In the evolutionary arms race between phage and bacteria, mutations can allow phages to escape CRISPR-Cas-mediated destruction. However, bacteria are quick to acquire new spacer sequences which allow them to retarget phage mutants. Anti-CRISPR proteins are another defense mechanism for phages to escape destruction from CRISPR-Cas systems. Anti-CRISPR technology has applications in the enhancement of control and precision in gene editing with CRISPR/Cas systems and in augmenting phage therapy approaches to treat bacterial infections.
Anti-CRISPR proteins act by disrupting DNA binding or by inhibiting the cleavage of target sequences.The first anti-CRISPR proteins were identified in 2013 by a research group lead by Alan R. Davidson at University of Toronto . Anti-CRISPR proteins have been identified that target type I and type II CRISPR-Cas systems.Anti-CRISPR proteins that target the CRISPR-Cas9 system, commonly used for genome editing were identified in 2016 and 2017 by Davidson’s group and Joseph Bondy-Denomy’s group at University of California, San Francisco. In genome editing for therapeutic uses, anti-CRISPR proteins may provide a valuable “off switch” for better control of Cas9 activity. Anti-CRISPR proteins have been shown to reduce off-target cutting by CRISPR-Cas9 in human cells.
Anti-CRISPR proteins are discovered using bioinformatics, experimental and metagenomic screening. In addition to being found in phages, Anti-CRISPR proteins are also found in prophages. A prophage is a phage genome integrated into its host genome during what is called the lysogenic cycle. Anti-CRISPR proteins can also be encoded by non-phage elements, mobile genetic elements, including plasmids and integrative and conjugative elements, transposons, integrons and other uncharacterized elements. Researchers at University of California, San Francisco, discovered sequences of anti-CRISPR genes, aacrIIA, in E. faecalis, which frequently spreads antibiotic resistance genes despite having CRISPR-Cas systems. CRISPR-Cas systems are thought to provide a barrier to horozontal gene transfer. Their work demonstrated that AcrIIA proteins through CRISPR-Cas9 inhibition, can enhance the spread of antibiotic resistance plasmids that encode them.
Defense Advanced Research Projects Agency (DARPA) supported two research studies published in Science in 2018 that identified new anti-CRISPR proteins, including one that inhibits CRISPR-Cas12a, which is becoming more popular for genome editing applications. DARPA supported this research because of the potential for anti-CRISPR proteins to be used as a countermeasure against nefarious use of CRISPR.The two research groups are Jennifer Doudna’s at University of California, Berkeley and Bondy-Denomy’s group.Jennifer Doudna previously founded the company Caribou Biosciences which is developing various applications for CRISPR-Cas technology.
The former DuPont subsidiary DuPont Nutrition and Health, at their US and France locations, collaborated with researchers at Université Laval (Canada) in research on anti-CRISPR proteins. In 2018 Université Laval and Dupont Nutrition Bioscences applied for a patent on methods and compositions for use of anti-CRISPR (ACR) proteins in plants. DuPont Nutrition & Health merged with Industrial Biosciences to form DuPont Nutrition & Biosciences in 2019 to form the subsidiary, DuPont Nutrition Biosciences ApS based in Denmark.
