Viewing a Cascade — The Bailey Lab

CRISPR systems are diverse, and different Types use different complexes to identify and destroy target nucleic acid sequences. The wide-spread (in nature) Type I CRISPR systems use a multi-subunit complex of proteins and crRNA called Cascade (CRISPR-associated complex for antiviral defense) to find and identify DNA targets that match the crRNA sequence, before recruiting Cas3 protein to do the cutting.

In 2014, three labs completed structures of different versions of the Cascade complex, in three papers published at the same time – including one led by Sabin Mulepati, a then-PhD student in the Bailey lab.

Sabin used x-ray crystallography to determine the structure of Cascade bound to a single strand of target DNA that matched the Cascade crRNA. He was able to determine the structure to a resolution of 3 angstroms.

The complex, shown below with color-coded subunits, looks like a top-heavy seahorse – a fat head curving into the body, with a stubby tail. The guide crRNA and single strand of target DNA spirals around from top to tail in one wide loop.

2 views of Cascade model, like a top-heavy seahorse, a fat head curving into a body with stubby tail. Green crRNA & orange ssDNA spiral down in wide loop; backbone is blue Cas7 subunits. Topped by pink Cas6e; tail is a purple Cse1 and red Cas5.

Sabin’s high-resolution structure showed that Cascade aligns the 32-base-long matching sections of the guide crRNA and target DNA not by coiling them together like a double stranded DNA helix, but by holding them alongside each other like two sides of flat ladder. The normally coiled strands are flattened by adding kinks - every sixth base gets a quarter twist, pointing away from the “rungs” of the “ladder.” This explains why most of the bases – the “rungs” – need to match for the target to be cut, but the sixth ones can sometimes vary, in either the seed sequence at the start of the crRNA or throughout the sequence.

The image below has two views of the crRNA and ssDNA structure - to the left, a side view, showing where the two strands have 5-bp segments of binding, separated by 1-bp gaps. To the right, a top-down view shows the two lined up in a scalloped circle - asterisks where the scalloped edges pinch in indicate the location of the flipped out bases.

2 views of crRNA-ssDNA. Left, side view: loose spiral with short segments of base pairing, with small 1-base gaps between. Right, top-down view: strands line up in a scalloped curve, with flipped out bases where the scallops pinch in.

The guide crRNA is held in place by six repeating Cas7 proteins, that span the head and body of the seahorse shape. The Cas7 subunits are hand-shaped, with a palm at the center, a thumb, and fingers. The crRNA strand lays along these subunits with the thumbs curled over the twisted sixth base. The thumbs also overlap the target DNA strand at each twisted base, sandwiching the strand against Cse1 and Cse2, two proteins across the seahorse shape’s belly and tail.

Sabin used a single stranded DNA target for the structure because that combination produced better crystals than Cascade with double stranded DNA targets. So to get clues to how Cascade would look bound to its normal target of double stranded DNA, he modeled the interactions, based on his structure and a cryo-EM structure that was lower resolution but used double stranded DNA. This modeled Cascade bound to double stranded target DNA revealed some likely sites for Cas3 binding. Sabin also compared his structure to a structure of apo Cascade, not bound to a target. He observed that in his structure, the Cse1 and Cse2 subunits were shifted, in a way that may lock the DNA strand into the Cascade complex and positioning the non-target DNA strand so it can be cut.

The other two papers published at the same time, Jackson et al. and Zhao et al., solved the apo structures of Cascade – complete with a crRNA but without a DNA target. They also showed the crRNA with every sixth base flipped out.

To learn more about the details of Sabin’s structure, check out the paper.

The work was funded by the NIH’s National Institutes of General Medical Sciences.

Want to learn more about the work this paper builds on? Check out these papers:

Other Cascade structures published at the same time

Jackson RN, Golden SM, van Erp PB, Carter J, Westra ER, Brouns SJ, van der Oost J, Terwilliger TC, Read RJ, Wiedenheft B. Structural biology. Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science. 2014 Sep 19;345(6203):1473-9. doi: 10.1126/science.1256328. Epub 2014 Aug 7. PMID: 25103409; PMCID: PMC4188430.

Zhao H, Sheng G, Wang J, Wang M, Bunkoczi G, Gong W, Wei Z, Wang Y. Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli. Nature. 2014 Nov 6;515(7525):147-50. doi: 10.1038/nature13733. Epub 2014 Aug 12. PMID: 25118175.

Perspective in Science

Zhang Y, Sontheimer EJ. Structural biology. Cascading into focus. Science. 2014 Sep 19;345(6203):1452-3. doi: 10.1126/science.1260026. PMID: 25237089.

Previous Cascade structures

Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Zhou K, Snijders AP, Dickman MJ, Doudna JA, Boekema EJ, Heck AJ, van der Oost J, Brouns SJ. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol. 2011 May;18(5):529-36. doi: 10.1038/nsmb.2019.

Wiedenheft B, Lander GC, Zhou K, Jore MM, Brouns SJJ, van der Oost J, Doudna JA, Nogales E. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature. 2011 Sep 21;477(7365):486-489. doi: 10.1038/nature10402.

Hochstrasser ML, Taylor DW, Bhat P, Guegler CK, Sternberg SH, Nogales E, Doudna JA. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference. Proc Natl Acad Sci USA. 2014 May 6;111(18):6618-23. doi: 10.1073/pnas.1405079111.

Tolerance of mismatches at certain base positions

Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10098-103. doi: 10.1073/pnas.1104144108.

Fineran PC, Gerritzen MJ, Suárez-Diez M, Künne T, Boekhorst J, van Hijum SA, Staals RH, Brouns SJ. Degenerate target sites mediate rapid primed CRISPR adaptation. Proc Natl Acad Sci USA. 2014 Apr 22;111(16):E1629-38. doi: 10.1073/pnas.1400071111.