Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing
Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing
Anabela S. Ramalho
Kris De Boeck
Marianne S. Carlon
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene.
The 3272–26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR
gene, generating new acceptor and donor splice sites respectively. Here we develop a
genome editing approach to permanently correct these genetic defects, using a single crRNA and
the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise,
showing very strong discrimination between the wild-type and mutant sequence and a
complete absence of detectable off-targets. The efficacy of this gene correction strategy is
verified in intestinal organoids and airway epithelial cells derived from CF patients carrying
the 3272–26A>G or 3849+10kbC>T mutations, showing efficient repair and complete
functional recovery of the CFTR channel. These results demonstrate that allele-specific
genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way
for a permanent splicing correction in genetic diseases.
C disorder with an approximate frequency of 1 in 2500
ystic fibrosis (CF) is a lethal autosomal recessive inherited
births. CF is linked to mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene1, which
encodes a chloride/bicarbonate channel expressed in the apical
membrane of epithelial cells. The lack of ion conductance across
the membrane of these cells leads to impaired ion and liquid
homeostasis, generating a multi-organ disorder. The primary
cause of mortality in CF patients is bacterial infections of the
airways, provoking chronic lung disease and ultimately
respiratory failure2. Current CF treatments are not curative and limited
to the reduction of clinical symptoms including intestinal-airway
blockages and chronic bacterial infections. Recent therapeutic
advances were obtained in CF treatments through the
development of CFTR correctors and potentiators3,4, which however
target exclusively few types of mutations including the highly
In search for a cure for CF, several gene therapy approaches
have been explored5, mostly based on CFTR cDNA gene addition
through viral or non-viral vectors6,7. Despite promising results
obtained in the respiratory tract of animal models8–10 and
advancements in gene therapy clinical trials11–14, curative goals
were hampered mainly by low expression levels of the delivered
The recent advances in genome editing, with the development
of precise and efficient CRISPR-nucleases, have highly accelerated
the progress of gene correction for genetic diseases, including
CF15. Following initial discovery of the Streptococcus pyogenes
Cas9 (SpCas9), several additional CRISPR-nucleases have been
discovered with different functional, mechanistic and structural
features, expanding the genome editing tool-box16. Among these,
AsCas12a has been widely used for its different PAM
requirements and a natural very high specificity17–20.
By means of genome editing, in contrast to the classical gene
addition strategies, the correction of the mutated CFTR holds the
promise to restore physiological levels of CFTR expression and
function. In CF cellular models CFTR genetic repair was obtained
through strategies exploiting the cellular homology-directed
repair (HDR) pathway21,22. Nevertheless, the HDR pathway is
not highly active in human cells, thus strongly limiting the clinical
efficacy of this application23,24. Alternatively, genetic
modification by non-homologous end joining (NHEJ) is more efficient
and does not require the delivery of a donor DNA template.
CRISPR-nucleases have been successfully used to induce NHEJ to
knockout genes or genomic regulatory elements25,26. This
includes permanent repair of splicing defects in CFTR minigene
models27 as an alternative to the transient and inefficient use of
oligonucleotides or spliceosome-mediated RNA trans-splicing
Here we develop a genome editing strategy to repair
327226A>G (c.3140-26A>G) and 3849+10kbC>T (c.3718-2477C>T)
CFTR mutations. The 3242-26A>G is a point mutation that
creates a new acceptor splice site causing the abnormal inclusion
of 25 nucleotides within exon 2030,31. The resulting mRNA
contains a frameshift in CFTR, producing a premature
termination codon and consequent expression of a truncated
nonfunctional CFTR protein. The 3849+10kbC>T mutation creates a
novel donor splice site inside intron 22 of the CFTR gene, leading
to the insertion of the new cryptic exon of 84 nucleotides, which
results in an in-frame stop codon and consequent production of a
truncated non-functional CFTR protein32,33.
In this study, we harness the AsCas12a nuclease with a single
CRISPR RNA (crRNA) to repair the CFTR 3272–26A>G and
3849+10kbC>T splicing defects in different cell types including
primary CF patients’ airway epithelial cells and intestinal
organoids. The genome editing strategy that we develop is highly
specific, as demonstrated by a preserved second allele and
complete absence of off-target cleavages. CFTR functional recovery by
the AsCas12a single crRNA strategy is validated in
intestinal organoids derived from CF patients carrying the
327226A>G or the 3849+ 10kbC>T mutations, thus highlighting the
power of this approach for the permanent correction of genetic
diseases caused by deep intronic splicing mutations.
Splicing correction of a 3272-26A>G minigene model.
Minigenes are modeled exon–intron genetic constructs that are useful
for studying RNA splicing regulation34–36. These constructs are
commonly used to study specific cis-acting elements and their
binding factors involved in constitutive or alternative splicing
We generated minigene models to mimic the splicing pattern
of the CFTR gene corresponding to the region encompassing part
of exon 18, full length exons 19 and 20, and intron 19 (legacy
name: exon 16, 17a, 17b, and intron 17a), either wild-type
(pMG3272-26WT) or carrying the 3272-26A>G mutation
(pMG3272-26A>G) (Fig. 1a). The altered or correct splicing
pattern produced by the mutated or wild-type minigenes,
respectively, was evaluated by RT-PCR and sequencing analyses
in transfected HEK293T cells (Supplementary Fig. 1a, b)30.
We then designed single guide RNA (sgRNA)16 and crRNA,
for SpCas9 and AsCas12a, respectively (Supplementary Data 1),
to generate single or double cleavages to obtain either isolated
indels or deletions within intron 19, near the 3272-26A>G
mutation. The splicing pattern of the pMG3272-26A>G was
evaluated after its transient co-transfection with the sgRNAs/
crRNAs in combination with either SpCas9 or AsCas12a
(Supplementary Fig. 2a, b). We observed increased levels of
the correct splicing product by using either SpCas9 with at least
four sgRNA pairs (−52/+9, −47/−0, −47/+9, −47/+10)
(Supplementary Fig. 2a) or AsCas12a in with crRNAs −2 or
+11 used individually or in combination with another crRNA
(Supplementary Fig. 2b). Analysis of the deletions induced by
sgRNA pairs showed that SpCas9 efficiently cuts the expected
DNA fragments (Supplementary Fig. 2c). Conversely, weak
deletion products were detected with AsCas12a in samples
where splicing was not repaired (compare Supplementary
Fig. 2b and d).
To further validate the activity of SpCas9 or AsCas12a with the
selected sgRNAs/crRNAs within a more physiological chromatin
context, we tested the splicing correction of CFTR intron 19 in
HEK293 cells stably transfected with the pMG3272-26A>G
minigene (HEK293/3272-26A>G). Unexpectedly, all the
SpCas9–sgRNA pairs failed to correct the splicing defect, suggesting
inefficient cleavage at the chromosomal level (Supplementary
Fig. 2e, f). Conversely, the AsCas12a-crRNA+11 generated high
amounts of correct transcripts from the pMG3272-26A>G
transgene (more than 60%, Fig. 1b, c and Supplementary Fig. 2g)
and efficient DNA editing (67.4%, Fig. 1d).
The tracking of indels by decomposition (TIDE) analysis of the
integrated minigenes38, following editing with AsCas12a-crRNA+
11, revealed a heterogeneous pool of deletions (Supplementary
Fig. 3a). The edited variants were cloned into the
pMG327226A>G minigene to analyze the individual editing events and
their derived splicing products (Fig. 1e). Sequence analysis of the
edited sites showed a high frequency of 18 nucleotides deletions,
which is consistent with the editing profile of AsCas12a (deletions
bigger than four bases20), along with the persistence of the
327226A>G mutation (Fig. 1e and Supplementary Fig. 3a). Notably,
the splicing analysis revealed that the frequent 18 nucleotides
deletion (9/34 clones) fully restored the correct splicing (Fig. 1e
pMG3272-26WT ex 19
Ctr +11 –2
% correct splicing
20 40 60 80 100
CFTR intron 19 minigene models ex 20 ex 20 A>G
and Supplementary Fig. 3b). Most of the remaining edited sites,
occurring at low frequency (1/34 clones), generated a correct
splicing, accompanied in few cases by an additional transcript
product (Supplementary Fig. 3b). Overall, the large majority
(68%) of analyzed editing events contributed to the effective
restoration of normal splicing in the bulk cell population. In silico
analysis39,40 of the most frequent editing events (above 1% indel
frequency) shows that the large majority of the indels decrease the
strength of the cryptic splice sites activated by the 3272-26A>G
CF mutation (Supplementary Fig. 3c).
In conclusion, AsCas12a in combination with a single guide
RNA (crRNA+11) generates small deletions upstream of the
3272-26A>G mutation in a minigene model, producing efficient
recovery of the CF splicing defect.
Precision of the AsCas12a-based 3272-26A>G correction. The
large majority of CF patients are compound heterozygous for the
3272-26A>G mutation, thus requiring a careful evaluation of the
potential AsCas12a-crRNA+11 modifications within the other
mutant allele, having the wild-type 3272–26 sequence.
The cleavage properties of the AsCas12a-crRNA+11 were
analyzed in stable cell lines expressing either pMG3272-26WT or
pMG3272-26A>G (HEK293/3272-26WT and
HEK293/327226A>G cells, respectively). As shown in Fig. 2a, cleavage
efficiency of crRNA+11 dropped from 77%, detected in
HEK293/3272-26A>G, to 7.5% in HEK293/3272-26WT,
demonstrating at least 10-fold differential cleavage between the mutant
and wild-type allele. Reciprocal experiments with crRNA+11/wt,
targeting the CFTR 3272-26WT sequence showed high cleavage
efficiency (86.5%) in HEK293/3272-26WT and low indels
formation (12%) in HEK293/3272-26A>G (Fig. 2a), thus
demonstrating high allelic discrimination by AsCas12a with the
The specificity of the AsCas12a-crRNA+11 delivered by
lentiviral vectors towards the wild type intron, was further
confirmed in Caco-2 epithelial cells endogenously expressing the
wild type CFTR gene. Long-term nuclease expression (10 days
after transduction), which has been demonstrated to highly favor
non-specific cleavages41, did not generate any unspecific CFTR
editing above TIDE background levels (about 1%)38; whereas
AsCas12a-crRNA+11/wt efficiently edited the CFTR gene
(86.3%, Fig. 2b).
To exclude splicing alterations following potential wild-type
intronic cleavages, the splicing pattern was evaluated in HEK293/
3272-26WT and Caco-2 cells: no major alterations were observed
following AsCas12a treatment in combination with either crRNA+
11/wt or crRNA+11 (Supplementary Fig. 4a, b).
The specificity of the AsCas12a-crRNA+11 was also tested in
terms of off-target cleavages by a genome-wide survey,
GUIDEseq35,36. Off-target profiling of AsCas12a-crRNA+11 genome editing
in HEK293/3272-26A>G cells18,42 showed very high specificity, as
demonstrated by exclusive editing of the 3272-26A>G CFTR locus,
while non-specific cleavages in the second allele, or any other
genomic loci, could not be detected (Fig. 2c and Supplementary
3272-26A>G splicing correction in primary airway cells. The
efficacy of the 3272-26A>G correction by AsCas12a-crRNA+11
was further validated in primary airway epithelial cells derived
from a patient compound heterozygous for the 3272-26A>G
splicing mutation (3272-26A>G/ΔF508). Human primary airway
epithelial cells are a physiologically relevant 2-D model for CF
disease modeling and preclinical testing of CF therapies43,44. As
expected, two different transcripts were detected in these cells
(Fig. 3a), whose difference in size and abundance is consistent
with the cells heterozygosity for the 3272-26A>G mutation and in
agreement with previous data30. A 13-fold correction of the
aberrant 3272-26A>G splicing was obtained by lentiviral delivery
of AsCas12a-crRNA+11 either with or without puromycin
selection (+25nt isoform: 18.8% control sample, 1.4% crRNA+11,
0.3% crRNA+11 puro; Fig. 3a and Supplementary Fig. 5a–c).
The editing efficiency was evaluated by TIDE analysis revealing
30% of indels (up to 42% following puromycin selection, Fig. 3b
and Supplementary Fig. 5d). Of note, since TIDE sequencing does
not distinguish between the two CFTR alleles (3272-26A>G/
ΔF508), the allelic discrimination of our genome editing approach
was evaluated by deep sequencing, revealing 78.7% editing of the
3272-26A>G allele and complete absence of indels in the second
CFTR allele (3272-26WT allele, Supplementary Fig. 6a, b). To
further evaluate the off-target profile in primary airway epithelial
cells, computationally predicted off-target sites with up to four
mismatches (12 sites) were analyzed by deep sequencing.
Consistent with GUIDE-seq results (Fig. 2c) no off-target cleavages
were observed (Supplementary Fig. 6c).
Splicing correction in 3272-26A>G intestinal organoids.
Human organoids represent a near-physiological model for
translational research45. Intestinal organoids from CF patients are
+25 nt (%)
valuable tools to evaluate CFTR channel activity and functional
Encouraged by the splicing correction and allele specificity
obtained in minigene cell models and in primary airway epithelial
cells, we next evaluated the rescue potential of the CF phenotype
by AsCas12a-crRNA+11 in human intestinal organoids
compound heterozygous for the 3272-26A>G mutation
As observed in primary epithelial airway cells, the splicing
pattern of CFTR intron 19 in the crRNA control and untreated
organoids showed two transcript variants (Fig. 3c). Lentiviral
delivery of AsCas12a-crRNA+11 showed nearly complete
disappearance of the altered splicing product generated by the
3272-26A>G allele (+25nt), indicating efficient correction of
the aberrant intron 19 splicing (Fig. 3c and Supplementary
Fig. 7a, b). The amount of indels induced by AsCas12a-crRNA
+11 was initially evaluated by the T7 Endonuclease I assay,
resulting in approximately 30% editing of the CFTR locus
(Fig. 3d), which is consistent with the degree of restored
splicing observed in Fig. 3c.
Deep sequencing analysis revealed 40.25% indels in the CFTR
locus (39.77% within the 3272-26A>G allele and 0.48% within the
other allele, Fig. 3e), thus confirming the high efficiency of
AsCas12a-crRNA+11 editing observed with the T7 Endonuclease
I assay (Fig. 3d). Further sequence analysis revealed that 84.9% of
the sequencing reads including the 3272-26A>G mutation
contained variable length deletions, while sequencing reads
corresponding to the other allele (3272-26WT) contained only
0.9% indels, thus indicating a 94-fold allelic discrimination
In agreement with previous reports49,50, and despite the
heterogeneity of the observed editing, the repair events in
patient’s organoids were largely similar to those observed in
pMG3272-26A>G model, with the 18 nucleotide deletion as the
most frequent repair (compare Fig. 3e with Fig. 1e). Notably, this
18 nucleotides deletion, as well as most of the other reported
indels (with a frequency above 0.5% of total DNA repair
events, Fig. 3e), generated splicing correction when cloned in
the pMG3272-26 model (Fig. 1e).
Lumen formation and increased organoid size of intestinal
organoids (swelling) depends on the activity of the CFTR anion
channel46 (schematized in Fig. 3g) and thus can be used to measure
the restoration of CFTR function after AsCas12a-crRNA+11
genome editing. Fourteen days post AsCas12a-crRNA+11
treatment the patient’s organoids showed a 2.5-fold increased
organoid area at steady-state compared to the organoids of
control and untreated samples, thus indicating restored channel
function following repair of the CFTR 3272-26A>G allele (Fig. 3h,
i). Noteworthy, there was no significant difference in organoid
area between treatment with AsCas12a-crRNA+11 or
transduction of WT CFTR cDNA (Fig. 3i), further supporting the
remarkable efficiency of the AsCas12a-crRNA+11 genetic editing
in 3272-26A>G phenotypic reversion.
In addition to demonstrating a rescue in steady-state CFTR
function (organoid swelling after treatment with
AsCas12acrRNA+11), CFTR function was also assessed by the
wellestablished forskolin-induced swelling (FIS) assay46 (Fig. 3h, j).
Consistent with the data in Fig. 3i, the FIS assay revealed an
increase in AsCas12a-edited organoid area of 2.8-fold, which is
similar to the results obtained with lentiviral delivery of WT
CFTR cDNA (Fig. 3j and Supplementary Fig. 7c).
In light of these results, we conclude that AsCas12a-crRNA+11
modifications of the 3272-26A>G defect in patient’s organoids
allows the repair of the intron 19 splicing defect, leading to full
recovery of the endogenous CFTR protein function.
Genetic correction of the CFTR 3849+10kbC>T splicing
defect. To further evaluate the broader application of the
developed AsCas12a-crRNA editing strategy, the CFTR 3849
+10kbC>T splicing mutation was investigated. This genetic
variant generates the inclusion of a cryptic exon of 84 nucleotides
in the CFTR mature mRNA, which is translated into a truncated
defective anion channel33.
We generated minigene models (pMG3849+10kbWT and
pMG3849+10kbC>T) containing exon 22, part of intron 22 and
exon 23 (legacy name: exon 19, intron 19 and exon 20)
(schematized in Fig. 4a) that were demonstrated to mimic either
wild-type or defective CFTR splicing (Supplementary Fig. 8a–c).
The crRNA+14, targeting the 3849+10kbC>T mutation, showed
a complete correction of the altered splicing in combination with
AsCas12a in the minigene model (Fig. 4b). Moreover, lentiviral
transduction of AsCas12a-crRNA+14 in Caco-2 cells, generated
indels (3.5%) near background levels in the wild-type CFTR gene,
while the AsCas12a-crRNA+14/wt, targeting the wild-type
sequence in the same region, produced 64% CFTR editing, thus
indicating specificity of the AsCas12a-crRNA+14 towards the
mutant allele (Fig. 4c).
To further verify the AsCas12a-crRNA+14 specificity, also in
terms of genome-wide off-target activity, GUIDE-seq analysis was
performed in HEK293T cells, showing the complete absence of
sequence reads in the CFTR locus or in any other off-target site;
the 631 sequencing reads corresponding to spontaneous DNA
breaks are indicative of the proper execution of the GUIDE-seq
assay (Supplementary Fig. 4c).
The CFTR splicing pattern was then analyzed in airway
epithelial cells derived from a compound heterozygous patient
carrying both the 3849+10kbC>T and the ΔF508 mutations. The
aberrant splicing generated by the 3849+10kbC>T mutation
(+84 nt transcript) could be reversed following lentiviral delivery
of the mutation specific AsCas12a-crRNA+14 (Fig. 4d), which
correlated with 20% indels (30% after puromycin selection,
Fig.4e). Allelic discrimination of crRNA+14 was evaluated by
deep sequencing, revealing 70.7% cleavages in the 3849+10kbC>T
locus and fully preserved second CFTR allele (3849+10kbWT
allele, Supplementary Fig. 9a, b). The editing precision was
further evaluated through deep-sequencing analysis of three
predicted off-target sites (up to four mismatches), showing
complete absence of non-specific cleavages (Supplementary
Fig. 9c) as observed with the GUIDE-seq analysis in
HEK293Tcells (Supplementary Fig. 4c).
The genome editing strategy was verified in intestinal
organoids, derived from a compound heterozygous patient
carrying the 3849+10kbC>T mutation (3849+10kbC>T/
ΔF508). Lentiviral delivery of AsCas12a crRNA+14 produced
30% of indels in the CFTR loci modifying the aberrant splicing
site (Fig. 4f, g and Supplementary Fig. 10a, b), leading to the
rescue of organoid swelling as strong as the one observed after
CFTR cDNA addition (Fig. 4h, i). Sequencing analysis aimed at
evaluating allelic discrimination, confirmed the extreme precision
of this genome editing approach (Fig. 4g and Supplementary
Finally, we evaluated the editing efficacy of SpCas9 using the
pMG3849+10kbC>T minigene. We found that, consistently with
the most commonly used genome editing strategies27,51, SpCas9
reversed the splicing defects exclusively with sgRNA pairs which
deleted the intronic region containing the mutation
(Supplementary Fig. 11a, b); in contrast to AsCas12a, SpCas9 combined with
an individual sgRNA targeting the mutation (+1, +5, +18) did
not repair the splicing defect (Supplementary Fig. 11a).
The best identified sgRNAs pair, −95/+119, was selected
among those generating the expected deletion in the CFTR locus
and specifically repairing the aberrant splicing (Supplementary
Fig. 11c–e). This SpCas9–sgRNA pair induced an increase in
organoid area which was significantly lower than the increase
observed in organoids after lentiviral delivery of the CFTR cDNA
(Supplementary Fig. 11f, g), thus suggesting a lower efficacy than
the one obtained with AsCas12a (Fig. 4h, i). Moreover, in contrast
to the allele specificity of the single AsCas12-crRNA+14, the
SpCas9–sgRNA pairs, which are necessary for functional
correction, produced deletions also in the non-targeted second
allele (ΔF508). Therefore, the final genome editing efficacy should
be considered diluted over the two alleles. In addition, although
our sgRNA pool was designed in silico to minimize the
probability of SpCas9 off-target activity52, the GUIDE-seq assay
for sgRNA+119 revealed 11 off-target sites throughout the
genome (Supplementary Figs. 11h and 4d).
In conclusion, similarly to the splicing repair of the
327226A>G variant, the correction of the CFTR 3849+10kbC>T
splicing defect was efficiently and precisely obtained by using
AsCas12a combined with a single allele specific crRNA in CF
patient-derived organoids. This strategy was proven superior to
9+ pGM38 sa+AC
G r 4
M t 1
p C +
caCFTR intron 22 minigene models
The most advanced strategies so far developed for CF gene
therapy are based on the delivery, preferably in the lungs, of a
copy of the CFTR cDNA to compensate patients’ defective CFTR
gene. The main limitation of this gene therapy strategy is the low
and non-permanent CFTR expression obtained in the affected
tissues7,53. CFTR expression below therapeutic benefit is mainly
due to loss of the trans-gene during the rapid turnover of
pulmonary epithelial cells and inefficient lung transduction;
conditions that are further worsened by disease symptoms in CF
patients. Viral and non-viral vectors have proven valid to deliver
the CFTR cDNA in cell and animal models of CF8,10. Among
these are the adenoviral and adeno-associated-viral (AAV)
delivery systems which, however, are either associated with
immune responses or necessitate multiple treatments due to their
transient cellular persistence54,55. Lentiviral vectors recently
reached the clinic for the treatment of a severe combined
immunodeficiency56. This delivery system with improved genome
safety features over the original retroviral vectors, offer significant
advantages in terms of deliverability in both dividing and
nondividing cells. In the CF clinical field recent pre-clinical studies
provided encouraging results on lentiviral-mediated CFTR
delivery which are leading to the preparation of a first-in-man
lentivirus trial in patients with CF11,57.
Latest advances in genome editing, mainly represented by
CRISPR-nucleases, offer the unprecedented opportunity to
efficiently repair genetic defects within the endogenous CFTR locus
to permanently restore the physiological expression of the gene.
Furthermore, genetic correction does not require continuous
expression of the nuclease thereby bypassing the need for a stable
transgene expression58. Several methods have been described to
limit continuous expression of lentiviral transgenes as natural
promoter silencing and self-limiting CRISPR-Cas genetic
The CRISPR-nuclease SpCas9 was harnessed to correct the
ΔF508 mutation by homology-directed repair (HDR)22.
Nevertheless, the low efficiency of HDR in human cells and the
requirement of the additional delivery of a DNA donor template
pose several hurdles for a future clinical translation. However, the
delivery of CRISPR-SpCas9 with multiple sgRNAs was shown to
correct CFTR intronic splicing mutation in cellular minigene
models by inducing deletions, although quite large in size (around
150–200 bp), of the intronic mutation and surrounding
In our study, we developed a genome editing strategy using one
of the most precise programmable nucleases18, AsCas12a, that
permanently corrects CFTR splicing defects of at least two
relevant splicing mutations (3272-26A>G and 3849+10kbC>T) in
combination with a single crRNA. This approach is based on
small deletions (about 4–26 nt) within intronic sequences which
remove essential splicing regulatory elements forming aberrant 3′
or 5′ cryptic splice sites. Indeed, our sequencing data indicate that
the AsCas12a strategy likely inactivates the cryptic 3′ splice site
(poly-pyrimidine-T-rich-tract and last essential 3′-intronic
nucleotides) activated by the CFTR 3272-26A>G mutation and
the 5′ cryptic donor splice site in intron 22 activated by the 3849
+10kbC>T. This was confirmed by the in silico analysis of the
As opposed to AsCas12a, SpCas9 splicing repair required
multiple sgRNAs thus strictly depending on efficient concomitant
cleavages of the target sites and efficient co-delivery by the
The high-fidelity AsCas12a genetic repair18 applied in this
study to CFTR splicing defects, resulted in an allele specific and
genome-wide off-target free editing. Allelic discrimination was
obtained by designing the crRNA target sequence on the
mutations (3272-26A>G and 3849+10kbC>T), thus providing the
relevant advantage of minimizing the potential risk of on-target
We demonstrated that our AsCas12a-based gene correction
strategy efficiently corrects the splicing pattern in human primary
airway epithelial cells and rescued endogenous CFTR function in
patient derived intestinal organoids, which are recognized as a
highly valuable preclinical model to predict ex vivo therapeutic
efficacy in CF patients47, providing an important milestone for
future clinical trials. Even though CFTR modulators (i.e.
potentiator VX-770, Kalydeco, Vertex) are used in clinic also for CF
patients carrying the splicing mutations of this study, these drugs
are associated with side effects and strongly depend on the
residual correct splicing of mutant CFTR, which is extremely reduced
and variable among patients30–33. This explains the urgent need
for a permanent correction of physiologic levels of CFTR,
potentially reachable with the genome editing approach described
in our study.
In conclusion, this work sets a robust proof-of-concept to treat
two different deep intronic mutations by genome editing using
the AsCas12a-crRNA nuclease system, which can be broadened
to other splicing defects in CF and even to other genetic diseases
caused by splicing alterations.
Plasmids. Wild type and mutated minigenes for 3272-26A>G and 3849+10kbC>T
mutations were cloned into previously published pcDNA3 and pCI plasmid,
respectively62,63. Wild type minigenes (pMG3272-26WT and pMG3849
+10kbWT) were obtained by PCR amplification and cloning of target regions from
CFTR gene of HEK293T cell. Primers used are listed in Supplementary Data 4. The
pMG3272-26WT plasmid includes the last 23 bases of exon 18, full length exons 19
and 20, and intron 19. The addition of the last part of exon 18 was performed by
semi-nested PCR: the first PCR was done with oligo TM exon 18 exon 19 hCFTR
fw and oligo exon 20 hCFTR rev; the second PCR was done with the forward
primer oligo KpnI-AgeI exon 18–19 hCFTR fw and the same reverse oligo. The
pMG3849+10kbWT plasmid contains full length exons 22 and 23, and portions of
intron 22 as previously described35. Mutated minigenes (pMG3272-26A>G and
pMG3849+10kbC>T) were obtained by site-directed mutagenesis of
pMG327226WT and pMG3849+10kbWT, for both mutations. Plasmid sequences are
described in Supplementary Data 5. Guide RNAs were cloned into pY108
lentiAsCas12a (Addgene Plasmid 84739) or lentiCRISPR v1 (Addgene Plasmid 49535)
using BsmBI restriction sites as previously described64. These plasmids allow
simultaneous delivery of the RNA-guided nuclease and the sgRNA to target cells
and contain puromycin as selection marker.
Cell lines. Human colorectal adenocarcinoma cells (Caco-2), HEK293T cells and
HEK293 cells stably expressing pMG3272-26WT (HEK293/pMG3272-26WT) or
3272-26A>G cells (HEK293/pMG3272-26A>G) were cultured in Dulbecco's
modified Eagle's medium (DMEM; Life Technologies) supplemented with 10%
fetal bovine serum (FBS; Life Technologies), 10 U/ml antibiotics (PenStrep, Life
Technologies) and 2 mM L-glutamine at 37 °C in a 5% CO2 humidified atmosphere.
Puromycin selection was performed using 10 μg/ml for Caco-2 cells, and 2 μg/ml
for HEK293T or HEK293 cells. HEK293T, HEK293, and Caco-2 cells were
obtained from American Type Culture Collection (ATCC; www.atcc.org). Stable
minigene cell lines (HEK293/3272–26A>G and HEK293/3272-26WT) were
produced by transfection of Bgl-II linearized minigene plasmids (pMG3272-26WT or
pMG3272-26A>G) in HEK293 cells. Cells were selected with 500 µg/ml of G418,
48 h after transfection. Single cell clones were isolated and characterized for the
expression of the minigene construct.
Transfection and lentiviral transduction of cell lines. Transfection experiments
were performed in HEK293T cells seeded (150,000 cells/well) in a 24-well plate and
transfected using polyethylenimine (PEI) with 100 ng of minigene plasmids and
700 ng of plasmid encoding for nuclease and sgRNA/crRNA (pY108 lentiAsCas12a
or lentiCRISPR v1). After 16 h incubation cell medium was changed, and samples
were collected at 3 days from transfection.
Lentiviral particles were produced in HEK293T cells at 80% confluency in
10 cm plates. 10 µg of transfer vector (pY108 lentiAsCas12a or lentiCRISPR v1)
plasmid, 3.5 µg of VSV-G and 6.5 µg of Δ8.91 packaging plasmid were transfected
using PEI. After over-night incubation the medium was replaced with complete
DMEM. The viral supernatant was collected after 48 h and filtered in 0.45 μm PES
filter. Lentiviral particles were concentrated and purified with 20% sucrose cushion
by ultracentrifugation for 2 h at 4 °C and 150,000 × g. Pellets were resuspended in
appropriate volume of OptiMEM. Aliquots were stored at −80 °C. Vector titres
were measured as reverse transcriptase units (RTU) by SG-PERT method65.
For transduction experiments HEK293/pMG3272-26WT or HEK293/
pMG3272-26A>G and Caco-2 cells were seeded (300,000 cells/well) in a 12-well
plate and the day after were transduced with three RTU of lentiviral vectors. 48 h
later, cells were selected with puromycin (2 μg/ml for HEK293 or 10 μg/ml for
Caco-2 cells) and collected 10 days from transduction.
Transcripts analysis. RNA was extracted using TRIzol™ Reagent (Invitrogen) and
resuspended in DEPC-ddH2O. cDNA was obtained starting from 500 ng of RNA
using RevertAid Reverse Transcriptase (Thermo Scientific), according to
manufacturer’s protocol. Target regions were amplified by PCR with Phusion High
Fidelity DNA Polymerase (Thermo Fisher). Oligonucleotides are listed in
Supplementary Data 4. Uncropped and unprocessed scans are available in the Source
Detection of nuclease-induced genomic mutations. Genomic DNA was
extracted using QuickExtract DNA extraction solution (Epicentre) and the target
locus amplified by PCR using Phusion High Fidelity DNA Polymerase (Thermo
Fisher). Oligos are listed in Supplementary Data 4. To evaluate indels resulting
from cleavage of one gRNA, purified PCR products were sequenced and analyzed
using the TIDE or the SYNTHEGO ICE software38,66. In some experiments DNA
editing was measured also by T7 Endonuclease 1 (T7E1) assay (New England
BioLabs) following manufacturer’s instructions and as previously described41.
Primary airway epithelial cell culture and transduction. Primary airway
(bronchial) epithelial cells were derived from CF patients compound heterozygous
for 3272-26A>G splicing mutation (3272-26A>G/ΔF508, n = 1) and for 3849
+10Kb C>T splicing mutation (3849+10Kb C>T/ΔF508, n = 1) (kindly provided
by the Primary Cell Culture Service of the Italian Cystic Fibrosis Research
Foundation). The Ethics Committee of the Istituto Giannina Gaslini approved this study
and informed consent was obtained from all participating CF subjects. Cells were
cultured in LHC9/RPMI 1640 (1:1) without serum67 and the day before the
transduction, 50,000 cells at passage 3, were seeded into a 24-well plate previously
treated with collagen. Transduction was performed with two RTU of lentiviral
vectors. Puromycin selection, where indicated, was performed with 2 μg/ml, 48 h
post transduction for 72 h. Cells were collected for analysis after 15 days.
Human intestinal organoids culture and transduction. Human intestinal orga
noids of CF subjects compound heterozygous for 3272-26A>G splicing mutation
(3272-26A>G/4218insT, n = 1, CF-86) and for 3849+10Kb C>T mutation (3849
+10Kb C>T/ΔF508, n = 1, CF-110) were derived from fresh rectum suction
biopsies and cultured as previously described46. The Ethics Committee of the
University Hospital Leuven approved this study and informed consent was
obtained from all participating CF subjects. Organoids cultures, at passages 10–15,
were trypsinized to single cell using trypsin 0.25% EDTA (Gibco),
30,000–40,000 single cells were resuspended with 25 µl of lentiviral vector (0.25–1
RTU) and incubated for 10 min at 37 °C8. The same amount of Matrigel (Corning)
was added and the mix plated in a 96-well plate. After polymerisation of the
Matrigel drops (37 °C for 7 min), they were covered with 100 µl of complete
organoid medium46 containing 10 µM of Rock inhibitor (Y-27632 2HCl, Sigma
Aldrich, ref. Y0503) for the first 3 days to ensure optimal outgrowth of single stem
cells48. Medium was replaced every 2–3 days until the day of organoid analysis.
Analysis of CFTR activity in intestinal organoids. Fourteen days after viral
vector transduction, organoids were incubated for 30 min with 0.5 µM
calceingreen (Invitrogen, ref. C3-100MP) and analyzed by confocal live cell microscopy
with a ×5 objective (LSM800, Zeiss, with Zen Blue software, version 2.3).
Steadystate organoids area was determined by calculating the absolute area (xy plane,
µm2) of each organoid using ImageJ software through the Analyze Particle
algorithm. Defective particles with an area <1500 or 3000 µm for 3272-26A>G or 3849
+10Kb C>T, respectively, were excluded from the analysis. Data were averaged for
each different experiment and plotted in a box plot representing means ± SD.
The FIS assay was performed by stimulating organoids with 5 µM of forskolin
and analyzed by confocal live cell microscopy at 37 °C for 60 min (one image every
10 min). The organoid area (xy plane) at different time points was calculated using
ImageJ, as described above.
GUIDE-seq. GUIDE-seq experiments were performed as previously described42.
Briefly, 2 × 105 HEK293T cells were transfected using Lipofectamine 3000
transfection reagent (Invitrogen) with 1 µg of lenti Cas12a plasmid (pY108) and 10 pmol
of dsODNs42. The day after transfection cells were detached and selected with 2 µg/
ml puromycin. Four days after transfection cells were collected and genomic DNA
extracted using DNeasy Blood and Tissue kit (Qiagen) following manufacturer’s
instructions. Using Bioruptor Pico sonicatin device (Diagenode) genomic DNA
was sheared to an average length of 500 bp. Library preparations were performed
with the original adapters and primers according to previous work. Libraries were
quantified with the Qubit dsDNA High Sensitivity Assay kit (Invitrogen) and
sequenced with the MiSeq sequencing system (Illumina) using an Illumina Miseq
Reagent kit V2-300 cycles (2 × 150 bp paired-end). Raw sequencing data (FASTQ
files) were analyzed using the GUIDE-seq computational pipeline42. After
demultiplexing, putative PCR duplicates were consolidated into single reads.
Consolidated reads were mapped to the human reference genome GrCh37 using
BWA-MEM; reads with mapping quality lower than 50 were filtered out. Upon the
identification of the genomic regions integrating double-stranded
oligodeoxynucleotide (dsODNs) in aligned data, off-target sites were retained if at most
seven mismatches against the target were present and if absent in the background
controls. Visualization of aligned off-target sites is available as a color-coded
sequence grid23,26. GUIDE-seq data are listed in Supplementary Data 3.
Targeted deep sequencing. The loci of interest were amplified using Phusion
high-fidelity polymerase (Thermo Scientific) from genomic DNA extracted from
human intestinal organoids (3272-26A>G/4218insT) 14 days after transduction
with lentiAsCas12a-crRNA +11, +14 or Ctr, either in organoids (on-target) or
airway cells (on and off-target). Amplicons were indexed by PCR using Nextera
indexes (Illumina), quantified with the Qubit dsDNA High Sensitivity Assay kit
(Invitrogen), pooled in near-equimolar concentrations and sequenced on an
Illumina Miseq system using an Illumina Miseq Reagent kit V3−150 cycles (150 bp
single read). Primers used to generate the amplicons are reported in Supplementary
Data 4. Raw sequencing data (FASTQ files) were analyzed using CRISPResso
online tool68, by setting Windows size = 3, Minimum average read quality
(phred33 scale) = 30 and minimum single bp quality (phred33 scale) = 10
(Supplementary Data 2).
In silico off-target analysis. Off-target for crRNA +11 and +14 were analyzed by
Cas-OFFinder online algorithm, by selecting: AsCpf1 from Acidaminococcus or
LbCpf1 from Lachnospiraceae 5'-TTTV-3', mismatch number≤4, DNA bulge size
= 0 and as a target genome the Homo sapiens (GRCh38/hg38)—Human.
In silico splicing prediction. For wild-type, mutated and edited CFTR gene
sequences, a region of 400 bp spanning either the 3272-26A>G or 3849+10Kb C>T
locus was analyzed by HSF and MaxEnt prediction algorithms39,40 available at the
Human Splicing Finder website (www.umd.be/HSF3/). Splice sites score were
normalized to the score of the 3272-26A>G, or 3849+10Kb C>T splice site
(Supplementary Data 6).
Statistical analyses. Statistical analyses were performed by GraphPad Prism
version 6. For organoids experiments ordinary one-way analysis of variance
(ANOVA) was performed. For the in silico prediction analyses Shapiro–Wilk test
was used to verify the distribution of the data. Significance of the data was
calculated by two-tailed Wilcoxon signed-rank test. Differences were considered
statistically different at P < 0.05.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.
GUIDE-seq and targeted deep-sequencing data have been deposited at BioProject
(https://www.ncbi.nlm.nih.gov/bioproject/) under the accession number PRJNA551109.
The source data underlying Figs. 1b–e, 2a, b, 3a–d, j, k, 4b–f, i and Supplementary
Figs. 1a, 2e-f, 3a, b, 4a, b, 5a–c, 7b, c, 8a, 11a–e, g are provided as a Source Data file. All
other relevant data are available from the authors upon reasonable request.
The authors are grateful to Daniele Arosio for helpful discussion throughout the
development of this study and to Liesbeth De Keersmaecker for her excellent technical
assistance. We thank Francesca Demichelis and Davide Prandi for continuing support in
GUIDE-seq analyses. This work was supported by the Italian Cystic Fibrosis Foundation
grant FFC#1/2017 adopted by Associazione Trentina Fibrosi Cistica in ricordo di Maria
Cainelli e Romana Petrolli, delegazioni FCC di Imola e Romagna, di Alborello, Lucca,
and by intramural funding from the University of Trento. We are grateful to Primary
Cell Culture Serviceof the Italian Cystic Fibrosis Research Foundation at the Laboratory
of Medical Genetics, G. Gaslini Institute, Genova, Italy, for CF primary cells. Work at the
KU Leuven was supported by grants from the King Baudouin Foundation, Belgium
[Fund Alphonse and Jean Forton, 3M140231] and a KU Leuven C3 grant OPIT-CF.
G.M., G.P., and A.C. conceived the study and designed the experiments; G.M. performed
most experiments; M.S.C., A.S.R., K.D.B., and Z.D. contributed to organoid experiments;
G.M., G.P., A.Ca., C.M., M.S.C., and A.C. analyzed the data; G.M., G.P., M.S.C., Z.D., and
A.C. wrote and edited the paper. All authors read, corrected, and approved the final
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Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-11454-9.