CRISPR-Inspired Method Targets Large, Repetitive DNA Elements

Target capture through PCR has been a mainstay in genomics for years, but scientists working on especially repetitive, poorly characterized, or rapidly evolving regions continue to struggle to fish out those stretches of DNA for further study. However, whole genome sequencing, the only other alternative for these regions, can force researchers to pay for much more information than they actually need. Fortunately, the development of the CRISPR/Cas9 system has dramatically changed the landscape of genome editing. Advances with this system have inspired new methods for targeting a specific genomic region. One of those techniques is called CATCH, for Cas9-assisted targeting of chromosome segments. Developed by Yuval Ebenstein at Tel Aviv University, Ting Zhu and Chunbo Lou from Tsinghua University, and collaborators, the protocol incorporates concepts from CRISPR to target extremely large —50 kb and longer — DNA regions without a priori knowledge of their DNA sequence.

CATCH Methodology

The method involves using RNA-guided Cas9 to make two cuts in the flanking region on either side of the target of interest. Because the approach can scoop up some off-target DNA, the team implemented a size-selection step to remove the unwanted fragments. The original paper describing the process reported generating custom BACs by pairing CATCH with Gibson assembly to target the region of interest and clone it into a vector. The authors wrote, “The procedure takes ?8?h of bench time over 1–2 days to accomplish using standard laboratory equipment at reasonable costs, which drastically simplifies and accelerates efforts to clone large bacterial genomic sequences.” The paper also presented data from validation studies using E. coli and several other bacterial genomes. Targeting experiments were most effective for regions ranging from 50 kb to 100 kb. Efficiency decreased beyond that point, and the team suggested that the upper limit for this approach might be 150 kb.

DNA Size Selection and CATCH

Size selection is important to the CATCH protocol, and the original method involved using pulsed-field gel electrophoresis. This is a cumbersome process, so scientists have been testing automated DNA extraction and sizing, including using the new SageHLS platform. For example, this particular instrument was designed to purify extremely large DNA fragments and sort them by size. By recovering a high fraction of DNA and requiring little hands-on time, it offers a more streamlined workflow than conventional gel electrophoresis. The team is still working to optimize the entire process, with early results in bacterial and human genomes presented in this 2017 AGBT poster. Since the CATCH paper came out, many other labs have adopted the method, particularly for repetitive plant genomes. Other scientists have also paired the CATCH protocol with automated size selection (like the SageHLS platform). At the AGBT conference this year, GiWon Shin from Stanford University presented a method for targeting almost megabase-size regions for analysis. Targeting is performed with custom Cas9 guides, and DNA is extracted from intact cells using the SageHLS platform. In his talk, Shin presented results from evaluations of this process targeting BRCA1, the MHC locus, and 38 candidate structural variants.

Adaptations of CATCH

The adaptation of the CRISPR/Cas9 system has undoubtedly been one of the greatest biological achievements this century. It has inspired new technological advances in genome editing, in particular the development of CATCH. The ability to target regions even in large genomes can be monumental for our understanding of biological systems that once seemed out of reach. As the authors of the original paper so eloquently wrote, “Adaptations of CATCH to larger genomes would make it a powerful tool, enabling numerous applications such as targeted cloning of large genes from plants and mammals, targeted sequencing of disease-specific genes in humans or identification of copy-number variations.” To learn more about CATCH and DNA size selection, visit HLS-CATCH.