CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.

Conventional transformation induced plasticity (TRIP) steel (0.17C-1.52Si-1.61Mn-0.03Al, wt%) was produced via strip casting technology simulated in the laboratory. Effects of holding temperature, holding time and cooling rate on ferrite formation were studied via analysis of the continuous cooling transformation diagram obtained here. A typical microstructure for conventional TRIP steels consisting of ~ 0.55 fraction of polygonal ferrite with bainite, retained austenite and martensite was obtained. However, coarse prior austenite grain size of ~80 μm led to large polygonal ferrite grain size of ~17 μm, coarse second phase regions of ~21 μm size, small amount of retained austenite (0.02-0.045) and the presence of Widmanstätten ferrite. Optimisation of the microstructure-property relationship was reached via a variation in the isothermal bainite transformation temperature. The highest retained austenite fraction of 0.045±0.003 with medium carbon content of 1.23±0.01 wt% was obtained after holding at 400 °C, resulting in the highest ultimate tensile strength of 590±35 MPa and largest total elongation of 0.27±0.05. The presence of TRIP effect in the studied steel was revealed through the analysis of strain hardening exponent and modified Crussard-Jaoul model. Effect of processing parameters on retained austenite retention and stress-strain behaviour was discussed. Conventional transformation induced plasticity (TRIP) steel (0.17C-1.52Si-1.61Mn-0.03Al, wt. %) was produced via strip casting technology simulated in the laboratory. Effects of holding temperature, holding time and cooling rate on ferrite formation were studied via analysis of the continuous cooling transformation diagram obtained here. A typical microstructure for conventional TRIP steels consisting of ~ 0.55 fraction of polygonal ferrite with bainite, retained austenite and martensite was obtained. However, coarse prior austenite grain size of ~ 80 μm led to large polygonal ferrite grain size of ~ 17 μm, coarse second phase regions of ~ 21 μm size, small amount of retained austenite (0.02 -0.045) and the presence of Widmanstätten ferrite. Optimisation of the microstructure-property relationship was reached via a variation in the isothermal bainite transformation temperature. The highest retained austenite fraction of 0.045±0.003 with medium carbon content of 1.23±0.01 wt. % was obtained after holding at 400 °C, resulting in the highest ultimate tensile strength of 590±35 MPa and largest total elongation of 0.27±0.05. The presence of TRIP effect in the studied steel was revealed through the analysis of strain hardening exponent and modified Crussard -Jaoul model. Effect of processing parameters on retained austenite retention and stress-strain behaviour was discussed.

Conventional dual phase (DP) steel (0.08C-0.81Si-1.47Mn-0.03Al wt.%) was manufactured using simulated strip casting schedule in laboratory. The average grain size of prior austenite was 117. ±. 44. μm. The continuous cooling transformation diagram was obtained. The microstructures having polygonal ferrite in the range of 40-90%, martensite with small amount of bainite and Widmanstätten ferrite were observed, leading to an ultimate tensile strength in the range of 461-623. MPa and a corresponding total elongation in the range of 0.31-0.10. All samples exhibited three strain hardening stages. The predominant fracture mode of the studied steel was ductile, with the presence of some isolated cleavage facets, the number of which increased with an increase in martensite fraction. Compared to those of hot rolled DP steels, yield strength and ultimate tensile strength are lower due to large ferrite grain size, coarse martensite area and Widmanstätten ferrite. Abstract: Conventional dual phase (DP) steel (0.08C-0.81Si-1.47Mn-0.03Al wt. %) was manufactured using simulated strip casting schedule in laboratory. The average grain size of prior austenite was 117±44 μm. The continuous cooling transformation diagram was obtained. The microstructures having polygonal ferrite in the range of 40-90 %, martensite with small amount of bainite and Widmanstätten ferrite were observed, leading to an ultimate tensile strength in the range of 461 -623 MPa and a corresponding total elongation in the range of 0.31 -0.10. All samples exhibited three strain hardening stages. The predominant fracture mode of the studied steel was ductile, with the presence of some isolated cleavage facets, the number of which increased with an increase in martensite fraction. Compared to those of hot rolled DP steels, yield strength and ultimate tensile strength are lower due to large ferrite grain size, coarse martensite area and Widmanstätten ferrite. Disciplines Engineering | Science and Technology Studies

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