High-throughput methods for CRISPR editing evaluation and m1A RNA modification tools development

Before CRISPR-Cas9, genome engineering struggled with inaccurate gene targeting, low efficiency, off-target effects, and labor intensity. CRISPR-Cas9 revolutionized this field, enabling precise gene targeting, efficiency improvement, reduced off-target effects, and transformative applications in research and various industries. While the CRISPR-Cas9 gene editing system appears straightforward, significant variability exists in its precision and effectiveness. To enhance our comprehension of the factors influencing CRISPR accuracy and efficiency, and to facilitate the development of improved CRISPR prediction utilities, a comprehensive dataset demonstrating on- and off-target CRISPR activity in cells is now available. These studies employed synthetic and self-targeting gRNA libraries to achieve this, collectively termed “synthetic gene editing”. This strategy involves capturing CRISPR-induced editing events at both synthetically surrogate and genomically integrated target sites.
The initial objective of this Ph.D. project was to develop synthetic surrogate library-based methods for high-throughput evaluation of CRISPR editing in cells. Additionally, develop tools for CRISPR-guided m1A RNA modification, for extending CRISPR technology in RNA modification field applications.
This thesis is structured in two principal parts: the first part describes our use of synthetic library-based CRISPR high-throughput tools to evaluate the application of CRISPR guided RNA on CRISPR on and CRISPR off, and the detailed protocol of this tool; the second part aims to further enhance the applicability of CRISPR tools in the realm of RNA modification, focusing on the development of CRISPR-guided RNA modification tools.
We establish a high-throughput method to generate high-quality CRISPR on- targeted gRNA activity data. We constructed a surrogate library for CRISPR on-target targeting, comprising 10,000+ gRNAs with corresponding target sites. Each vector within the on-target surrogate library contains a gRNA expression cassette and the surrogate on-target site. Following Cas9 induction in the library-carrying cells, CRISPR editing efficiency was quantified through deep sequencing, and indels introduced at the surrogate target sites were analyzed. A strong correlation was found between surrogate and endogenous genomic loci target sites by comparing CRISPR efficiency and indel profiles. This extensive dataset of CRISPR on-target editing efficiency offers deeper insights into the impact of DNA contexts on CRISPR efficacy, including factors like base preferences along each gRNA spacer position, gRNA spacer secondary structure, melting temperature, sequence motifs, and binding free energy. Crucially, these CRISPR targeting efficiency data sets are essential for refining computational prediction tools. Based on these data collaborators have developed a more accurate prediction tool for CRISPR gRNA editing efficiency (Paper 1).
For high-throughput quantification of CRISPR off-targets in cells, we have further refined the synthetic surrogate library method, called SURRO-Seq. CRISPRmediated deletions at the surrogate locus result in the excision of nucleotides that distinguish similar off-target sites, making the alignment of indel reads to their respective off-target loci infeasible. To address this challenge associated with indel assignment, a solution is proposed wherein a barcoding approach is implemented. This strategy entails the unique molecular tagging of each distinct off-target site using a specific 10-nucleotide barcode. By employing the SURRO-seq, we systematically assessed over 8000 putative off-target loci for 110 therapeutic CRISPR guide RNAs, facilitating the applications of CRISPR gene editing tools in clinical applications (Paper 2).
To better apply synthetic surrogate library-based methods for broad applications of high-throughput assessment of CRISPR-Cas9 efficiency, specificity, and PAM compatibility in cells, I wrote a detailed protocol for high-throughput assessment of CRISPR gene editing (Paper 3).
To advance the use of CRISPR techniques in RNA modification, I started the CRISPR-guided RNA modification project. First, I attempted to create a fusion enzyme called dCas13-NpmA by combining the 16S rRNA methyltransferases NpmA with catalytically inactive Cas13. This fusion enzyme was designed to introduce m1A modifications into mRNA. Unfortunately, subsequent experiments failed to detect any methylase activity of the resulting fusion protein. Consequently, the next step involves an exploration of novel m1A methylases to construct new fusion proteins (Draft paper).