Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System

<u>C</u>lustered <u>R</u>egularly <u>I</u>nterspaced <u>S</u>hort <u>P</u>alindromic <u>R</u>epeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9

Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition

<div><p>Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from <i>Staphylococcus epidermidis</i> has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from <i>Escherichia coli</i> we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.</p></div

Synonymous mutations of the crRNA and the PAM do not affect self versus non-self discrimination.

<p><b>A</b>) Infectivity of a library of M13 phage containing PAM mutants adjacent to the g8 protospacer was tested against cells expressing Cascade, Cas3 and g8 CRISPR containing mutations at the −1, −2 and −3 nucleotides of the CRISPR repeats. PAM mutations are shown on the left, with the PAM sequences indicated in the 3′ to 5′ direction. CRISPR repeat mutations at positions −1, −2 and −3 are indicated on the top in the 5′ to 3′ direction. Underscored sequences have been tested for binding affinity by EMSA. Base pairing potential between the PAM positions and the repeat is indicated using numbers (0–7) that correspond to a base pairing pattern that is shown in the panel on the right. A 0 signifies no base pairing, a 1 signifies base pairing at the −3 position, a 2 signifies base pairing at the −2 position, etc. Black circles with white digits indicate resistance against phage infection (e.o.p.<10⁻⁴), grey circles indicate partial resistance (e.o.p.∼10⁻²) and red digits without circle indicate susceptibility to phage infection (e.o.p. = 1), as determined by phage spot assays. Letters B, C, D, E indicate combinations shown in detail in the corresponding panels. <b>B</b>) Combination of g8^C-3AC-2T CRISPR and M13 phage with CAT PAM, gives rise to full base pairing and a lack of resistance (red font). <b>C</b>) Combination of g8^C-3AC-2T CRISPR and M13 phage with CTC PAM, gives rise to only base pairing at the −1 position and yields a lack of resistance (red font). <b>D</b>) Combination of g8^C-3AC-2A CRISPR and M13 phage with CTT PAM gives rise to full base pairing and a lack of resistance (red font). <b>E</b>) Combination of g8^C-3AC-2A CRISPR and M13 phage with CTC PAM gives rise to only two potential base pairs at the −1 and −2 positions and yields a lack of resistance (red font). Note in (A–E) PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat.</p

Base pairing at the −2 and −3 positions does not interfere with CRISPR-immunity.

<p><b>A</b>) Cells expressing g8^C-2A-Cascade and Cas3 are partially resistant to M13 phage containing the CTT PAM adjacent to the g8 protospacer (white font/grey bar, e.o.p.∼10⁻²), but not when containing the CAT, CCT, CTC or CGT PAM (red font/red bars). Note that in the figure the PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat. The <i>in vitro</i> binding affinity of purified WT g8^C-2A-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. <b>B</b>) Assays as in (A) using cells expressing the g8^C-3G CRISPR, Cascade and Cas3, show that cells are not resistant to M13 phage containing the CAT, CTT, CCT, CTC or CGT PAMs adjacent to the g8 protospacer (red font/red bars). The <i>in vitro</i> binding affinity of purified WT g8^C-3G-Cascade for dsDNA containing the M13 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. Asterisks indicate that the Kd value is >>1000 nM and the error bars represent the standard deviation of the mean.</p

Potential base pairing between the crRNA repeat regions and protospacer flanking regions does not affect CRISPR-interference.

<p><b>A</b>) Model of the R-loop formed by Cascade during dsDNA binding. <b>B</b>) Cells expressing WT g8-Cascade and Cas3 are resistant to plasmids containing the CAT PAM adjacent to the g8 protospacer (black bars, transformation efficiency 6.7±1.5×10⁵ cfu/µg DNA for plasmid pWUR690 and 6.8±0.9×10⁵ cfu/µg DNA for plasmid pWUR688), but are susceptible to plasmid transformation when the g8 protospacer is flanked by a CGG PAM, which is fully complementary to the 5′-handle (red bars, transformation efficiency 4.2±0.9×10⁸ cfu/µg DNA for plasmid pWUR687 and 4.5±0.8×10⁸ cfu/µg DNA for plasmid pWUR689). Transformation efficiency for a control pUC19 plasmid is 6.2±1.1×10⁸ cfu/µg DNA. The histogram shows the <i>in vitro</i> binding affinity of purified WT g8-Cascade for dsDNA containing the g8 protospacer flanked by sequences with a varying base pairing potential, as shown on the right. Asterisks indicate that the Kd value is >>1000 nM and the error bars represent the standard deviation of the mean.</p

Model of self versus non-self discrimination by Type III-A systems and target versus non-target discrimination by Type I-E systems.

<p><b>A</b>) Model of the mechanism employed by Type III-A systems. Type III-A systems presumably target DNA through R-loop formation. The crRNA (here depicted as the 37 nt species) could be part of a ribonucleoprotein complex consisting of Cas proteins and a single crRNA (Csm complex). R-loop formation and subsequent interference is inhibited when base pairing occurs between the −2, −3 and −4 positions of the repeat sequence at the 5′ end of the crRNA and the sequence flanking the 3′ end of the protospacer. How this base pairing is monitored is currently unknown, but it might involve a Cas protein. <b>B</b>) Model of target versus non-target discrimination by Type I-E systems. Type I-E systems target DNA through R-loop formation. The 61 nt crRNA is part of the Cascade ribonucleoprotein complex. R-loop formation and subsequent Cas3-mediated cleavage of the target DNA are activated when a PAM is present at the −1, −2 and −3 positions, in the sequence flanking the 3′ end of the protospacer. The presence of a PAM is monitored through the Cse1 subunit.</p

Base pairing at the −1 position is not required for CRISPR-interference.

<p><b>A</b>) Model of the R-loop formed by Cascade during dsDNA binding. The nucleotide adjacent to the spacer sequence (the −1 position) has the potential to base pair with the first nucleotide of the PAM in the target strand of the DNA. <b>B</b>) Cells expressing WT g8-Cascade and Cas3 are resistant to M13 phage containing the CAT, CTT, CCT or CTC PAMs adjacent to the M13 protospacer (white font/black bars, e.o.p.<10⁻⁴), but not when containing the CGT PAM (red font/red bars, e.o.p. = 1). Note that in the figure the PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat. The <i>in vitro</i> binding affinity of purified WT g8-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. <b>C</b>) Assays as in (B) using cells expressing the g8^G-1T CRISPR, Cascade and Cas3, show that cells are resistant to M13 phage containing the CAT, CTT, CCT or CTC PAMs adjacent to the g8 protospacer (white font/black bars), but not when containing the CGT PAM (red font/red bars). The <i>in vitro</i> binding affinity of purified WT g8^G-1T-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. In (B) and (C) error bars represent the standard deviation of the mean.</p

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In <i>Escherichia coli</i>, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions <i>in vivo</i> and <i>in vitro.</i> Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine

Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In <i>Escherichia coli</i>, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions <i>in vivo</i> and <i>in vitro.</i> Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine