Optimized base editors enable efficient editing in cells, organoids and mice

Abstract

CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.

In situ CRISPR‐Cas9 base editing for the development of genetically engineered mouse models of breast cancer

“…Somatic base conversion for cancer modeling has previously been achieved in situ by hydrodynamic injection of plasmids encoding BE3 and an sgRNA in the mouse tail vein, which led to oncogenic C‐to‐T editing at the β‐catenin gene in the adult liver (Zafra et al , ). This approach is not applicable in the mammary gland, as we previously observed that de novo expression of Cas9 in adult mice elicits strong immune infiltration in this compartment, which could be circumvented by expressing the bacterial endonuclease from a conditional knock‐in allele (Annunziato et al , ).…”

“…Recently, base editors encoding alternative Cas9 orthologs or engineered SpCas9 variants that recognize a broader range of PAMs have been optimized (Hu et al , ; Nishimasu et al , ; Huang et al , ; Kleinstiver et al , ). In parallel, CBEs have been developed with reduced or expanded width of the editing window, to minimize bystander editing at non‐target cytosines or to enlarge the repertoire of targetable bases, respectively (Kim et al , ; Jiang et al , ; Zafra et al , ; Tan et al , ; Thuronyi et al , ). Finally, base editors that efficiently convert target cytosines to a mixture of the other three bases have also been established (Hess et al , ) and might be particularly appealing for localized sequence diversification and mutagenesis in vivo .…”

“…The sgRNAs were cloned as described (Sanjana et al , ) into Lenti‐U6‐tdTomato‐P2A‐BlasR vectors (Lenti‐sgRNA, Zafra et al , , Addgene plasmid # 110854) or pGIN backbones (Evers et al , ). All vectors were validated by Sanger sequencing.…”

“…For cloning of the Lenti‐sg Pik3ca E545K /sg Trp53 Q97* ‐ Myc tandem vector, a fragment encoding sg Pik3ca E545K was amplified by PCR from Lenti‐sg Pik3ca E545K ‐ Myc using XbaI‐containing primers and cloned in the XbaI digested backbone of Lenti‐sg Trp53 Q97* ‐ Myc . pLenti‐FNLS‐P2A‐Puro was a gift from Lukas Dow (Zafra et al , , Addgene plasmid # 110841). Concentrated stocks of VSV‐G pseudotyped lentivirus were produced by transient co‐transfection of four plasmids in 293T as previously described (Follenzi et al , ).…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…Naturally occurring loss-of-function mutations in the angiopoietin-like 3 (ANGPTL3) gene are associated with reduced blood triglycerides and low-density lipoprotein cholesterol. By employing the latest versions of BEs with improved activity [128,129], rather than the suboptimal BE3 in their study, it is conceivable that higher editing efficacy is likely to be achieved. Another study performed by the same research group targeted the Pcsk9 gene using a similar in vivo method in mice [127].…”