Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis

Abstract

CRISPR–Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR–Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR–Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1–Cas2–prespacer complexes, and negative stained electron microscopy structure of the Cas4–Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1–Cas2–prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4–Cas1 and Cas1–Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.

DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism

“… 3 Notably, the above two CRISPR-Cas systems both lack Cas4. In contrast, in the three available Cas1-Cas2-prespacer structures of Cas4-containing CRISPR-Cas systems ( E. faecalis , 21 Synechocystis , 22 and Geobacter sulfurreducens 10 ), the Cas2 dimers were all oriented perpendicular to the axis of the prespacer ( Figure 2 D).…”

“…The overall architecture of the S. thermophilus Cas1-Cas2-prespacer complex was similar to that of previous Cas1-Cas2-prespacer structures. 3 , 10 , 21 , 22

Figure 2 Cryo-EM structure of the S. thermophilus Cas1-Cas2-prespacer complex (A) Schematic diagram of the prespacer DNA, including a 23-bp duplex and two 15-nt 3′ overhangs on both ends. The PAM-containing and PAM-deficient overhangs are colored red and blue, respectively.

…”

Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types

“… 41 For example, in type I-C and I-D, one Cas4 protein interacts with a Cas1 dimer. 14 , 48 Therefore, the spacer trimming effects might be specific to systems with unfused, stand-alone Cas4 proteins. However, even though the effects of spacer trimming by Cas4/1 from V-B was not observed in this study, we cannot exclude a role for Cas4/1 in prespacer processing, as observed in other Cas4/1 systems.…”

Unique properties of spacer acquisition by the type III-A CRISPR-Cas system

“…Cas1 and Cas2 interact to form an integrase complex ( 23 , 25 , 28 ) found to be essential for acquiring new spacers in all adaptation-competent CRISPR-Cas systems tested thus far ( 23 , 24 , 26–28 , 31 , 32 , 48 , 100 ). Many systems also require additional Cas proteins in order to adapt.…”