Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Abstract

Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two novel strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing lead to increased levels of by-products, it rescued pegRNAs that performed poorly with a nickase-based prime editor. We also present a small molecule approach that yielded increased product purity of PEn editing. Next, we developed a homology-independent PEn editing strategy by engineering a single primed insertion gRNA (springRNA) which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

Pendekatan Berbasis Genomik Dalam Mengatasi Permasalahan Abnormalitas Klon Kelapa Sawit

“…Perbaikan yang terjadi akan menyebabkan insersi atau delesi (indel) secara acak, yang dapat mengakibatkan mutasi karena pergeseran pembacaan open reading frame (orf) pada wilayah pengkodean gen. Indel yang terjadi secara efektif menciptakan gen knock out yang mengakibatkan gen menjadi tidak terekspresi atau memiliki ekspresi lain dibanding tipe liarnya (Bortesi dan Fischer, 2015). Berbeda dengan perbaikan kerusakan DSB yang menggunakan mekanisme HDR, ketika templat homolog tersedia di wilayah sekitar DSB, mekanisme ini tidak menghasilkan indel sehingga dapat dimanfaatkan untuk penyisipan gen dengan sifat baru (gen knock in) (Peterka et al, 2022).…”

Prime editing in hematopoietic stem cells—From ex vivo to in vivo CRISPR-based treatment of blood disorders

“…Frontiers in Genome Editing frontiersin.org Kweon et al, 2021;Nelson et al, 2021;Liu N. et al, 2022;Happi Mbakam et al, 2022;Hong et al, 2022;Kweon et al, 2022;Levesque et al, 2022;Peterka et al, 2022;Schene et al, 2022;Simon et al, 2022;Tao et al, 2022;Velimirovic et al, 2022;Wang et al, 2022;Zhang et al, 2022;Zhuang et al, 2022). We and others have previously utilized plasmid-based prime editors allowing stable integration of the system into the genome of human cells using piggyBac transposase, taking advantage of the prolonged expression to achieve highly effective prime editing (Eggenschwiler et al, 2021;Wolff et al, 2021).…”