Concurrent Scientific Session (Genomics): CRISPR/Cas Technology
Engineered AsCas12a variants with enhanced activity and broadened PAM compatibility
CRISPR-Cas12a (Cpf1) is a type V CRISPR effector RNA-guided DNA endonuclease with utility in synthetic biology and genome engineering. As an alternative to the commonly used Streptococcus pyogenes Cas9 (SpCas9), Cas12a recognizes TTTV (V = A/G/C) PAM sequences, which permits editing in AT-rich regions of the human genome. However, the extended length of AsCas12a PAM sequence relative to SpCas9 (TTTV vs. NGG) restricts its utility in genome editing. To improve the targeting range and enzymatic activity of Cas12a, we developed a bacterial-based selection assay to select for Cas12a mutants that demonstrate increased cleavage activity and reduced PAM specificity. We first selected Cas12a variants with enhanced activity at non-conical TTTT PAM, since this specific motif is significantly more prevalent throughout the human genome than the other three TTTV motifs. After multiple rounds of selections, we successfully enriched several variants from a Cas12a library with random mutations. We characterized the top enriched variants and revealed their enhanced cleavage activities at both TTTV and TTTT PAM sites when delivered into human cells as ribonucleoprotein (RNP) complexes. Combining mutations from these variants further enhanced cleavage activity, which globally enhanced the total genome editing efficiency of Cas12a over 96 sites found within several unique genomic loci. Early off-target site detection studies with GUIDE-Seq suggest that these mutants retain high on-target specificity of WT Cas12a with ribonucleoprotein (RNP)-based delivery. Overall, we anticipate that this new variant with enhanced activity and broadened PAM compatibility will allow for broader application of the CRISPR-Cas12a system for genome editing.
Genome Editing Session: Trends and core functions for CRISPR-directed gene editing
While this CRISPR-directed gene editing is revolutionizing genetics, gene therapy and even cancer diagnostics, its application is not without challenges. We’ll open the session with an overview of a gene editing core that carries out pharmacogenomics as a service business and provides educational opportunities. We will then address several scientific challenges in both execution and analyses that can impede workers in the field, providing information as to how core directors are working to address these problems. These challenges include 1) Specificity: fidelity of gene editing is a centerpiece of this technology yet target range remains a challenge. To improve the targeting range and enzymatic activity of Cas12a, Michael Collingwood will describe a bacterial-based selection assay to select for Cas12a mutants that demonstrate increased cleavage activity and reduced PAM specificity. Overall, this new variant with enhanced activity and broadened PAM compatibility could allow for broader application of the CRISPR-Cas12a system for genome editing; 2) Analyses of Genomic Changes: quantifying editing rates in pools of cells or identifying correctly edited clones can be complex and is always tedious. Although targeted NGS provides a high-throughput platform for optimizing editing reagents and identifying correctly modified clones, the large amount of data produced can be difficult to analyze. Shondra Miller will talk about a new system, CRIS.py, a simple and highly versatile python-based program, which can concurrently analyze next-generation sequencing data for both knock-out and multiple user-specified knock-in modifications from one or many edited samples; 3) Gene Editing in a Mouse Model; The mouse remains a preferred organism for creating genetically modified animal models especially for testing novel and innovative gene editing strategies. C. (Guru) Gurumurthy will cover the paradigm shifts caused by the CRISPR tool, as well as the latest CRISPR trends in mouse genome engineering. This comprehensive overview will be interspersed with a dynamic dialogue with the audience.