Project description:We report the PAMs of phylogenetically-diverse Cas12a nucleases using a cell-free TXTL-based PAM screen. By adding a 5N randomized PAM library and Cas12a-gRNA in vitro, recognized PAM sequences were cleaved, while non-recognized PAMs remained. By amplifying the non-cleaved DNA, we used next-generation sequencing to analyze the depletion of functional PAMs of these Cas12a nucleases.
Project description:The use of CRISPR/Cas proteins for the creation of multiplex genome-engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR/Cas9 system for multiplex genome-engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome-editing in Arabidopsis thaliana. We identified optimized promoter sequences for driving expression of single transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome-engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome-editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex-genome-engineering in plant-based systems.
Project description:In this work, we analyzed recognized PAM sequences from four CRISPR-Cas systems: E. coli I-E, B. halodurans I-C, S. thermophilus CR1 II-A, and F. novicida V. Cells containing functional PAMs were sorted using FACS and subsequently sequenced.
Project description:we characterized a novel compact Cas12a ortholog, EbCas12a, from the Erysipelotrichia bacterium with activities in mammalian cells. It is with the PAM sequence of 5’-TTTV-3’ (V=A, G, C) and the smallest size of ~3.47kb among reported Cas12a orthologs so far. Moreover, enhanced EbCas12a (enEbCas12a) was also developed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells. With the help of the compact enEbCas12a, all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo. Altogether, with the help of the novel smallest high fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
Project description:In this work, we analyzed recognized PAM sequences from four CRISPR-Cas systems: E. coli I-E, B. halodurans I-C, S. thermophilus CR1 II-A, and F. novicida V. Cells containing functional PAMs were sorted using FACS and subsequently sequenced. Ranking of recognized PAMs from a positive screen
