Role of nucleotide identity in effective CRISPR target escape mutations

Künne, Tim; Zhu, Yifan; Silva, Fausia da; Konstantinides, Nico; McKenzie, Rebecca E.; Jackson, Ryan N.; Brouns, Stan J. J.

doi:10.1093/nar/gky687

Cited by 11 publications

(7 citation statements)

References 73 publications

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“…region of the protospacer flanking the PAM; A>T at position +1; Figure 5A). The seed mutation in pTarget +1 compromises interference activity and promotes priming (13,26). Second, plasmid pCas4/1–2LRSR, containing cas4/1, cas2 and a minimal array (with leader , two repeats and a spacer), was used instead of pLRSR, and combined with pCas3-8-7-5/6.…”

Section: Resultsmentioning

confidence: 99%

Cas4–Cas1 fusions drive efficient PAM selection and control CRISPR adaptation

Almendros

Nóbrega

McKenzie

et al. 2019

Nucleic Acids Research

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Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR–Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.

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Section: Resultsmentioning

confidence: 99%

Cas4–Cas1 fusions drive efficient PAM selection and control CRISPR adaptation

Almendros

Nóbrega

McKenzie

et al. 2019

Nucleic Acids Research

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“…1c) suggests that spacers 1-5 are likely to be the more recently acquired spacers, while the remaining spacers 6-22 represent the core structure of the CRISPR arrays. We also note that several reads (3) contain CRISPR arrays with a loss of four spacers (14)(15)(16)(17), as shown in Fig. 1b, which is shown as a separating node providing an alternative route in the graph from node (10)(11)(12)(13)(14) to node (19)(20)(21)(22) in Fig.…”

Section: Spacer Graphs Provide Visual Summaries and Are Useful For Stmentioning

confidence: 92%

“…The diversity of CRISPR-Cas systems have been suggested to be attributed to the evolutionary arms-race between prokaryotes and their invaders [11][12][13]. Similarly to the evolutionary diversity of CRISPR-Cas systems, invaders such as phages have also been observed to evolve in tandem to evade host defense mechanisms, such as anti-CRISPR genes which are among some of the recently discovered mechanisms [1,2,[14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Long reads reveal the diversification and dynamics of CRISPR reservoir in microbiomes

Lam

2019

BMC Genomics

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Background: Sequencing of microbiomes has accelerated the characterization of the diversity of CRISPR-Cas immune systems. However, the utilization of next generation short read sequences for the characterization of CRISPR-Cas dynamics remains limited due to the repetitive nature of CRISPR arrays. CRISPR arrays are comprised of short spacer segments (derived from invaders' genomes) interspaced between flanking repeat sequences. The repetitive structure of CRISPR arrays poses a computational challenge for the accurate assembly of CRISPR arrays from short reads. In this paper we evaluate the use of long read sequences for the analysis of CRISPR-Cas system dynamics in microbiomes. Results: We analyzed a dataset of Illumina's TruSeq Synthetic Long-Reads (SLR) derived from a gut microbiome. We showed that long reads captured CRISPR spacers at a high degree of redundancy, which highlights the spacer conservation of spacer sharing CRISPR variants, enabling the study of CRISPR array dynamics in ways difficult to achieve though short read sequences. We introduce compressed spacer graphs, a visual abstraction of spacer sharing CRISPR arrays, to provide a simplified view of complex organizational structures present within CRISPR array dynamics. Utilizing compressed spacer graphs, several key defining characteristics of CRISPR-Cas system dynamics were observed including spacer acquisition and loss events, conservation of the trailer end spacers, and CRISPR arrays' directionality (transcription orientation). Other result highlights include the observation of intense array contraction and expansion events, and reconstruction of a full-length genome for a potential invader (Faecalibacterium phage) based on identified spacers. Conclusion: We demonstrate in an in silico system that long reads provide the necessary context for characterizing the organization of CRISPR arrays in a microbiome, and reveal dynamic and evolutionary features of CRISPR-Cas systems in a microbial population.

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“…2b). This discrepancy can be due to the different nucleotide sequences used, as the spacer sequence and the identity of the mismatch can influence the sensitivity to the mismatch 24,25 . Additionally, the differences could be explained by the measured parameters; that is, cleavage rate in vitro and end-point for bacterial survival.…”

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confidence: 99%

Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches

et al. 2020

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C as9 is an RNA-guided endonuclease that specifically binds and cleaves DNA with complementarity to the spacer sequence of CRISPR RNA (crRNA) located upstream of a protospacer adjacent motif (PAM) 1,2. Sufficient base pairing of the crRNA spacer of either dual-RNA (trans-activating crRNA (tracrRNA) in complex with crRNA) or single-guide RNA (sgRNA, tracrRNA fused to crRNA) with the target DNA strand leads to displacement of the nontarget strand and R-loop formation 1-5. On completion of this process, Cas9 endonuclease domains, HNH and RuvC, cleave the target and nontarget strand, respectively, resulting in a double-strand DNA break 1,2. Due to its programmability, CRISPR-Cas9 was adapted for a wide variety of genome engineering approaches 6-9. However, a main concern for the application of the CRISPR-Cas9 technology is off-target cleavage that occurs when Cas9 cleaves mismatched targets; that is, targets with imperfect complementarity to the dual-RNA/sgRNA spacer. It is therefore crucial to identify regions and amino acids that determine Cas9 specificity. Streptococcus pyogenes Cas9 (SpCas9) requires a seed sequence of 10-12 base pairs (bp) adjacent to the PAM for successful cleavage 2. The REC3 domain senses PAM-distal mismatches and controls the transition of the HNH domain into an active conformation, which serves as a mechanism to block the cleavage in the presence of mismatches 10,11. Alongside other strategies to reduce off-target cleavage 12 , both structure-guided and random mutagenesis have been used to generate enhanced specificity Cas9 variants 10,13-19. Three of the improved variants 10,15,17 contain mutations that increase the energetic barrier for transition of the HNH domain to the cleavage-competent state 10. eSpCas9 (ref. 17) and SpCas9-HF1 (ref. 15) require one additional base pair between crRNA and target DNA for stable binding, which makes target unwinding more sensitive to mismatches and slows cleavage in the presence of mismatches 20. It was shown that mutation of two residues in the loop region of the bridge helix impairs target cleavage and improves specificity 21. The conformational checkpoint through the HNH domain before cleavage and the seed sequence are the only known specificity determinants for Cas9. In addition, stable R-loop formation is an essential prerequisite for cleavage 5 , but residues involved in this process have not been identified. Therefore, we investigated whether other Cas9 domains are involved in mismatch sensing and R-loop stabilization focusing on the PAM-adjacent region of the target. We show that arginines of the Cas9 bridge helix influence guide RNA binding, and target DNA binding and cleavage. We identify two groups of arginines with opposite effects on Cas9 mismatch sensitivity. We demonstrate that R63, R66 and R70 stabilize the R-loop in the presence of PAM-adjacent mismatches thus reducing the Cas9 specificity. Additionally, we observe that Q768 mediates Cas9 insensitivity to a mismatch in target position 15 and is involved in sensitivity to PAM-distal mism...

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Role of nucleotide identity in effective CRISPR target escape mutations

Cited by 11 publications

References 73 publications

Cas4–Cas1 fusions drive efficient PAM selection and control CRISPR adaptation

Cas4–Cas1 fusions drive efficient PAM selection and control CRISPR adaptation

Long reads reveal the diversification and dynamics of CRISPR reservoir in microbiomes

Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches

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