Improved genome editing by an engineered CRISPR-Cas12a

Abstract

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing

“…The recent discovery and development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), together known as the CRISPR/Cas system, has sparked a boom in the fields of genome editing and medical diagnosis. − Particularly, as a well-known member of the CRISPR/Cas family, CRISPR/Cas12a is a class 2 type V CRISPR-associated enzyme that binds to a single-stranded guide CRISPR RNA (crRNA). Compared with the other members of the CRISPR/Cas family, CRISPR/Cas12a has some unique characteristics. , It is able to cleave not only double-stranded DNA (dsDNA) with high sequence-specificity ( cis -cleavage activity) but also single-stranded DNA (ssDNA) in a sequence-independent manner ( trans -cleavage activity). ,− In view of the unique properties, numerous Cas12a-based biosensors have been developed for the detection of different targets. − Despite remarkable progress in recent years, − CRISPR/Cas12a-based biosensors still mainly target at nucleic acids and are rarely used in live cells. Scientists are curious about whether the CRISPR/Cas system can be deployed as a general live cell biosensing tool for a wide range of intracellular biomolecules and not just nucleic acid-associated targets.…”

Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA

“…Finally, as structure-guided mutants have improved the activity and fidelity of CRISPR-Cas-based nucleic acid detection and genome editing tools [51][52][53][54] , we expect that this work provides a framework for further development of SPARTA-based nucleic acid detection tools 12 . In conclusion, this study provides critical insights into the structural architecture of SPARTA systems and the molecular mechanisms that control the catalytic activation of their TIR domains.…”

CRISPR‐Cas Biochemistry and CRISPR‐Based Molecular Diagnostics

“…Yet, the improvement is not significant, and its activities are heavily spacer region‐dependent and sometimes lower than wild‐type LbCas12a. A recent study aiming to increase genome editing activities of LbCas12a resulted in three constructs with superior cis‐ and trans‐cleavage activities by combinations of rational mutagenesis and directed evolution [79] . On the other hand, to specifically improve nucleic acid detection, a 7‐mer DNA extension on the 3′ end of crRNA stabilizes the protein complex and increases the kinetics of detection [80] .…”