Functional Versatility of CRISPR-Cas Systems in a Bacterial Pathogen Restricted; Files Only

Ratner, Hannah (Fall 2019)

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CRISPR-Cas systems are widespread in prokaryotes and function as adaptive immune systems that use small RNAs (CRISPR RNAs; crRNAs) to guide Cas proteins to recognize and cleave foreign nucleic acids. There is increasing evidence for broader roles of these systems. The bacterial pathogen Francisella novicida naturally encodes a CRISPR-Cas9 system that plays a critical role in bacterial virulence. We determined that Cas9 enables virulence by repressing the transcription of four endogenous genes and providing protection from phagosome produced antimicrobials. This regulation is mediated by a non-canonical small RNA (scaRNA), rather than a crRNA, that guides Cas9 to bind DNA targets and block transcription. scaRNA binds endogenous targets without lethally cleaving the bacterial chromosome due to reduced scaRNA:DNA complementarity. We harnessed this activity to reprogram scaRNA to repress other genes. Furthermore, with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also be licensed to direct cleavage of the invading DNA. These findings highlight that a cleavage-competent Cas9 complex can exist in two distinct functional states in bacteria: binding to endogenous DNA as a transcriptional repressor and cleaving foreign DNA to prevent infection. With this knowledge, we investigated Cas12a activity, a second CRISPR system in F. novicida. Despite the differences in timing of expression, Cas12a prevented infection with foreign DNA as efficiently as Cas9 when both systems were reprogrammed to prevent infection by the same DNA target. Synthesizing the requirements for DNA targeting and scaRNA-based repression, we demonstrated that the crRNAs of the F. novicida CRISPR-Cas9 and CRISPR-Cas12a systems can be commandeered to direct transcriptional repression in addition to DNA targeting, ultimately reprogramming Cas12a to repress endogenous targets. The functional versatility of F. novicida Cas9 and Cas12a indicates that subtle, single base changes in the crRNAs can direct the mechanics of CRISPR protein function. The shift between DNA targeting and transcriptional repression via DNA binding likely underpins a broad class of underappreciated CRISPR functions, one that is potentially critical to the physiology of the numerous Cas9- and Cas12a-encoding pathogenic and commensal organisms.

Table of Contents

Chapter 1. Introduction     (1)

CRISPR-Cas Biology (1)

             History        (1)

             Overview    (2)

             Figure 1. The three stages of adaptive immunity by CRISPR-Cas9 systems       (3)

             Classification and Mechanistic diversity       (4)

Adaptive Immunity by CRISPR-Cas9 Systems        (9)

             crRNA maturation    (9)

             Target interference     (10)

             Spacer acquisition     (11)

             Figure 2. Schematic of Cas9:gRNA interactions   (12)

Francisella     (14)

             Introduction to Francisella spp          (14)

             Francisella intracellular lifecycle       (16)

             Innate Immune Evasion and Survival (17)

             Model for CRISPR-Cas System Biology     (20)

Chapter 2. CRISPR-Cas Functions Beyond Adaptive Immunity      (23)

Abstract          (24)

Introduction   (25)

Activation and function of CRISPR-Cas systems in response to envelope stress     (27)

             Figure 1. Activation of CRISPR-Cas systems in response to environmental changes.      (28)

CRISPR-Cas control of population behaviors (29)

CRISPR-Cas mediated regulation of host-pathogen interactions      (31)

              Figure 2. CRISPR-Cas mediated physiological changes   (34)

Are CRISPR-Cas systems more broadly involved in stress responses?       (36)

Conclusion    (38)

References     (39)

Chapter 3. Catalytically active Cas9 mediates transcriptional interference to facilitate bacterial virulence     (48)

Abstract          (49)

Introduction   (50)

Results (53)

             FnoCas9 has a highly specific regulon         (53)

             FnoCas9 represses transcript levels by targeting the 5’ UTR of target genes (53)

             Figure 1. FnoCas9, scaRNA, and tracrRNA regulate transcript levels in a specific genomic region of F. novicida (54)

             Figure 2. FnoCas9 targets sequences coding for 5’ untranslated regions (UTRs) leading to transcriptional interference    (56)

             scaRNA has complementarity to the 1104 and 1101 5’ UTRs          (57)

             FnoCas9 uses a PAM to interact with target 5’ UTR DNA   (57)

             Extent of complementarity to scaRNA modulates transcriptional interference (59)

             Figure 3. A PAM motif is required for FnoCas9 transcriptional interference       (60)

             Proximity of the scaRNA binding site to the TSS is required for transcriptional interference (63)

             Figure 4. FnoCas9 transcriptional interference is controlled by degree of scaRNA complementarity and target proximity to the TSS       (64)

             Cleavage-capable FnoCas9 binds competing RNAs to form two distinct complexes with different functions         (66)

             Figure 5. FnoCas9 forms complexes with two different RNA duplexes  (68)

             Repression of each gene in the FnoCas9 regulon contributes to virulence (69)

             scaRNA can be reprogrammed to guide FnoCas9 to repress non-native targets       (69)

             Figure 6. Deletion of 1104-1101 restores virulence of a cas9 mutant     (70)

             Figure 7. scaRNA can be reprogrammed to repress new targets  (72)

Discussion    (74)

Supplemental Figures. (80)

             Figure S1. Validation of the 1104-1103-1102 and 1101 Transcripts, Related to Figure 1 (80)

             Figure S2. scaRNA is Required for FnoCas9 Interaction with 1104 and 1101 DNA, Related to Figures 2 and 3 (82)

             Figure S3. Parameters Governing FnoCas9-mediated Transcriptional Repression and Transformation Restriction, Related to Figure 4    (84)

             Figure S4. Identity to scaRNA Determines Repression Level From a Plasmid-based Reporter Construct, Related to Figure 4      (85)

             Figure S5. crRNA Restriction of Foreign DNA is scaRNA-independent, Related to Fig. 5       (87)

             Figure S6. Repression of Each Gene in the FnoCas9 Regulon Contributes to Virulence of F. novicida, Related to Figure 6 (89)

             Figure S7. Reprogrammed scaRNA Represses Transcription by Binding Promoter Regions, Related to Figure 7 (90)

STAR Methods         (91)

References      (101)

Chapter 4. F. novicida CRISPR-Cas systems can functionally complement each other in DNA defense while providing target flexibility  (107)

Abstract         (108)

Introduction    (109)

Results (111)

             FnoCas9 contributes to F. novicida virulence independently of FnoCas12a  (111)              

             Endogenous F. novicida CRISPR systems function independently in DNA defense (112)

             Figure 1. FnoCas9 contributes to virulence independently of FnoCas12a (113)

             Figure 2. Cas12a & Cas9 have distinct targets that they inhibit with similar efficiencies      (115)

             Cas12a exhibits PAM promiscuity in native host     (116)

             Cas12a and Cas9 follow different patterns of expression during transformation      (117)

             Figure 3. Cas12a requires a 5’ protospacer-adjacent motif (PAM)          (118)

             Cas9 and Cas12a have the same baseline ability to restrict foreign DNA in F. novicida       (119)

             Figure 4. cas12a and cas9 have different expression patterns during transformation        (120)

             Figure 5. Cas9 and Cas12a exhibit similar endogenous DNA targeting efficiencies when reprogrammed for the same artificial target       (122)

Discussion     (123)

Materials and methods (127)

Chapter 5. Extent of crRNA complementarity shifts CRISPR-Cas systems between transcriptional repression and DNA defense  (131)

Abstract         (132)

Introduction    (133)

Results (135)

             FnoCas9 crRNAs with reduced target complementarity can repress transcription     (135)

             Figure 1. Reduced crRNA:target complementarity shifts Cas9 function from DNA cleavage to transcriptional repression (137)

             Cas12a crRNAs can direct different functions based on target complementarity       (138)

             Figure 2. crRNA complementarity to target DNA shifts Cas12a from transcriptional repression to DNA cleavage (139)

             Cas12a can be reprogrammed to repress endogenous Cas9 targets    (140)

             Figure 3. Cas12a can be reprogrammed to repress endogenous targets    (141)

Discussion     (143)

Materials and methods         (146)

Chapter 6. Discussion and Relevance (150)

BLP repression in F. novicida virulence       (150)

             Cas9 protects against combinatorial killing by host antimicrobials     (150)

             Connection between the bacterial envelope and sensitivity to ROS    (151)

             Implications of the ecological distribution of CRISPR-Cas9 systems for human health       (152)

Case to Broaden the Search for New CRISPR functions   (153)

             Considerations for the identification of RNAs that direct non-cleavage CRISPR-Cas9 functions  (153)

             crRNA-directed Cas9 and Cas12a functional versatility       (155)

             A need to re-analyze spacer targets    (156)

             Experimental evidence of non-cleavage and endogenous crRNA functions   (158)

             Evolution of non-cleavage functions  (159)

             Multifunctional CRISPR effectors in research and engineering      (160)

Conclusion    (161)

Appendix (163)

Appendix A.  (163)

               Figure: Cas9 protects against combinatorial killing by antimicrobial peptides and ROS   (163)

Appendix B.   (164)

               Figure: Virulence of Δcas9 in host antimicrobial knockout mice  (164)

Appendix C.   (165)

               Figure: BLP repression underlies Cas9-mediated resistance to H2O2      (165)

Appendix D.  (166)

             Figure: The absence of ROS production from the host (cyBB-/ ) does not alter signaling from WT or Δcas9 F. novicida to TLR2 (166)

Appendix E.   (167)

             Figure: Cas9 helps evade ROS stress during infection to promote dissemination through a mammalian host        (167)

Appendix F. (168)

             Figure: Self-targeting spacers result in the selection of a subtle target variation that alters Cas9 function from restriction to transcriptional repression         (168)

Appendix G.  (169)

Appendix H. Cas9-mediated targeting of viral RNA in Eukaryotic Cells     (172)

             Figure 1: FnCas9 can be reprogrammed to inhibit viral protein production in euk. cells      (187)

             Figure 2: FnCas9 targets and associates with HCV RNA (189)

             Figure 3: Molecular requirements for FnCas9 mediated HCV inhibition  (190)

             Figure 4: RNA sequence requirements for FnCas9 inhibition of HCV    (191)

             Figure 5: Cas9 can inhibit an established viral infection     (192)

References     (193)

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