing success.
But no matter how accurately and reliably those edits are performed, their fidelity must still be verified, increasingly by next-generation sequencing (NGS), enabling wide-scale assessment at nucleotide-level resolution. In addition, it’s prudent to identify other possible (off-target) sites that may have been unintentionally edited. The efficient, automatable, and scalable CIRCLE-seq is an in vitro NGS assay that allows for sensitive off-target detection of CRISPR-mediated cleavage.
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. These repetitive stretches of DNA were originally derived from viral pathogens, and serve as templates to guide the CRISPR associated protein 9 (Cas9) endonuclease to newly invading viruses. When Cas9 encounters a homologous viral sequence it creates a proximal double-stranded break (DSB). Similarly, when Cas9 and guide RNA (gRNA) are transfected into a mammalian cell, homologous sequences proximal to the gRNA are cleaved. The DSB is then repaired by joining the ends back together by an endogenous error-prone non-homologous end joining (NHEJ) process, or by using an exogenously provided donor (repair) template for patching by homology-directed repair (HDR).
Researchers use HDR to change a genetic sequence in a known and predictable manner. The gRNA determines where the edit will take place, while the donor template DNA (which shares homology at its ends with the DSB site) dictates what the edit will look like.
As these are critical components, it’s important to warrant that the gRNA and the repair template are error-free. Using quality reagents such as KAPA HiFi high-fidelity DNA polymerase for amplification can help to ensure accuracy and specificity.
Even when using the best polymerase to generate an accurate repair template for CRISPR HDR, labs need a way to verify that gene editing has been successful—that the edited site contains the expected sequence. NGS offers a high-throughput, automation-friendly method for verifying on-target gene editing in clonal isolates.
To get the best results from NGS, a robust library preparation is essential. The Molecular Biology Core Facility (MBCF) at the Dana-Farber Cancer Institute found that the KAPA HyperPrep Kit provides a workflow able to accommodate a wide range of amplicon input amounts and sizes, thus minimizing the need for input QC and sample-specific workflow modifications. Adapting the workflow to their automated liquid handling system allowed for amplicon-based library preparation of 96 clonal samples in about 3-1/2 hours of instrument time.
It is one thing to verify that the intended site has been successfully modified, but no matter how thorough the in-silico design is, it’s possible for the Cas9 nuclease used in CRISPR/Cas9-based gene editing to cleave and change unintended targets with similar sequences. Thus, labs need a method to identify possible off-target cleavage sites, and to be able to differentiate these from simple errors introduced as a result of amplification. There are several NGS workflows that can accomplish this.
Digenome-seq, for example, is a relatively simple in vitro assay in which the Cas9 nuclease is used to cut genomic DNA extracted from cells of interest, NGS sequencing adapters are ligated to all the free ends, and the resulting library is sequenced to identify the cuts. Yet because the library is not enriched for cut sites there is a high background of non-modified DNA, necessitating a large number of sequencing reads and making it difficult to find rare cleavage events.
The GUIDE-seq method reduces sequencing costs by first enriching for an oligonucleotide incorporated into Cas9-introduced DSBs in vivo. But it is less sensitive, and as a cell-based assay, it is time-consuming, labor-intensive, and limited to cells that can be readily transfected.
CIRCLE-seq, which stands for Circularization for In vitro Reporting of CLeaveage Effects by SEQuencing, combines a flexible in vitro workflow with all the enrichment benefits of selectively sequencing only Cas9-cleaved DNA.1 Randomly sheared genomic DNA is circularized and then cleaved with Cas9. Only molecules with a cleavage site are carried through to the next steps of library preparation, PCR amplification, and NGS, to reveal the sequences susceptible to Cas9 cleavage.
As an in vitro assay, CIRCLE-seq avoids the lengthy cell culture steps of cell-based methods for CRISPR QC. In addition, CIRCLE-seq is a scalable, sensitive technique requiring fewer sequencing reads—and thus lower sequencing costs—than other in vitro methods. Success in CIRCLE-seq requires efficient library preparation using reagents such as the KAPA DNA Library Prep Kit, as well as a high-fidelity DNA polymerase that minimizes amplification bias during library amplification. The KAPA HiFi DNA Polymerase used in the CIRCLE-Seq method also ensures that any mutations detected are genuine—not the result of errors in DNA amplification—and that all genomic regions are analyzed for off-target cleavage.
NGS-based tools afford nucleotide-level verification of CRISPR/Cas9-mediated editing at every stage.
Tsai, S.Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nature Methods volume 14, pages607–614(2017).
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