Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system.

Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for modulating CRISPR-based applications. However, characterizing novel anti-CRISPR proteins and exploiting Acr-related technologies is a rather long and tedious process. Here, we established a versatile plasmid interference with CRISPR interference (PICI) system in Escherichia coli for rapidly characterizing Acrs and developing Acr-based technologies. Utilizing the PICI system, we discovered two novel type II-A Acrs (AcrIIA33 and AcrIIA34), which can inhibit the activity of SpyCas9 by affecting DNA recognition of Cas9. We further constructed a circularly permuted AcrIIA4 (cpA4) protein and developed optogenetically engineered, robust AcrIIA4 (OPERA4) variants by combining cpA4 with the light-oxygen-voltage 2 (LOV2) blue light sensory domain. OPERA4 variants are robust light-dependent tools for controlling the activity of SpyCas9 by approximately 1000-fold change under switching dark-light conditions in prokaryotes. OPERA4 variants can achieve potent light-controllable genome editing in human cells as well. Together, our work provides a versatile screening system for characterizing Acrs and developing the Acr-based controllable tools.

Discovery of potent and versatile CRISPR-Cas9 inhibitors engineered for chemically controllable genome editing.

Anti-CRISPR (Acr) proteins are encoded by many mobile genetic elements (MGEs) such as phages and plasmids to combat CRISPR-Cas adaptive immune systems employed by prokaryotes, which provide powerful tools for CRISPR-Cas-based applications. Here, we discovered nine distinct type II-A anti-CRISPR (AcrIIA24-32) families from Streptococcus MGEs and found that most Acrs can potently inhibit type II-A Cas9 orthologs from Streptococcus (SpyCas9, St1Cas9 or St3Cas9) in bacterial and human cells. Among these Acrs, AcrIIA26, AcrIIA27, AcrIIA30 and AcrIIA31 are able to block Cas9 binding to DNA, while AcrIIA24 abrogates DNA cleavage by Cas9. Notably, AcrIIA25.1 and AcrIIA32.1 can inhibit both DNA binding and DNA cleavage activities of SpyCas9, exhibiting unique anti-CRISPR characteristics. Importantly, we developed several chemically inducible anti-CRISPR variants based on AcrIIA25.1 and AcrIIA32.1 by comprising hybrids of Acr protein and the 4-hydroxytamoxifen-responsive intein, which enabled post-translational control of CRISPR-Cas9-mediated genome editing in human cells. Taken together, our work expands the diversity of type II-A anti-CRISPR families and the toolbox of Acr proteins for the chemically inducible control of Cas9-based applications.

Anti-CRISPRs: The natural inhibitors for CRISPR-Cas systems.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene-editing tools. As the natural inhibitors of CRISPR-Cas systems, anti-CRISPRs (Acrs) can be used as the "off-switch" for CRISPR-Cas systems to limit the off-target effects caused by Cas9. Since the discovery of CRISPR-Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.

AcrIIA5 Inhibits a Broad Range of Cas9 Orthologs by Preventing DNA Target Cleavage.

CRISPR-Cas9 is an adaptive immune system for prokaryotes to defend against invasive genetic elements such as phages and has been used as a powerful tool for genome editing and modulation. To overcome CRISPR immunity, phages encode anti-CRISPR proteins (Acrs) to inhibit Cas9, providing an efficient "off-switch" tool for Cas9-based applications. Here, we characterized AcrIIA5, which is a Cas9 inhibitor discovered in a virulent phage of Streptococcus thermophilus. We found that AcrIIA5 is a potent and broad-spectrum inhibitor of CRISPR-Cas9, which can inhibit diverse Cas9 orthologs of type II-A, type II-B, and type II-C. AcrIIA5 inhibits Cas9 by preventing DNA target cleavage, but DNA target binding of Cas9 is unaffected. Importantly, it can affect the activity of the RuvC nuclease domain of Cas9 independent of the HNH nuclease domain. Our work expands the diversity of the inhibitory mechanisms used by Acrs and provides the guidance for developing controlling tools in Cas9-based applications.

An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.