Albert Cheng, PhD 郑瑚博士
Professor
Institute of Zoology, CAS
研究员
中国科学院动物研究所
Preprints
Publication Types:
Targeted directional kilobase sequence insertion by combining prime editing with recombinases or integrases
Targeted insertion of exogenous sequences to genomes is useful for therapeutics and biological research. While CRISPR/Cas technologies have been very efficient in gene knockouts by double-strand breaks (DSBs) followed by indel formation through non-homologous end-joining (NHEJ) repair pathway, the precise introduction of new sequences mainly rely on inefficient homology directed repair (HDR) pathways following Cas9-induced DSBs and are restricted to dividing cells. The recent invention of Prime Editing allows short sequences to be precisely inserted at target sites without DSBs. Here, we combine Prime Editing and sequence-specific recombinases and integrases to insert kilobase sequences directionally at target sites. This technique, called PRIMAS for Prime editing, Recombinase, Integrase-mediated Addition of Sequence, will expand our genome editing toolbox for targeted insertion of long sequences up to kilobases and beyond.
Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold
RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3′ end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. We show that CREST is capable of simultaneously manipulating RNA alternative splicing and A-to-G or C-to-U base editing. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce more than 90% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology and the development of RNA-focused therapeutics.
Local recruitment of DNA repair proteins enhances CRISPR-ssODN-HDR editing
CRISPR-Cas technologies enable precise editing of genomic sequences. One major way to introduce precise editing is through homology directed repair (HDR) of DNA double strand breaks (DSB) templated by exogenously supplied single-stranded oligodeoxyribonucleotides (ssODN). Competing pathways determine the outcome of edits. Non-homologous end-joining pathways produce destructive insertions/deletions (indels) at target sites and are dominant over the precise homology directed repair pathways. In this study, we aim to favor HDR and use two strategies to recruit DNA repair proteins (DRPs) to Cas9 cut site, the Casilio-DRP approach that recruits RNA-binding protein-tethered DRPs to target site via aptamers appended to guide RNA; and the 53BP1-DRP approach that recruits DRPs to DSBs via DSB-sensing activity of 53BP1. We conducted two screens using these approaches and identified DRPs such as FANCF and BRCA1 that when recruited to Cas9 cut site, enhance ssODN-templated HDR and increase the proportion of precise edits. This study provides not only new constructs for enhanced CRISPR-ssODN-HDR but also a collection of DRP fusions for studying DNA repair processes.