Investigation of target specificity of CRISPR-Cas nucleases
- TTo study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in 'seed' sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. This initial study provided an in vitro understanding of the complexities of Cas9-gRNA interaction and cleavage beyond the general paradigm of site determination based on the 'seed' sequence and PAM. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions. In addition to studies where large numbers of targets are compared with a single spacer (gRNA/crRNA trigger), substantial useful data on effective effector design can come from comparisons of a large number of different spacer sequences in their ability to target homologous chromosomal sequences. In chapter 3, we describe a general, high-throughput procedure to test the efficacy of thousands of targets, applying this to the Escherichia coli type I-E Cascade (CRISPR-associated complex for antiviral defense) system. These studies were followed with reciprocal experiments in which the consequence of CRISPR activity was survival in the presence of a lytic phage. From the combined analysis of the Cascade system, we found that (i) type I-E Cascade PAM recognition is more expansive than previously reported, with at least 22 distinct PAMs, with many of the noncanonical PAMs having CRISPR-interference abilities similar to the canonical PAMs; (ii) PAM positioning appears precise, with no evidence for tolerance to PAM slippage in interference; and (iii) while increased guanine-cytosine (GC) content in the spacer is associated with higher CRISPR-interference efficiency, high GC content (> 62.5%) decreases CRISPR-interference efficiency. Our findings provide a comprehensive functional profile of Cascade type I-E interference requirements and a method to assay spacer efficacy that can be applied to other CRISPR-Cas systems. The description of CRISPR-Cas specificity from this work and several parallel studies in diverse groups has served as a key foundation of recent advances in genome editing. Understanding the basic recognition and targeting ability of the CRISPR-Cas systems, the work of this thesis, is important to understanding how to utilize these systems for genome editing and other applications and toward understanding the limits of such application. Each CRISPR-Cas system has its own unique target specificity profile for each distinct to each target sequence, with the differences observed within the same CRISPR nuclease (e.g. in vitro vs. in vivo, various in vivo systems, etc.) likely due to the rate limiting step in each condition. In providing precise high throughput assays for specificity, the approaches here allow assessment and optimization of target specificity as influenced by the diversity of specific in vivo or in vitro condition in which the CRISPR-Cas system is being utilized.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Fu, Becky Xu Hua
|Stanford University, Department of Genetics.
|Fire, Andrew Zachary
|Fire, Andrew Zachary
|Statement of responsibility
|Becky Xu Hua Fu.
|Submitted to the Department of Genetics.
|Thesis (Ph.D.)--Stanford University, 2017.
- © 2017 by Xu Hua Fu
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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