Engineering of CRISPR-Cas12b for human genome editing

Abstract

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

A Survey of Genome Editing Activity for 16 Cas12a Orthologs

“…[11][12][13][14] Finally, Cas12a is more specific than Cas9 in certain contexts, making it well-suited to applications in which high specificity is critical. 15,16 Given previous work showing that different Cas enzyme orthologs exhibit a range of activity in eukaryotic cells 2,16,17 and indicating the potential advantages of Ca-s12a, we sought to identify additional Cas12a orthologs with high activity in eukaryotic cells. Here we examine 16 new Cas12a-family proteins for nuclease activity in human cells.…”

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

“…6) . It is also interesting to note that several proteins in the Cas12 family have strong nickase activities against target DNA, but can only weakly linearize dsDNA targets (Yan et al 2019;Strecker et al 2019).…”

CRISPR-Cas nucleases and base editors for plant genome editing

“…Cas12b (formerly C2c1) is a Class II Type V-B endonuclease that targets double-stranded DNA (Strecker et al 2019). Cas12b specifically cuts DNA with its single RuvC domain and produces staggered ends with * 7-nt overhangs (Wu et al 2017;Liu et al 2017).…”

“…Teng et al (2018) identified a Cas12b ortholog from Alicylobacillus acidiphilus (AaCas12b) that kept its nuclease activity between the temperatures of 31°C and 59°C, enabling genome editing in mammalian cell cultures. Recently, a Cas12b ortholog from Bacillus hisashii (BhCas12b) was characterized in detail and its engineered mutant, BhCas12b v4 was tested for cleavage activity in human cells under lower temperatures than the wild-type form (Strecker et al 2019). Artificial crRNAs have also been successfully engineered to work with multiple Cas12b orthologs for genome editing in human cells (Teng et al 2019).…”