De novo PAM generation to reach initially inaccessible target sites for base editing

ABSTRACT Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed ‘inception’ that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.

Highlighted nucleotide changes represented as mean ± standard deviation.

Fig. S4 .
Fig. S4.Range of possible codon outcomes after base editing in kcnh6a via inception Expected range of all potential translated codon outcomes after inception editing using the ABE8e base editor at the kcnh6a locus.The anticipated de novo PAM (green outlined box) introduced by kcnh6a-step1 guide RNA and K506R/T507A edits as well as the kcnh6a-step2-adjusted guide RNA induced I502V mutations are indicated by black arrows.Resulting codon range per amino acid position given.AA pos, amino acid position; canonical PAM (orange); de novo PAM (green); PAM, protospacer adjacent motif; WT, wild-type

Fig. S5 .
Fig. S5.No inception editing events using wild-type kcnh6a-step2 guide RNA (A) Inception mix containing the kcnh6a-step2-wt guide RNA fails to edit p.I502 despite the efficient de novo PAM generation validated via Sanger sequencing and EditR (Kluesner et al., 2018).The 96 % c.1519A>G editing is in position 1 of the kcnh6a-step2-wt target-site preventing the second editing event.(B) At 4 days post fertilization the two-chambered embryonic heart shows Diastole/Systole periodicity.Phenotypically the inception editants were indifferent from wild-type (WT) controls.AA pos, amino acid position; canonical PAM (orange); de novo PAM (green); red box, sequence difference to edited locus in kcnh6a-step2-wt guide RNA

Fig. S8 .
Fig. S8.Range of possible codon outcomes after base editing in rx3 via inception Expected range of all potential translated codon outcomes after inception editing using the ABE8e base editor at the rx3 locus.The anticipated de novo PAM (green outlined box) introduced by rx3-step1 guide RNA and splice acceptor edits (ΔSA) as well as the rx3-step2-adjusted guide RNA induced A84A mutations are indicated by black arrows.Resulting codon range per amino acid position given.Further possible edits are indicated by white arrows.AA pos, amino acid position; canonical PAM (orange); de novo PAM (green); PAM, protospacer adjacent motif; SA, splice acceptor; WT, wild-type

SA
: Supplementary information

Allele frequency table and translation of aligned targeted Illumina amplicon sequencing reads of oca2 inception and control editants
Development: doi:10.1242/dev.201115:Supplementary information Development • Supplementary information Fig. S3.Canonical (step 1) and inception (step 2) guide RNA target sites indicated.Analysis based on the Allele frequency table output files derived from CRISPResso2 tool (Clement et al., 2019), cut off at 0.2 % read abundance per replicate.Alleles sorted by frequency (reads in parentheses).Alignment differences to wild-type reference indicated by color: adenine, red; guanine, yellow; thymidine, green; cytosine, blue; -, deletions.Injection mix components and replicate numbers provided.Anticipated alleles, red outlined box; base editing window, black outlined box; canonical PAM (orange); de novo PAM (green); non-synonymous codon changes, purple box; pre-termination STOP codon, black box Development: doi:10.1242/dev.201115:Supplementary information Development • Supplementary information

Fig. S7. Allele frequency table and translation of aligned targeted Illumina amplicon sequencing reads of kcnh6a inception and control editants
Anticipated alleles, red outlined box; base editing window, black outlined box; canonical PAM (orange); de novo PAM (green); non-synonymous codon changes, purple box Development: doi:10.1242/dev.201115:Supplementary information Development • Supplementary information