Paper of the Month March 2019

Engineering of CRISPR-Cas12b for human genome editing

Clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR-associated protein 9 (Cas9) has revolutionized the field of genome engineering. Discovered in bacteria, the CRISPR/Cas system serves as an adaptive defense system against foreign DNA, such as virus DNA. Numerous, highly diverse Cas-proteins have been described. Up until now, scientists have mainly been harnessing type II-a family members of Cas9 enzymes for genome editing in human cells. To increase the efficiency of intracellular delivery smaller Cas-variants are desired. Cas12b proteins are known to often be smaller than Cas9. However, the previously described Cas12b enzyme discovered from thermophilic bacterium only works at higher temperatures.

In this paper, Strecker and colleagues applied bioinformatic tools to identify new Cas12b family members, which could be active at lower temperatures, such as those found in the human body. They selected several promising candidates from the initial computational search and applied a combination of rational engineering strategies to generate a version of Cas12b capable of efficiently and specifically editing genomes. In detail:
As all known class II family members, the Cas12b enzymes requires a protospacer adjacent motif (PAM) for DNA cleavage. The PAM is a 2-6 base pair long DNA sequence immediately following the DNA sequence targeted by the Cas-protein. The sequence is mandatory for successful binding and cutting of DNA. The PAM motif does not exist in the bacterial genome and hence enables to distinguish bacterial self from non-self-DNA. To identify the optimal PAM for the Cas12b-variants, the authors generated a randomized PAM-library followed by the target-sequence. A DNA-library is a mixture of different DNA-sequences. The library contains all possible nucleotide combinations of a certain length, which might serve as a PAM for the Cas12b variants. If the combination serves as a PAM, the nuclease can cleave a DNA sequence, which will not be visible in a sequencing-based readout. The researchers were able to detect depleted PAMs for 4 enzymes and therefore, continued to evolve those nucleases further.
Another strategy to increase the efficiency of newly discovered Cas-variants is to optimize the scaffold of the trans-activation CRISP RNA (tracrRNA). The tracrRNA acts as a scaffold linking the CRISPR RNA (crRNA) to Cas-enzymes. The crRNA defines the genomic target for Cas-nucleases and is specific to the target site of interest. By altering the tracrRNA, the authors were able to increase the efficiency of the Cas-enzymes up to 30-fold.

Interestingly, the researchers observed that Cas12b preferentially nicks the non-target strand, whereas often a double-strand break is the preferred outcome. An approach to change the interaction between the enzymes and DNA-binding is to introduce amino-acids with a positive charge, which leads to a stronger binding to the negatively charged DNA. Additionally, the researchers changed different amino acids on the surface to increase protein flexibility and activity near the catalytic centrum. These procedures progressively led to a final Cas12b variant, Bacillus hisashii Cas12b, which showed efficient DNA cleavage activity and introduced DNA double-strand breaks at moderate temperature.

Besides high efficiency, nuclease specificity is required for genome editing. Specificity means that the nucleases cleave only the on-target (gene of interest), but not potential off-targets. Off-targets are loci in the genome that show sequence similarities to the on-target sequence. The authors addressed this question by using a method called GUIDE-seq. This technique relies on the integration of double-stranded oligodeoxynucleotides into a DNA double-strand break occurring at an off-target site. The integrated double-stranded oligodeoxynucleotide and therefore, the off-target, is detected by amplification and Next Generation Sequencing. No off-target integration was detected at the investigated sites for BhCas12b.  

The authors concluded that the high target specificity and the small size of BhCas12b makes this new Cas-variant a suitable tool for in vivo genome editing.