Right tool for the right job: Exploring the diversity of type V CRISPR-Cas systems

Wen Ying Wu

Research output: Thesisinternal PhD, WU

Abstract

CRISPR-Cas is an extraordinary prokaryotic adaptive immune system, divided into two classes that each contain three types and a wide variety of subtypes. Class 2 effector proteins are the most exploited because of their compact structures with multi-functional properties. Amongst the class 2 systems, the more recently discovered type V systems appears to be the most diverse type, with new mechanistic features still to be uncovered. The research described in this thesis focusses on the characterization and subsequent development of applications of type V CRISPR-Cas systems, more specifically types V-A and V-U1.

Chapter 1 introduces CRISPR-Cas as a sophisticated prokaryotic adaptive immune system. CRISPR-Cas systems. Each CRISPR-Cas system has unique features, but all actively participate in adaptive immunity through a three-step mechanism: adaptation, expression and interference. Although all variants follow the same steps, the different CRISPR-Cas (sub)types are highly diverse with unique structural and functional features at all levels of the mechanism.

After describing the underlying molecular mechanism of CRISPR-Cas, an overview is given in chapter 2 on the different genome editing applications. The main focus is on DNA-targeting class 2 effector proteins, such as Cas9, Cas12a and Cas12b. Apart from natural variants, engineered CRISPR-Cas nuclease variants that increase editing precision or regulate nuclease activity were presented as well. One of the biggest bottle necks of genome editing in eukaryotes is the delivery of a specific nuclease. Therefore, different approaches of nuclease/guide delivery were discussed. In addition, various host repair pathways were examined based on the type of DNA damage and the type of repair template available.

In chapter 3, the first step of the CRISPR-Cas adaptive immunity, adaptation of the CRISPR memory, is studied in two type V systems, namely type V-A and V-B from Francisella novicida  tularensis subsp. novicida U112 and Alicyclobacillus acidoterrestris ATCC 49025, respectively. The type V-A locus encodes Cas12a, Cas4, Cas1 and Cas2, whereas that of V-B encodes Cas12b, a Cas4/1 fusion protein and Cas2. It was found that in type V-A, only Cas1 and Cas2 are required for adaptation, and in type V-B, Cas4/1 and Cas2 are required for adaptation, but Cas4 activity is dispensable. Spacers acquired without a functional Cas4, appeared to target protospacers containing mostly non-conical PAMs. Thus, Cas4 activity is required for PAM selection and acquisition of suitable spacers in both type V-A and V-B. The role of Cas12a in the adaptation process has not been elucidated yet in this chapter, but will be addressed in future studies.

Following adaptation, chapter 4 describes crRNA maturation in type V-A. Type V-A crRNA maturation is distinct to that found in type II, where Cas9 requires both a crRNA and a tracrRNA, and gets processed by endogenous RNaseIII after ribonucleoprotein complex formation. This study demonstrated that Cas12a does not require a tracrRNA nor RNase III for crRNA maturation. Instead Cas12a itself is able to process pre-crRNA into mature crRNA using a previously unknown RNase domain found in Cas12a. Having Cas12a able to process its own crRNA is greatly advantageous for genome editing applications and allows for simple simultaneous multi-gene (multiplex) editing using a single CRISPR-array. Using a single CRISPR-array containing four spacers, Cas12a was able to simultaneously edit up to four genes in mammalian cells (ex vivo) and up to three genes in mouse brain cells (in vivo).

Apart from processing its own pre-crRNA, another distinct feature of Cas12a is the generation of staggered ends after cleavage of dsDNA. These staggered ends were exploited in chapter 5 to create in a novel genome editing approach in E. coli, termed “cut and paste. Cleavage by Cas12a generates double-stranded DNA breaks with 4-5 nt compatible staggered ends. These staggered ends can be repaired by ligation using T4 ligase. Using cut & paste, a genomic deletion in E. coli was successfully achieved, albeit with a relatively low editing efficiency.

To further explore other type V systems, chapter 6 focusses on the characterization of a novel compact type V systems, type V-U1 from Mycolicibacterium mucogenicum CCH10. The type V-U1 CRISPR-Cas locus express a small effector protein MmuCas12u1. MmuCas12u1 is roughly half the size of Cas12a. Despite its small size, MmuCas12u1 seems to retain some functional features also found in Cas12a. Features such as processing its own pre-crRNA, targeting dsDNA and recognizing a 5’-TTN-3’ PAM. The RuvC domain of MmuCas12u1 does not cleave dsDNA, but instead is hypothesized to be involved dsDNA-activated transcriptional silencing. By leveraging this property, MmuCas12u1 has been used for single- and multiplex- transcriptional silencing in E. coli.

Chapter 7 described how the fundamental knowledge gained on MmuCas12u1 is used to develop small Mmu base editors (MmuBE). MmuBEs are fusion proteins consisting of MmuCas12u1, cytidine deaminase and uracil glycosylase inhibitor, which is a tool for RNA-guided targeted nucleotide (C à T) substitution. Several MmuBE variants were constructed and characterized in E. coli. Most variants are relatively efficient, with a base editing window consisting of two regions, a PAM-proximal (2-5) and a PAM-distal (13-19) region, with the PAM-proximal region having more edits. In addition, a small-scale pilot experiment also demonstrated on-target base-editing by MmuBE in eukaryotic cells, namely in Saccharomyces cerevisiae. MmuBEs are currently the smallest base editors (genes ~2.8 kb) known, further expanding the current toolbox for prokaryotic base editing, and with great promise for eukaryotic base editing.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • van der Oost, John, Promotor
  • Staals, Raymond, Co-promotor
Award date12 Feb 2021
Place of PublicationWageningen
Publisher
Print ISBNs9789463956680
DOIs
Publication statusPublished - 12 Feb 2021

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