Internally inlaid SaCas9 base editors enable window specific base editing

Abstract

Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary. Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N. Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.

Powerful Microbial Base-Editing Toolbox: From Optimization Strategies to Versatile Applications

“…The BE-PIGS system was invented by inserting deaminase (APOBEC1) into the PAM-interaction (PI) domain of the SpyCas9 protein (Figure C), therefore boosting an editing window ranging from the 2nd to 12th nucleotide . Besides, by inserting a cytosine deaminase into SaCas9 between amino acids 693 and 694 (Figure C), the obtained variant Sa-CBE-693 exhibited an PAM-adjacent editing window ranging from the 2nd to 18th nucleotide …”

“…41 Besides, by inserting a cytosine deaminase into SaCas9 between amino acids 693 and 694 (Figure 2C), the obtained variant Sa-CBE-693 exhibited an PAM-adjacent editing window ranging from the 2nd to 18th nucleotide. 42 Apart from being directly fused by linkers, a series of novel approaches to couple deaminases with Cas9 proteins have been developed. By fusing the deaminases with a specific molecule and modifying the sgRNA with matched binding sites, the deaminases could be recruited by the Cas domain through the noncovalent intermolecular interactions.…”

Application of CRISPR-Cas System to Mitigate Superbug Infections

“…YEE-BE2 and YEE-BE3 are two cytidine deaminase mutants that have been developed to improve DNA selectivity and minimize off-target editing. YEE-BE3, a SaCas9 mutant, is the most efficient at DNA editing in a two-nucleotide window width, compared with YEE-BE2 and other noncanonical PAMs [68][69][70].…”